Valérie Chopin

Epigallocatechin (EGC) of green tea induces apoptosis of human breast cancer cells but not of their normal counterparts.

Abstract(−)-Epigallocatechin (EGC), one of green tea polyphenols, has been shown to inhibit growth of cancer cells. However its mechanism of action is poorly known. We show here that EGC strongly inhibited the growth of breast cancer cell lines (MCF-7 and MDA-MB-231) but not that of normal breast epithelial cells. The inhibition of breast cancer cell growth was due to an induction of apoptosis, without any change in cell cycle progression. MCF-7 cells are known to express a wild-type p53 whereas MDA-MB-231 cells express a mutated p53. The fact that EGC induced apoptosis in both these cell lines suggests that the EGC-triggered apoptosis is independent of p53 status. Moreover, neutralizing antibodies against the death receptor Fas and inhibitors of caspases, such as caspase-8 and -10, efficiently inhibited the EGC-triggered apoptosis. In addition, immunoblotting revealed that EGC treatment was correlated with a decrease in Bcl-2 and an increase in Bax level. These results suggest that EGC-triggered apoptosis in breast cancer cells requires Fas signaling.

P21 WAF1/CIP1 is dispensable for G1 arrest, but indispensable for apoptosis induced by sodium butyrate in MCF-7 breast cancer cells

Sodium butyrate (NaB) has been proposed as a potential anticancer agent. However, its mechanism of action is not totally elucidated. Here, we showed that NaB-induced cell cycle arrest and apoptosis were associated with an increase of P21waf1/cip1 in MCF-7 breast cancer cells. This increase was more important in the nuclei, as revealed by immunofluorescence analysis. Transient transfections of MCF-7 cells with p21 deficient for interaction with CDK, but not with p21 deficient for interaction with PCNA (p21PCNA−), abrogated NaB-induced cell cycle arrest. This indicated that cell cycle blockage involved the interaction of P21waf1/cip1 with CDK. However, P21waf1/cip1 was dispensable, since p21 antisense did not modify cell cycle arrest. On the other hand, NaB-induced apoptosis was abolished by p21 antisense or p21PCNA−. In addition, NaB decreased PCNA levels, but increased the association of PCNA with P21waf1/cip1. These results suggested that NaB-induced apoptosis required P21waf1/cip1 and its interaction with PCNA.

Sodium butyrate induces P53-independent, Fas-mediated apoptosis in MCF-7 human breast cancer cells

This study was performed to determine the effect and action mechanisms of sodium butyrate (NaB) on the growth of breast cancer cells. Butyrate inhibited the growth of all breast cancer cell lines analysed. It induced cell cycle arrest in G1 and apoptosis in MCF‐7, MCF‐7ras, T47‐D, and BT‐20 cells, as well as arrest in G2/M in MDA‐MB‐231 cells. Transient transfection of MCF‐7 and T47‐D cells with wild‐type and antisense p53 did not modify butyrate‐induced apoptosis. Pifithrin‐α, which inhibits the transcriptional activity of P53, did not modify cell growth or apoptosis of MCF‐7 and T47‐D cells treated with butyrate. These results indicate that P53 was not involved in butyrate‐induced growth inhibition of breast cancer cells. Treatment of MCF‐7 cells with anti‐Fas agonist antibody induced cell death, indicating that Fas was functional in these cells. Moreover, butyrate potentiated Fas‐induced apoptosis, as massive apoptosis was observed rapidly when MCF‐7 cells were treated with butyrate and anti‐Fas agonist antibody. In addition, butyrate‐induced apoptosis in MCF‐7 cells was considerably reduced by anti‐Fas antagonist antibody. Western blot analysis showed that butyrate increased Fas and Fas ligand levels (Fas L), indicating that butyrate‐induced apoptosis may be mediated by Fas signalling. These results demonstrate that butyrate inhibited the growth of breast cancer cells in a P53‐independent manner. Moreover, it induced apoptosis via the Fas/Fas L system and potentiated Fas‐triggered apoptosis in MCF‐7 cells. These findings may open interesting perspectives in human breast cancer treatment strategy.

Human ether à‐gogo K+ channel 1 (hEag1) regulates MDA‐MB‐231 breast cancer cell migration through Orai1‐dependent calcium entry

Breast cancer (BC) has a poor prognosis due to its strong metastatic ability. Accumulating data present ether à go‐go (hEag1) K+ channels as relevant player in controlling cell cycle and proliferation of non‐invasive BC cells. However, the role of hEag1 in invasive BC cells migration is still unknown. In this study, we studied both the functional expression and the involvement in cell migration of hEag1 in the highly metastatic MDA‐MB‐231 human BC cells. We showed that hEag1 mRNA and proteins were expressed in human invasive ductal carcinoma tissues and BC cell lines. Functional activity of hEag1 channels in MDA‐MB‐231 cells was confirmed using astemizole, a hEag1 blocker, or siRNA. Blocking or silencing hEag1 depolarized the membrane potential and reduced both Ca2+ entry and MDA‐MB‐231 cell migration without affecting cell proliferation. Recent studies have reported that Ca2+ entry through Orai1 channels is required for MDA‐MB‐231 cell migration. Down‐regulation of hEag1 or Orai1 reduced Ca2+ influx and cell migration with similar efficiency. Interestingly, no additive effects on Ca2+ influx or cell migration were observed in cells co‐transfected with sihEag1 and siOrai1. Finally, both Orai1 and hEag1 are expressed in invasive breast adenocarcinoma tissues and invaded metastatic lymph node samples (LNM+). In conclusion, this study is the first to demonstrate that hEag1 channels are involved in the serum‐induced migration of BC cells by controlling the Ca2+ entry through Orai1 channels. hEag1 may therefore represent a potential target for the suppression of BC cell migration, and thus prevention of metastasis development. J. Cell. Physiol. 227: 3837–3846, 2012.

Normal breast epithelial cells induce p53-dependent apoptosis and p53-independent cell cycle arrest of breast cancer cells.

Cancer development depends not only on the nature of cancerous cells themselves, but also on the regulatory effects of various normal cells. The present study was performed to investigate the effect of normal breast epithelial cells (NBEC) on the growth of breast cancer cells under various conditions. We demonstrated that NBEC-conditioned medium (NBEC-CM) inhibited growth of breast cancer cell lines in monolayer culture and three-dimensional collagen gel culture, as well as in soft agar. In MCF-7 and T-47D cells which have a functional p53, NBEC-CM induced apoptosis without modifying cell cycle progression. In MDA-MB-231 and BT-20 cells that have a non-functional p53, NBEC-CM did not induce apoptosis, although a slight G1 blokage was observed in MDA-MB-231 cells. Transient transfections of MCF-7 and T-47D cells demonstrated that NBEC-triggered apoptosis was mediated by endogenous p53. Moreover, pifithrin-α which specifically inhibits the transcriptional activity of p53, completely abolished NBEC-induced apoptosis in both MCF-7 and T-47D cells, indicating that p53 mediated apoptosis via its transcriptional activity. Finally, orthovanadate, a protein tyrosine phosphatase inhibitor, completely inhibited NBEC-triggered apoptosis, indicating that NBEC-triggered apoptosis was regulated by tyrosine phosphatases.

Tamoxifen and TRAIL synergistically induce apoptosis in breast cancer cells

Tamoxifen (TAM), is widely used as a single agent in adjuvant treatment of breast cancer. Here, we investigated the effects of TAM in combination with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in estrogen receptor-α (ER-α)-positive and -negative breast cancer cells. We showed that cotreatment with TAM and TRAIL synergistically induced apoptosis regardless of ER-α status. By contrast, cotreatment did not affect the viability of normal breast epithelial cells. Cotreatment with TAM and TRAIL in breast cancer cells decreased the levels of antiapoptotic proteins including FLIPs and Bcl-2, and enhanced the levels of proapoptotic proteins such as FADD, caspase 8, tBid, Bax and caspase 9. Furthermore, cotreatment-induced apoptosis was efficiently reduced by FADD- or Bid-siRNA, indicating the implication of both extrinsic and intrinsic pathways in synergistic apoptosis induction. Importantly, cotreatment totally arrested tumor growth in an ER-α-negative MDA-MB-231 tumor xenograft model. The abrogation of tumor growth correlated with enhanced apoptosis in tumor tissues. Our findings raise the possibility to use TAM in combination with TRAIL for breast cancers, regardless of ER-α status.

Proteomics Demonstration That Normal Breast Epithelial Cells Can Induce Apoptosis of Breast Cancer Cells through Insulin-like Growth Factor-binding Protein-3 and Maspin

Normal breast epithelial cells are known to exert an apoptotic effect on breast cancer cells, resulting in a potential paracrine inhibition of breast tumor development. In this study we purified and characterized the apoptosis-inducing factors secreted by normal breast epithelial cells. Conditioned medium was concentrated by ultrafiltration and separated on reverse phase Sep-Pak C18 and HPLC. The proapoptotic activity of eluted fractions was tested on MCF-7 breast cancer cells, and nano-LC-nano-ESI-MS/MS allowed the identification of insulin-like growth factor-binding protein-3 (IGFBP-3) and maspin as the proapoptotic factors produced by normal breast epithelial cells. Western blot analysis of conditioned media confirmed the specific secretion of IGFBP-3 and maspin by normal cells but not by breast cancer cells. Immunodepletion of IGFBP-3 and maspin completely abolished the normal cell-induced apoptosis of cancer cells, and recombinant proteins reproduced the effect of normal cell-conditioned medium on apoptosis of breast cancer cells. Together our results indicated that normal breast epithelial cells can induce apoptosis of breast cancer cells through IGFBP-3 and maspin. These findings provide a molecular hypothesis for the long observed inhibitory effect of normal surrounding cells on breast cancer development.

Neurotrophin signaling in cancer stem cells

Cancer stem cells (CSCs), are thought to be at the origin of tumor development and resistance to therapies. Thus, a better understanding of the molecular mechanisms involved in the control of CSC stemness is essential to the design of more effective therapies for cancer patients. Cancer cell stemness and the subsequent expansion of CSCs are regulated by micro-environmental signals including neurotrophins. Over the years, the roles of neurotrophins in tumor development have been well established and regularly reviewed. Especially, nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) are reported to stimulate tumor cell proliferation, survival, migration and/or invasion, and favors tumor angiogenesis. More recently, neurotrophins have been reported to regulate CSCs. This review briefly presents neurotrophins and their receptors, summarizes their roles in different cancers, and discusses the emerging evidence of neurotrophins-induced enrichment of CSCs as well as the involved signaling pathways.

Intermediate Ca2+-Sensitive K+ Channels are Necessary for Prolactin-Induced Proliferation in Breast Cancer Cells

Prolactin (PRL) is a polypeptidic hormone which acts both systemically and locally to cause lactation by interacting with the PRL receptor, a Janus kinase (JAK2)-coupled cytokine receptor family member. Several studies have reported that serum PRL level elevation is associated with an increased risk for breast cancer, and evidence has suggested that PRL is one actor in the pathogenesis and progression of this cancer. We previously reported the involvement of hIKCa1 in breast cell cycle progression and cell proliferation. However, mechanisms by which PRL cooperates with these channels to modulate breast epithelial cell proliferation remain unknown. Our results showed that, in the MCF-7 breast cancer cell line, PRL increased hIKCa1 current density. These channels were functional and regulated the resting membrane potential. The PRL effects were inhibited by TRAM-34 and clotrimazole, the most used hIKCa1 blockers. Moreover, PRL increased proliferation in a dose-dependent manner without overexpressing hIKCa1. To determine whether PRL-induced proliferation and hIKCa1 activity involved the JAK2 pathway, we used pharmacological JAK2 inhibitors (AG490 and JAK inhibitor I). Indeed, PRL-induced JAK2 phosphorylation was required for both cell proliferation and hIKCa1 activity. In the presence of either hIKCa1 blockers or siRNA-hIKCa1, PRL failed to increase cell proliferation and hIKCa1 activity. Taken together, our results demonstrate that PRL plays a role in breast cancer cell proliferation by increasing hIKCa1 activity through the JAK2 signaling pathway.