Integration of Ca2+ signaling regulates the breast tumor cell response to simvastatin and doxorubicin.

Recent studies have suggested that the lipid-lowering agent simvastatin holds great promise as a cancer therapeutic; it inhibits the growth of multiple tumors, including triple-negative breast cancer. Doxorubicin- and simvastatin-induced cytotoxicity has been associated with the modulation of Ca2+ signaling, but the underlying mechanisms remain incompletely understood. Here we identify how Ca2+ signaling regulates the breast tumor cell response to doxorubicin and simvastatin. These two drugs inhibit cell survival while increasing apoptosis in two human breast cancer cell lines and five primary breast tumor specimens through the modulation of Ca2+ signaling. Signal transduction and functional studies revealed that both simvastatin and doxorubicin trigger persistent cytosolic Ca2+ release, thereby stimulating the proapoptotic BIM pathway and mitochondrial Ca2+ overload, which are responsible for metabolic dysfunction and apoptosis induction. Simvastatin and doxorubicin suppress the prosurvival ERK1/2 pathway in a Ca2+-independent and Ca2+-dependent manner, respectively. In addition, reduction of the Ca2+ signal by chelation or pharmacological inhibition significantly prevents drug-mediated anticancer signaling. Unexpectedly, a scratch-wound assay indicated that these two drugs induce rapid cell migration, while inhibiting cell invasion and colony formation in a Ca2+-dependent manner. Further, the in vivo data for MDA-MB-231 xenografts demonstrate that upon chelation of Ca2+, the ability of both drugs to reduce the tumor burden was significantly reduced via caspase-3 deactivation. Our results establish a calcium-based mechanism as crucial for executing the cell death process triggered by simvastatin and doxorubicin, and suggest that combining simvastatin with doxorubicin may be an effective regimen for the treatment of breast cancer.

Synergistic promoting effects of pentoxifylline and simvastatin on the apoptosis of triple-negative MDA-MB-231 breast cancer cells.

Pentoxifylline (PTX), a xanthine family molecule and simvastatin (SIM), an anti-hypercholesterolemic agent, have recently been considered as sensitizers to chemotherapy and radiotherapy. The present in vitro study evaluated their antitumor synergistic effects on MDA­MB­231 breast cancer cells characterized by the triple­negative phenotype (TNP). The anti-proliferative effects of these two agents were evaluated by MTT and clonogenic assays. Cell cycle progression was examined using propidium iodide staining. Apoptosis was investigated by Annexin V labeling, and by examining caspase 3 activity and DNA fragmentation. Autophagic vesicles and reactive oxygen species (ROS) levels were monitored by flow cytometry. Western blot analysis was performed to evaluate molecular targets. Our results revealed that when used alone, PTX and SIM exerted antitumor effects. Nevertheless, used in combination, the inhibition of cell proliferation was synergistically superior (80% vs 42%) than that observed following treatment with each agent alone after 48 h. PTX alone (0.5 mM) induced both apoptosis (25%) and autophagy (25%); however, when used in combination with SIM (0.5 µM), the balance between these processes was disrupted and the cells underwent apoptosis (>65%) as opposed to autophagy (<13%). This imbalance was associated with an increase in ERK1/2 and AKT activation, but not with an increase in mTOR phosphorylation, and with the suppression of the NF-κB pathway. In addition, in the cells treated with both agents, almost 78% of the cells were arrested at the G0/G1 phase and lost their colony-forming ability (38±5%) compared to the cells treated with PTX alone (115±5%). On the whole, these results suggest that the induction of autophagy may be a protective mechanism preventing MDA­MB­231 cancer cell death. The combined use of PTX and SIM may drive dormant autophagic cancer cells to undergo apoptosis and thus this may be a novel treatment strategy for breast cancer characterized by the TNP.

Pyr3, a TRPC3 channel blocker, potentiates dexamethasone sensitivity and apoptosis in acute lymphoblastic leukemia cells by disturbing Ca(2+) signaling, mitochondrial membrane potential changes and reactive oxygen species production.

Dexamethasone (Dex) is used as a chemotherapeutic drug in the treatment of acute lymphoblastic leukemia (ALL) because of its capacity to induce apoptosis. However, some ALL patients acquire resistance to glucocorticoids (GC). Thus, it is important to explore new agents to overcome GC resistance. The aim of the present work was to assess the ability of Pyr3, a selective inhibitor of transient receptor potential canonical 3 (TRPC3), to sensitize human ALL cells to Dex. We show here, for the first time, that Pyr3 enhances Dex sensitivity through the distraction of Dex-mediated Ca(2+) signaling in ALL cells (in vitro) and primary blasts (ex vivo) associated with mitochondrial-mediated reactive oxygen species production in ALL cells. Pyr3 alone induced Ca(2+) signaling via only endoplasmic reticulum-released Ca(2+) and exerted inhibitory effect on store-operated Ca(2+) entry in dose-dependent manner in ALL cell lines. Pre-incubation of cells with Pyr3 significantly curtailed the thapsigargin- and Dex-evoked Ca(2+) signaling in ALL cell lines. Pyr3 synergistically potentiated Dex lethality, as shown by the induction of cell mortality, G2/M cell cycle arrest and apoptosis in ALL cell lines. Moreover, Pyr3 disrupted Dex-mediated Ca(2+) signaling and increased the sensitivity of Dex-induced cell death in primary blasts from ALL patients. Additional analysis showed that co-treatment with Dex and Pyr3 results in mitochondrial membrane potential depolarization and reactive oxygen species production in ALL cells. Together, Pyr3 exhibited potential therapeutic benefit in combination with Dex to inverse glucocorticoid resistance in human ALL and probably in other lymphoid malignancies.

Rac3 induces a molecular pathway triggering breast cancer cell aggressiveness: differences in MDA-MB-231 and MCF-7 breast cancer cell lines.

BACKGROUND: Rho GTPases are involved in cellular functions relevant to cancer. The roles of RhoA and Rac1 have already been established. However, the role of Rac3 in cancer aggressiveness is less well understood. METHODS: This work was conducted to analyze the implication of Rac3 in the aggressiveness of two breast cancer cell lines, MDA-MB-231 and MCF-7: both express Rac3, but MDA-MB-231 expresses more activated RhoA. The effect of Rac3 in cancer cells was also compared with its effect on the non-tumorigenic mammary epithelial cells MCF-10A. We analyzed the consequences of Rac3 depletion by anti-Rac3 siRNA. RESULTS: Firstly, we analyzed the effects of Rac3 depletion on the breast cancer cells' aggressiveness. In the invasive MDA-MB-231 cells, Rac3 inhibition caused a marked reduction of both invasion (40%) and cell adhesion to collagen (84%), accompanied by an increase in TNF-induced apoptosis (72%). This indicates that Rac3 is involved in the cancer cells' aggressiveness. Secondly, we investigated the effects of Rac3 inhibition on the expression and activation of related signaling molecules, including NF-κB and ERK. Cytokine secretion profiles were also analyzed. In the non-invasive MCF-7 line; Rac3 did not influence any of the parameters of aggressiveness. CONCLUSIONS: This discrepancy between the effects of Rac3 knockdown in the two cell lines could be explained as follows: in the MDA-MB-231 line, the Rac3-dependent aggressiveness of the cancer cells is due to the Rac3/ERK-2/NF-κB signaling pathway, which is responsible for MMP-9, interleukin-6, -8 and GRO secretion, as well as the resistance to TNF-induced apoptosis, whereas in the MCF-7 line, this pathway is not functional because of the low expression of NF-κB subunits in these cells. Rac3 may be a potent target for inhibiting aggressive breast cancer.

Survival of cord blood haematopoietic stem cells in a hyaluronan hydrogel for ex vivo biomimicry.

Haematopoietic stem cells (HSCs) and haematopoietic progenitor cells (HPCs) grow in a specified niche in close association with the microenvironment, the so-called 'haematopoietic niche'. Scaffolds have been introduced to overcome the liquid culture limitations, mimicking the presence of the extracellular matrix (ECM). In the present study the hyaluronic acid scaffold, already developed in the laboratory, has been used for the first time to maintain long-term cultures of CD34⁺ haematopoietic cells obtained from human cord blood. One parameter investigated was the impact on ex vivo survival of CD34⁺ cord blood cells (CBCs) on the hyaluronic acid surface, immobilized with peptides containing the RGD motif. This peptide was conjugated by coating the hyaluronan hydrogel and cultured in serum-free liquid phase complemented with stem cell factor (SCF), a commonly indispensable cytokine for haematopoiesis. Our work demonstrated that these hyaluronan hydrogels were superior to traditional liquid cultures by maintaining and expanding the HPCs without the need for additional cytokines, and a colonization of 280-fold increment in the hydrogel compared with liquid culture after 28 days of ex vivo expansion.

Importance of local hypoxia on endothelial phenotype for an in vitro approach to bone marrow angiogenesis.

The vasculature of bone marrow differs from that in other organs, and its characteristics should be considered when exploring the medullar angiogenesis associated with hematological malignancies. We show here that the human bone marrow sinusoidal cell line HBME-1 has a specific expression pattern of angiogenic factors and receptors, characterized by a unique VEGFR3(+), Tie2(-) signature, that resembles the in vivo pattern. Moreover, the HBME-1 cultured for up to 3 days in hypoxic conditions, similar to those found in the bone marrow, specifically downregulated expression of VEGFR1, VEGFR2 and ETAR. Thus, a model using bone marrow sinusoidal cells cultured under reduced oxygen tension may be more relevant than classical in vitro endothelial cultures for understanding the interactions between endothelial and malignant cells in the medullar microenvironment.

Rac-1 GTPase controls the capacity of human leukaemic lymphoblasts to migrate on fibronectin in response to SDF-1α (CXCL12).

Acute lymphoblastic leukaemia (ALL) is characterized by malignant cell infiltration of bone marrow, requiring chemotactic response to SDF-1α. Using time-lapse video, we measured the velocity of ALL cells on fibronectin, and found that SDF-1α increased their migration activity for 2 h, but had no effect after 4h, following internalization of its receptor CXCR4. Transfection of ALL cells with dominant-negative Rac1 mutant significantly prolonged their chemotactic response to SDF-1α, and this effect was associated with an alteration of CXCR4 internalization. These data suggest a regulatory role for Rac1 in the chemotactic response of ALL cells to SDF-1α via receptor processing.