Proliferation Of MCF10A Cells Research Articles

Preparation, in vitro anti-tumour activity and in vivo pharmacokinetics of RGD-decorated liposomes loaded with shikonin

Shikonin (SHK) has been evidenced to possess effects against various cancer cells. However, poor aqueous solubility and high toxicity restrict its application. In the study, RGD-decorated liposomes loaded with SHK (RGD-Lipo-SHK) were prepared via thin-film hydration method. Characterization and cellular uptake of liposomes was evaluated. Cytotoxicity of blank liposomes and different SHK formulations was measured against breast cancer cells (MDA-MB-231, MCF-7, and MCF-10A). Anti-tumour effects and pharmacokinetic parameters of different SHK formulations were appraised in tumour spheroids and in rat model, respectively. Liposomes displayed a particle size of less than 127 nm with a polydispersity index about 0.21. The encapsulation efficiency was about 91% for SHK, and drug leakage rate of liposomes was less than 6%. RGD-Lipo-SHK showed superior cellular internalization in the αvβ3-positive MDA-MB-231 cells. Blank liposomes had no cytotoxicity to MDA-MB-231 and MCF-7 cells. Howbeit, different SHK formulations obviously inhibited proliferation of MCF-10A cells, especially free SHK. Meanwhile, RGD-Lipo-SHK significantly inhibited growth inhibition of tumour spheroids. The pharmacokinetics study indicated that the peak concentration, area under plasma concentration-time curves, half-life, and mean residence time of RGD-Lipo-SHK distinctly increased compared with those of free SHK. Altogether, these results demonstrated RGD-Lipo-SHK could reduce cytotoxicity, strengthen the antitumor-targeted effect, and prolong circulation time, which provides a foundation for further in vivo experimentations.

β2-Adrenoceptor Activation Favor Acquisition of Tumorigenic Properties in Non-Tumorigenic MCF-10A Breast Epithelial Cells.

Noradrenaline and adrenaline, and their cognate receptors, are currently accepted to participate in cancer progression. They may also participate in cancer initiation, although their role in this phase is much less explored. The aim of this work was to study the influence of adrenergic stimulation in several processes related to breast cancer carcinogenesis, using several adrenergic agonists in the MCF-10A non-tumorigenic breast cells. Activation of the β-adrenoceptors promoted an epithelial phenotype in MCF-10A cells, revealed by an increased expression of the epithelial marker E-cadherin and a decrease in the mesenchymal markers, N-cadherin and vimentin. MCF-10A cell motility and migration were also impaired after the β-adrenoceptors activation. Concomitant with this effect, β-adrenoceptors decrease cell protrusions (lamellipodia and filopodia) while increasing cell adhesion. Activation of the β-adrenoceptors also decreases MCF-10A cell proliferation. When the MCF-10A cells were cultured under low attachment conditions, activation the of β- (likely β2) or of α2-adrenoceptors had protective effects against cell death, suggesting a pro-survival role of these adrenoceptors. Overall, our results showed that, in breast cells, adrenoceptor activation (mainly through β-adrenoceptors) may be a risk factor in breast cancer by inducing some cancer hallmarks, providing a mechanistic explanation for the increase in breast cancer incidences that may be associated with conditions that cause massive adrenergic stimulation, such as stress.

Mechanism of anti-hyperplasia of mammary glands of Xihuang Pills blood-entering component based on UPLC-Q-TOF-MS and network pharmacology

In this study, based on network pharmacology and molecular docking method, the mechanism of anti-hyperplasia of mammary glands of Xihuang Pills blood-entering components was explored, and the efficacy and key targets of Xihuang Pills blood-entering components were experimentally verified by MCF-10A proliferation model of human mammary epithelial cells. In order to clarify the material basis and mechanism of Xihuang Pills in realizing anti-hyperplasia of mammary glands, the blood-entering components of Xihuang Pills were qualitatively analyzed by UPLC-Q-TOF-MS, and 22 blood-entering components were identified. By taking the blood-entering components as the research object, the network pharmacology prediction and molecular docking verification were carried out, and finally, three key targets were screened out, namely JAK1, SRC, and CDK1. In vitro experiments show that Xihuang Pills can inhibit the proliferation of MCF-10A cells, promote the apoptosis of MCF-10A cells, and reduce the expression of JAK1, SRC, and CDK1 targets in cells. To sum up, Xihuang Pills can promote the apoptosis of mammary epithelial cells by regulating the expression of JAK1, SRC, and CDK1 and then play an anti-hyperplasia role, which provides an experimental basis for clarifying the material basis of Xihuang Pills for anti-hyperplasia effect.

SEMA6D Differentially Regulates Proliferation, Migration, and Invasion of Breast Cell Lines.

Semaphorin 6D (SEMA6D), a member of the class 6 semaphorin family, is a membrane-associated protein that plays a key role in the development of cardiac and neural tissues. A growing body of evidence suggests that SEMA6D is also involved in tumorigenesis. In breast cancer, high SEMA6D levels are correlated with better survival rates. However, very little is known about the functional significance of SEMA6D in breast tumorigenesis. In the present study, we aimed to investigate the effects of SEMA6D expression on the normal breast cell line MCF10A and the breast cancer cell lines MCF7 and MDA MB 231. We demonstrated that SEMA6D expression increases the proliferation of MCF10A cells, whereas the opposite effect was observed in MCF7 cells. SEMA6D expression induced anchorage-independent growth in both cancer cell lines. Furthermore, migration of MCF10A and MCF7 cells and invasion of MDA MB 231 cells were elevated in response to SEMA6D overexpression. Accordingly, the genes related to epithelial-mesenchymal transition (EMT) were altered by SEMA6D expression in MCF10A and MCF7 cell lines. Finally, we provided evidence that SEMA6D levels were associated with the expression of the cell cycle, EMT, and Notch signaling pathway-related genes in breast cancer patients’ data. We showed for the first time that SEMA6D overexpression has cell-specific effects on the proliferation, migration, and invasion of normal and cancer breast cell lines, which agrees with the gene expression data of clinical samples. This study lays the groundwork for future research into understanding the functional importance of SEMA6D in breast cancer.

Oxocrebanine: A Novel Dual Topoisomerase inhibitor, Suppressed the Proliferation of Breast Cancer Cells MCF-7 by Inducing DNA Damage and Mitotic Arrest

BackgroundDNA topoisomerase (Topo) inhibition plays key role in breast cancer treatment. Stephania hainanensis H. S. Lo et Y. Tsoong (S. hainanensis), a Li nationality plant that has abundant aporphine alkaloids, can inhibit Topo. PurposeTo identify a dual Topo inhibitor, a deep and systematic study of active aporphine alkaloids in S. hainanensis and their mechanisms of inhibiting breast cancer proliferation and Topo activity are essential. Study designThis study aimed to assess the anti-breast cancer and Topo inhibitory activities of oxocrebanine and explore the underlying mechanisms. MethodsThe growth inhibitory activities of 12 compounds in S. hainanensis were screened by MTT assay in MCF-7, SGC-7901, HepG-2 cells, and compared with the effects on human normal mammary epithelial MCF-10A cells as non cancer control cells. The Topo inhibitory activity was assessed by DNA relaxation and unwinding assays, kDNA decatenation assay and western blot. Cell cycle and autophagy analyses were carried out with flow cytometry and staining. Acridine orange staining and α-tubulin morphology were observed by fluorescence microscopy. Western blot was used to examine microtubule assembly dynamics and the expression levels of key proteins associated with DNA damage, autophagy and mitotic arrest. ResultsOxocrebanine was the anti-breast cancer active alkaloid in S. hainanensis. It exhibited the best inhibitory effect on MCF-7 cells with an IC50 of 16.66 μmol/l, and had only weak effect on the proliferation of MCF-10A cells. Oxocrebanine inhibited Topo I and II α in a cell-free system and in MCF-7 cells. The DNA unwinding assay suggested that oxocrebanine intercalated with DNA as a catalytic inhibitor. Oxocrebanine regulated the levels of Topo I and IIα and DNA damage-related proteins. Oxocrebanine led to the mitotic arrest, and these effects occurred through both p53-dependent and p53-independent pathways. Oxocrebanine induced autophagy, abnormal α-tubulin morphology and stimulated enhanced microtubule dynamics. ConclusionOxocrebanine was the anti-breast cancer active aporphine alkaloid in S. hainanensis. Oxocrebanine was a Topo I/IIα dual inhibitor, catalytic inhibitor and DNA intercalator. Oxocrebanine caused DNA damage, autophagy, and mitotic arrest in MCF-7 cells. Oxocrebanine also disrupted tubulin polymerization. Accordingly, oxocrebanine held a great potential for development as a novel dual Topo inhibitor for effective breast cancer treatment.

Integration of proteomics and metabolomics reveals promotion of proliferation by exposure of bisphenol S in human breast epithelial MCF-10A cells

Bisphenol S (BPS) has been reported to have similar estrogenic effects as bisphenol A (BPA). Considering the endocrine disrupting effects of BPS, in this study, we investigated the effects of BPS exposure on normal human breast epithelial cell line MCF-10A by using mass spectrometry (MS)-based metabolomics and quantitative proteomics. We found that exposure to BPS for 24 h altered the proliferation of MCF-10A cells in a hormetic manner with the highest proliferation rate at the dosage of 1 μM. A total of 200 proteins were identified to be significantly changed by 1 μM of BPS exposure. The upregulation of epidermal growth factor receptor (EGFR) and Ras/mTOR-related proteins implied that EGFR-mediated pathways were involved in BPS-induced proliferation of MCF-10A cells. In addition, several proliferation-related protein markers were found to be elevated, such as MKI67 and CDH1, further indicating the promotion of proliferation by low dose of BPS exposure. Besides, 35 endogenous metabolites were found to be significantly changed. The joint pathway analysis of the altered metabolites and proteins suggested changes in pathways of tricarboxylic acid (TCA) cycle, purine metabolism, pyruvate metabolism and lipid metabolism, which were involved in sustaining cell proliferation and cellular signal transduction. Taken together, this study provides insights into the effects and the potential mechanisms of BPS on estrogen receptor α-negative normal breast cell line MCF-10A, broadening our knowledge about the risk of using BPS as the alternative of BPA.

Effects of phthalates on normal human breast cells co-cultured with different fibroblasts.

Whether or not phthalates play a role in breast carcinogenesis remains to be determined. The goal of this study was to explore the effects of phthalates on the growth of normal MCF-10A breast cells modulated by breast fibroblasts. Fibroblasts were derived from normal mammary tissue adjacent to both estrogen receptor (ER) positive and negative primary breast cancers, which were grown separately from nontumorigenic MCF-10A epithelial cells. MCF-10A co-culture cells were treated with 10 nM 17β-estradiol (E2), Butyl benzyl phthalate (BBP), di(n-butyl) phthalate (DBP), and di(20ethylhexyl) phthalate (DEHP) (10 and 100 nM). After incubation for 120 hours, the cells were harvested and extracted for MTT assay. Western blot analysis was used to evaluate the proliferative pathway proteins and the effects on ER α. Only fibroblasts from ER (+) breast cancer significantly stimulated proliferation of MCF-10A cells. Exposure of the co-culture to E2, BBP, DBP, DEHP, and E2 combined with one of these phthalates resulted in significantly increased cell proliferation, as well as proliferating cell nuclear antigen (PCNA) and ER α expressions. The present study demonstrates that phthalates express a significant influence in fibroblast–epithelial interactions, similarly to the effects of E2 on breast cells. The effects of phthalates on normal breast cells depend upon ER modulating actions. In breast carcinogenesis, phthalates should be considered as having endocrine disrupting potential, even at low concentrations.

Abstract 5478: The TRPA1 Ca2+-permeable channel mediates a non-canonical redox adaptation in cancer cells

Abstract Generation of reactive oxygen species (ROS), a natural byproduct of oxygen metabolism, occurs in all aerobic organisms at a controlled rate. Cancer cells are subjected to numerous cellular insults, including dysregulated oncogenes and dissociation from their natural extracellular matrix (ECM) niches, leading to the generation of high levels of ROS. Although cancer cells possess enhanced canonical antioxidant programs that neutralize ROS, they nevertheless exhibit prominent ROS levels in response to these insults, suggesting the existence of additional programs that allow cancer cells to tolerate elevated ROS. In this study, we provide evidence for an unconventional mechanism for redox adaptation involving the ROS-activated, Ca2+-permeable TRPA1 channel, which normally functions as an irritant receptor in sensory neurons but is highly upregulated in breast, lung, malignant neural sheath, and other tumors. We found that TRPA1 overexpression enhances survival and proliferation of MCF-10A cells under conditions of matrix-detachment and this effect was dependent on TRPA1 activation by ROS generated as loss of matrix anchorage. In MCF-10A acini, TRPA1 expression induced Ca2+ entry in ECM-deprived cells in the luminal space and promoted their survival and proliferation. Conversely, TRPA1 knockdown inhibited Ca2+ responses to ROS generated through ECM detachment and induced clearance of cells from the luminal space in breast cancer spheroids. TRPA1 was also activated by ROS-inducing chemotherapies and drove chemoresistance in breast, lung and malignant peripheral nerve sheath tumor cells, and its downregulation suppressed breast xenograft tumor growth and enhanced chemosensitivity. TRPA1 mediated these effects independent of antioxidant responses, but through activation of cellular survival and anti-apoptotic programs involving the RAS-ERK/AKT/mTOR pathways. Together, our findings describe an unexpected mechanism whereby cancer cells co-opt the neuronal TRPA1 channel in order to adapt to oxidative environments. This TRPA1-induced response is distinct from canonical redox adaptation mechanisms that rely on antioxidant program. As TRPA1 inhibitors are currently in clinical trials for pain and respiratory therapies, these studies raise the possibility of using these inhibitors as therapeutic chemosensitizers in TRPA1-enriched tumors. Citation Format: Nobuaki Takahashi, Hsing-Yu Chen, Isaac Harris, Daniel Stover, Roderick Bronson, Thomas Deraedt, Karen Cichowski, Alana Welm, Gordon Mills, Joan Brugge. The TRPA1 Ca2+-permeable channel mediates a non-canonical redox adaptation in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5478. doi:10.1158/1538-7445.AM2017-5478

MiRNA-1246 induces pro-inflammatory responses in mesenchymal stem/stromal cells by regulating PKA and PP2A.

The tumor microenvironment (TME) has an impact on breast cancer progression by creating a pro-inflammatory milieu within the tumor. However, little is known about the roles of miRNAs in cells of the TME during this process. We identified six putative oncomiRs in a breast cancer dataset, all strongly correlating with poor overall patient survival. Out of the six candidates, miR-1246 was upregulated in aggressive breast cancer subtypes and expressed at highest levels in mesenchymal stem/stroma cells (MSCs). Functionally, miR-1246 led to a p65-dependent increase in transcription and release of pro-inflammatory mediators IL-6, CCL2 and CCL5 in MSCs, and increased NF-κB activity. The pro-inflammatory phenotype of miR-1246 in MSCs was independent of TNFα stimulations and mediated by direct targeting of the tumor-suppressors PRKAR1A and PPP2CB. In vitro recapitulation of the TME revealed increased Stat3 phosphorylation in breast epithelial (MCF10A) and cancer cells (SK-BR-3, MCF7, T47D) upon incubation with conditioned medium (CM) of MSCs overexpressing miR-1246. Additionally, this stimulation enhanced proliferation of MCF10A cells, increased migration of MDA-MB-231 cells and induced attraction of THP-1 monocytic cells. Our data shows that miR-1246 acts as both key-enhancer of pro-inflammatory responses in MSCs and putative oncomiR in breast cancer, suggesting its influence on cancer-related inflammation and breast cancer progression.

Abstract A20: Insulin receptor signaling in mammary epithelial cells

Abstract Obesity has recently emerged as a major adverse factor in the prevalence and severity of many types of cancers, including breast cancer (BC). Obesity is often accompanied by hyperinsulinemia and the development of type 2 diabetes. Elevated circulating insulin levels are thought to mediate some of the adverse prognostic effects of obesity in BC (Goodwin et al., 2002). Insulin receptor (IR) is expressed in many human BCs and the activation of the IR/receptor tyrosine kinase signaling pathway is characteristic of most BCs (Mulligan et al., 2007). We hypothesize that ectopic expression of IR in BCs sensitizes BC cells to the action of insulin, leading to signaling events specific to epithelial cells, functionally distinct from the insulin responses in physiological insulin-target tissues. To further understand the role of IR in BC we have implemented the recently developed Mammalian Membrane Two Hybrid (MaMTH) (Petschnigg et al., 2014) screen to identify IR interactors in human mammary epithelial MCF10A cells. The ongoing screen has to date identified 99 IR protein-protein interactions (PPIs), 51 of which are insulin-dependent. Coimmunoprecipitation and shRNA knockdown analyses are being used to validate the PPIs and provide insight into the functional significance of IR associations for growth and proliferation of MCF10A cells. Ultimately, the epithelial cell IR-interactome is meant to provide an insight into the insulin contribution to the activation of RTK signaling in BC, and provide a roadmap for further exploration of IR signaling in cancer. Citation Format: Yekaterina Poloz, Mariam Z. El Sheikh, Julia Petschnigg, Bella Groisman, Max Kotlyar, Igor Jurisica, Igor Stagljar, Vuk Stambolic. Insulin receptor signaling in mammary epithelial cells. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A20.

Anti-proliferative actions of a synthetic REV-ERBα/β agonist in breast cancer cells

REV-ERBα and REV-ERBβ are nuclear receptors that are ligand-dependent transcriptional repressors. Heme is the natural ligand for these receptors, but several synthetic agonists and antagonists have been designed recently. The gene that encodes REV-ERBα, NR1D1, is closely associated with ERBB2, the gene that encodes the HER2 oncogene, which is amplified in HER2+ breast cancers. We examined the effect of a synthetic REV-ERB agonist, SR9011, on a range of estrogen receptor positive (ER+), ER−, HER2+, HER2− and triple negative breast cancer cell lines. We found that SR9011 suppressed proliferation of the breast cancer cell lines regardless of their ER or HER2 status. SR9011 had no effect on MCF10A cell proliferation. SR9011 appears to pause the cell cycle of the breast cancer cells prior to M phase. Cyclin A (CCNA2) was identified as a direct target gene of REV-ERB suggesting that suppression of expression of this cyclin by SR9011 may mediate the cell cycle arrest. These data indicate that synthetic REV-ERB ligands may hold utility in treatment of diseases associated with uncontrolled cellular proliferation such as cancer.

The chemokine CXCL9 exacerbates chemotherapy-induced acute intestinal damage through inhibition of mucosal restitution.

Acute intestinal damage induced by chemotherapeutic agent is often a dose-limiting factor in clinical cancer therapy. The aim of this study was to investigate the effect of chemokine CXCL9 on the intestinal damage after chemotherapy and explore the therapeutic potential of anti-CXCL9 agents. In vitro cell proliferation assay was performed with a non-tumorigenic human epithelial cell line MCF10A. Multiple pathway analysis was carried out to explore the pathway that mediated the effect of CXCL9, and the corresponding downstream effector was identified with enzyme-linked immunosorbent assays. Chemotherapy-induced mouse model of intestinal mucositis was prepared by a single injection of the chemotherapeutic agent 5-fluorouracil (5-FU). In vivo expression of cxcl9 and its receptor cxcr3 in intestinal mucosa after chemotherapy was determined by quantitative real-time PCR. Therapeutic treatment with anti-CXCL9 antibodies was investigated to confirm the hypothesis that CXCL9 can contribute to the intestinal epithelium damage induced by chemotherapy. CXCL9 inhibited the proliferation of MCF10A cells by activating phosphorylation of p70 ribosomal S6 kinase (p70S6K), which further promotes the secretion of transforming growth factor beta (TGF-β) as the downstream effector. A blockade of phospho-p70S6K with inhibitor abolished the effect of CXCL9 on MCF10A cells and reduced the secretion of TGF-β. The expression levels of cxcl9 and cxcr3 were significantly up-regulated in intestinal mucosa after 5-FU injection. Neutralizing elevated CXCL9 with anti-CXCR9 antibodies successfully enhanced reconstitution of intestinal mucosa and improved the survival rate of mice that received high-dose chemotherapy. CXCL9 inhibits the proliferation of epithelial cells via phosphorylation of p70S6K, resulting in the excretion of TGF-β as downstream mediator. CXCL9/CXCR3 interaction can exacerbate chemotherapeutic agent-induced intestinal damage, and anti-CXCL9 agents are potential novel therapeutic candidates for promoting mucosal restitution.

Phytoestrogens induce differential effects on both normal and malignant human breast cells in vitro

Objective To explore the effect and pathway of phytoestrogens in vitro on the growth of both normal and malignant breast cells.Methods Normal breast MCF-10A cells and breast cancer MCF-7 cells were incubated with 10 − 10–10 − 4 mol/l genistein, resveratrol, and quercetin (plasma concentrations in human: 10 nmol/l–10 μmol/l) for 48 h and were then extracted for a cell proliferation assay (MTT), and for a cell death assay (TUNEL) assay. The proteins involved in the proliferative and apoptotic pathways were evaluated by Western blot analysis. Additionally, a comparison with 17β-estradiol as well as an evaluation of the differential effects on estrogen receptors (ER) α and β were performed.Results MCF-7 cell proliferation was significantly inhibited at the concentrations greater than 10-4 mol/l for all three phytoestrogens and from 10 − 5 mol/l for resveratrol and quercetin. MCF-10A cell proliferation was significantly increased at the concentrations from 10 − 8 to 10 − 5 mol/l for genistein and resveratrol and only at 10 − 5 mol/l for quercetin. Apoptotic cells were significantly increased by these phytoestrogens in the MCF-7 cells. At a concentration of 10 − 7 mol/l of these phytoestrogens, a significant reduction of PI3K and Akt and an increase of Fas ligand, Fas-associated protein with death domain, cytochrome C, truncated Bid, caspase-9, and caspase-3 were noted in the MCF-7; PI3K and Akt were significantly increased in the MCF-10A. ERβ expression was significantly elevated in MCF-10A and MCF-7 with these phytoestrogens. The effects of estradiol on normal and malignant breast cells were completely opposite to those of phytoestrogens.Conclusions This study demonstrates that phytoestrogens have antiproliferative effects on breast cancer cells via an ER-dependent mechanism, even at low concentrations, but are also capable of maintaining the survival of normal breast cell via ER-independent or other mechanisms.

GPER mediates estrogen-induced signaling and proliferation in human breast epithelial cells and normal and malignant breast.

17β-Estradiol (estrogen), through receptor binding and activation, is required for mammary gland development. Estrogen stimulates epithelial proliferation in the mammary gland, promoting ductal elongation and morphogenesis. In addition to a developmental role, estrogen promotes proliferation in tumorigenic settings, particularly breast cancer. The proliferative effects of estrogen in the normal breast and breast tumors are attributed to estrogen receptor α. Although in vitro studies have demonstrated that the G protein-coupled estrogen receptor (GPER, previously called GPR30) can modulate proliferation in breast cancer cells both positively and negatively depending on cellular context, its role in proliferation in the intact normal or malignant breast remains unclear. Estrogen-induced GPER-dependent proliferation was assessed in the immortalized nontumorigenic human breast epithelial cell line, MCF10A, and an ex vivo organ culture model employing human breast tissue from reduction mammoplasty or tumor resections. Stimulation by estrogen and the GPER-selective agonist G-1 increased the mitotic index in MCF10A cells and proportion of cells in the cell cycle in human breast and breast cancer explants, suggesting increased proliferation. Inhibition of candidate signaling pathways that may link GPER activation to proliferation revealed a dependence on Src, epidermal growth factor receptor transactivation by heparin-bound EGF and subsequent ERK phosphorylation. Proliferation was not dependent on matrix metalloproteinase cleavage of membrane-bound pro-HB-EGF. The contribution of GPER to estrogen-induced proliferation in MCF10A cells and breast tissue was confirmed by the ability of GPER-selective antagonist G36 to abrogate estrogen- and G-1-induced proliferation, and the ability of siRNA knockdown of GPER to reduce estrogen- and G-1-induced proliferation in MCF10A cells. This is the first study to demonstrate GPER-dependent proliferation in primary normal and malignant human tissue, revealing a role for GPER in estrogen-induced breast physiology and pathology.

Comparison of cytotoxicity and genotoxicity of 4-hydroxytamoxifen in combination with Tualang honey in MCF-7 and MCF-10A cells

BackgroundThe Malaysian Tualang honey (TH) is not only cytotoxic to human breast cancer cell lines but it has recently been reported to promote the anticancer activity induced by tamoxifen in MCF-7 and MDA-MB-231 cells suggesting its potential as an adjuvant for the chemotherapeutic agent. However, tamoxifen produces adverse effects that could be due to its ability to induce cellular DNA damage. Therefore, the study is undertaken to determine the possible modulation of the activity of 4-hydroxytamoxifen (OHT), an active metabolite of tamoxifen, by TH in non-cancerous epithelial cell line, MCF-10A, in comparison with MCF-7 cells.MethodsMCF-7 and MCF-10A cells were treated with TH, OHT or the combination of both and cytotoxicity and antiproliferative activity were determined using LDH and MTT assays, respectively. The effect on cellular DNA integrity was analysed by comet assay and the expression of DNA repair enzymes was determined by Western blotting.ResultsOHT exposure was cytotoxic to both cell lines whereas TH was cytotoxic to MCF-7 cells only. TH also significantly decreased the cytotoxic effect of OHT in MCF-10A but not in MCF-7 cells. TH induced proliferation of MCF10A cells but OHT caused growth inhibition that was abrogated by the concomitant treatment with TH. While TH enhanced the OHT-induced DNA damage in the cancer cells, it dampened the genotoxic effect of OHT in the non-cancerous cells. This was supported by the increased expression of DNA repair proteins, Ku70 and Ku80, in MCF-10A cells by TH.ConclusionThe findings indicate that TH could afford protection of non-cancerous cells from the toxic effects of tamoxifen by increasing the efficiency of DNA repair mechanism in these cells.

Antiproliferative Effect of Portlandia Extracts and Their Fractions on Breast Cancer Cells

Aim: Rubiaceae is one of the largest families of plants and it includes the Jamaican genus Portlandia. This family has been used in several ethnomedicinal practices. Thus, in this study we investigated whether extracts of two species of Portlandia affect In vitro proliferation of breast cancer cells. Study Design: Metastatic (MDA-MB-231 and HeLa) cells were incubated in the absence or in the presence of Portlandia extracts. A human non-metastatic (MCF-10A) and normal (ATCC PCS-600-010) epithelial cell lines were used as control. Methodology: At the end of the study all cell lines were incubated with Portlandia extracts (-and its subfractions-) and then analyzed on proliferation, thymidine incorporation and mitogenic and survival signaling pathways on metastatic and non-metastatic cells. Results: It was found that Portlandia methanol extract inhibited proliferation of MDA-MB231 in a dose-dependent manner attenuating the phosphorylation of Erk1/2 and Akt1 Original Research Article European Journal of Medicinal Plants, 4(10): 1186-1199, 2014 1187 without affecting the proliferation of MCF-10A cells. [H] thymidine incorporation was also decreased by Portlandia methanol extract. This study suggests that Portlandia extracts selectively affected the proliferation of metastatic breast cancer cells through the modulation of Erk1/2 and Akt1 activities that play a critical role during cell survival and proliferation. Conclusion: Portlandia leaf extracts contain active compounds, which strongly repress cancer cell proliferation in a dose-dependent manner and selectively down-regulate phosphorylation of both Erk1/2 and Akt1 activities.

Role of Sphk1 in the malignant transformation of breast epithelial cells and breast cancer progression.

The ojective of the following study is to investigate the role of sphingosine kinase 1 (Sphk1) in the malignant transformation of breast epithelial cells and breast cancer progression and its mechanism. Immunohistochemistry was performed to detect Sphk1 and E-cadherin (E-cad) in resected breast samples. Sphk1 was transfected in normal human breast epithelial cell line (MCF-10A) by Lentivirus and silenced in breast cancer cell line (MCF-7) using small interfering ribonucleic acid. The effect of tumor necrosis factor alpha (TNF-α) and/or N, N-dimethylsphingosine (DMS) on the Sphk1 and E-cad expression, MCF-10A cell proliferation and invasion was investigated. Real time-polymerase chain reaction and western-blot was used to detect messenger ribonucleic acid and protein. Cell counting kit-8 and transwell were used to measure cell proliferation and invasion. Sphk1 was positive expression in 114 breast tumors (75.50%) but negative in fibroadenomas. The expression of E-cad and Sphk1 were negatively correlated and E-cad (-)/Sphk1 (+) carriers showed higher ratio of axillary lymph node metastasis than E-cad (+)/Sphk1 (-) carriers. Overexpression of Sphk1 in MCF-10A reduced E-cad expression and improved cell proliferation and invasion, but knockdown of Sphk1 in MCF-7 decreased cell proliferation and invasion. TNF-α increased Sphk1 expression, enhanced the ability of Sphk1 in decreasing E-cad expression, which could be blocked by DMS. TNF-α promoted MCF-10A cell proliferation and invasion. Sphk1 plays an important role in the malignant transformation of breast epithelial cells and modulates breast cancer metastasis through the regulation of E-cad expression. TNF-α can up-regulate Sphk1 expression and reduce E-cad expression through Sphk1, which can be blocked by DMS. TNF-α/Sphk1/E-cad pathway may be a newly discovered pathway and plays an important role in tumorigenesis and metastasis.

CXCL9 Contributes To Chemotherapy-Induced Acute Intestinal Damage Through Proliferative Inhibition Of Epithelial Cells

Apart from angiostasis and chemoattraction, CXCL9 can derange hematopoiesis by its influence on mesenchymal stroma cells. The receptor of CXCL9, CXCR3, is abundantly expressed intracellularly in epithelial cells, even though it is rarely present on the surface of these cells. Here, we hypothesized that CXCL9 influences the proliferative and degenerative activity of epithelial cells in vitro and in vivo. CXCL9 inhibited the proliferation of MCF10A cells in a dose-dependent manner. In vivo, rhCXCL9 caused an intestinal weight loss of 30% in normal mice (n=6, 0.59±0.05 g of rhCXCL9 treated mice versus 0.83±0.06 g controls, P = 0.0007 determined by 2-tailed student's t-test). Intestinal epithelial cells of 5-Fluorouracil (5-FU) treated mice developed a 2.55 fold higher level of cxcl9 expression, indicating that CXCL9 may participate in a chemotherapy-induced damage of the intestinal epithelium. Neutralization of the up-regulated endogenous CXCL9 by anti-CXCL9 monoclonal antibodies accelerated epithelial regeneration determined by villi length (317.5±19.9 μm versus no-antibody control 283.7±17.1 μm, P < 0.001) and crypt depth (78.0±8 μm versus control 67.9±10.9 μm, P = 0.326). CXCL9 function was highly associated with p70 ribosomal S6 kinase (p70S6K) activation (50.0±2.2 MFI versus control 27.9±1.4 MFI, P = 0.007), which was reversed by anti-CXCR3 (31.4±5.7 MFI, n=4). CXCL9 downstreamingly stimulated TGF-β secretion of epithelial cells through the mTOR/p70S6K pathway (66.3±17.1 pg/mL versus on-treated control 39.8±12.2 pg/mL, P <0.05), which was reversed by anti-CXCR3 (46.8±21.6 pg/mL, n=8). That explains the anti-proliferative effect of CXCL9 on these cells. Our results strongly suggest that anti-CXCL9 may help to mitigate a chemotherapy-induced intestinal damage. The work was supported by the National Science Foundation China (81273576, 30801419, 30901873), and the German Academic Exchange Service (A/09/90104). Disclosures:No relevant conflicts of interest to declare.

New Castanospermine Glycoside Analogues Inhibit Breast Cancer Cell Proliferation and Induce Apoptosis without Affecting Normal Cells

sp2-Iminosugar-type castanospermine analogues have been shown to exhibit anti-tumor activity. However, their effects on cell proliferation and apoptosis and the molecular mechanism at play are not fully understood. Here, we investigated the effect of two representatives, namely the pseudo-S- and C-octyl glycoside 2-oxa-3-oxocastanospermine derivatives SO-OCS and CO-OCS, on MCF-7 and MDA-MB-231 breast cancer and MCF-10A mammary normal cell lines. We found that SO-OCS and CO-OCS inhibited breast cancer cell viability in a concentration- and time-dependent manner. This effect is specific to breast cancer cells as both molecules had no impact on normal MCF-10A cell proliferation. Both drugs induced a cell cycle arrest. CO-OCS arrested cell cycle at G1 and G2/M in MCF-7 and MDA-MB-231cells respectively. In MCF-7 cells, the G1 arrest is associated with a reduction of CDK4 (cyclin-dependent kinase 4), cyclin D1 and cyclin E expression, pRb phosphorylation, and an overexpression of p21Waf1/Cip1. In MDA-MB-231 cells, CO-OCS reduced CDK1 but not cyclin B1 expression. SO-OCS accumulated cells in G2/M in both cell lines and this blockade was accompanied by a decrease of CDK1, but not cyclin B1 expression. Furthermore, both drugs induced apoptosis as demonstrated by the increased percentage of annexin V positive cells and Bax/Bcl-2 ratio. Interestingly, in normal MCF-10A cells the two drugs failed to modify cell proliferation, cell cycle progression, cyclins, or CDKs expression. These results demonstrate that the effect of CO-OCS and SO-OCS is triggered by both cell cycle arrest and apoptosis, suggesting that these castanospermine analogues may constitute potential anti-cancer agents against breast cancer.

Oxidative DNA Damage and Mammary Cell Proliferation by Alcohol-Derived Salsolinol

Drinking alcohol is a risk factor for breast cancer. Salsolinol (SAL) is endogenously formed by a condensation reaction of dopamine with acetaldehyde, a major ethanol metabolite, and SAL is detected in blood and urine after alcohol intake. We investigated the possibility that SAL can participate in tumor initiation and promotion by causing DNA damage and cell proliferation, leading to alcohol-associated mammary carcinogenesis. SAL caused oxidative DNA damage including 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), in the presence of transition metal ions, such as Cu(II) and Fe(III)EDTA. Inhibitory effects of scavengers on SAL-induced DNA damage and the electron spin resonance study indicated the involvement of H₂O₂, which is generated via the SAL radical. Experiments on scavengers and site specificity of DNA damage suggested ·OH generation via a Fenton reaction and copper-peroxide complexes in the presence of Fe(III)EDTA and Cu(II), respectively. SAL significantly increased 8-oxodG formation in normal mammary epithelial MCF-10A cells. In addition, SAL induced cell proliferation in estrogen receptor (ER)-negative MCF-10A cells, and the proliferation was inhibited by an antioxidant N-acetylcysteine and an epidermal growth factor receptor (EGFR) inhibitor AG1478, suggesting that reactive oxygen species may participate in the proliferation of MCF-10A cells via EGFR activation. Furthermore, SAL induced proliferation in estrogen-sensitive breast cancer MCF-7 cells, and a surface plasmon resonance sensor revealed that SAL significantly increased the binding activity of ERα to the estrogen response element but not ERβ. In conclusion, SAL-induced DNA damage and cell proliferation may play a role in tumor initiation and promotion of multistage mammary carcinogenesis in relation to drinking alcohol.