Elena Elstner

PPARγ Ligands and ATRA Inhibit the Invasion of Human Breast Cancer Cells in vitro

Invasion and metastasis are the main causes of death in breast cancer patients. Increased expression of matrix metalloproteinases (MMPs), especially gelatinases (MMP-2 and -9), has been closely associated with tumor progression. One of the nuclear hormone receptors (NHR), peroxisome proliferator-activated receptor γ (PPARγ), is a ligand-activated transcriptional factor that regulates cell proliferation, differentiation and apoptosis in both normal and cancer cells. Recent data indicate that PPARγ activation by its ligands can also lead to the inhibition of gelatinase B (MMP-9) and the blockage of migration in macrophages and muscle cells, implying the possibility that PPARγ ligands may possess anti-invasive activities on tumor cells. In this study, we showed that treatment of the highly aggressive human breast cancer cell line MDA-MB-231 with the synthetic PPARγ ligands pioglitazone (PGZ), rosiglitazone (RGZ), GW7845 or its natural ligand 15-deoxy-Δ 12, 14-prostaglandin J2(15d-PGJ2), at concentrations at which no obvious cytotoxicity was observed in vitro, led to a significant inhibition of the invasive capacities of this cell line through a reconstituted basement membrane (Matrigel®) in a Transwell® chamber model. All-trans-retinoic acid (ATRA), a ligand for retinoic acid receptor (RAR), was also studied and showed a similar inhibitory effect on invasion. Although no change was observed in the expression of MMP-9 after challenge with PPARγ ligands and/or ATRA on this cell line, the natural tissue inhibitor of gelatinases, namely the tissue inhibitor of MMP 1 (TIMP-1) was upregulated by these treatments and the gelatinolytic activities of gelatinases in the conditioned media were decreased. Since MMP-2 was not detectable in the conditioned media of MDA-MB-231 cells, and the gelatinolytic activities of the conditioned media were reduced only by MMP-9 neutralizing antibodies, it is most likely that the reduction of gelatinolytic activities by PPARγ ligands and/or ATRA was due to the decrease of MMP-9 activities. Because MMP-9 was absolutely required in the transmigration of this cell line through Matrigel® in our in vitro model as demonstrated by neutralizing antibodies against MMP-2 and -9, we concluded that down-regulation of gelatinase activities is, at least in part, responsible for the reduction of the invasive capacities of MDA-MB-231 cell line in vitro. Our results, for the first time, indicate that PPARγ ligands may have therapeutic value for the treatment of highly invasive breast cancer by targeting its invasive behavior.

Novel Therapeutic Approach: Ligands for PPARγ and Retinoid Receptors Induce Apoptosis in bcl-2-positive Human Breast Cancer Cells

Effective treatment of tumors is often associated with activation of the endogenous apoptosis pathways. We have studied eight breast cancer cell lines (MCF-7, BT20, BT474, MDA-MB-231, MDA-MB-436, SKBR3, T-47D, ZR-75-1) possessing a variety of genetic defects. The clonogenic growth of breast cancer cell lines was inhibited by a ligand for PPARγ (troglitazone, TGZ) combined with a ligand for either retinoid X receptor (RXR) (LG10069) (4/8 cell lines), RAR (ATRA) (5/8 cell lines) or RAR/RXR and RXR/RXR (9-cis-RA) (5/8 cell lines) independent of their expression of bcl-2, bag-1, ERα, and p53. The cell lines (MCF-7, T-47D, ZR-75-1), which expressed both BRCA1 and p27, were extremely sensitive to the inhibitory effect of the combination of TGZ and either ATRA or 9-cis-RA (ED90, 2-5 × 10−11 M). However, only MCF-7, MDA-MB-231, and ZR-75-1 cells, which expressed a high level of bcl-2 protein, underwent apoptosis when exposed to the combination of TGZ and either ATRA or 9-cis-RA. Importantly, this effect was independent of expression levels of p53, ERα, HER-2/neu, bag-1, and BRCA1. Therefore, the combination of ligands for PPARγ and retinoid receptors may have a therapeutic role for breast cancer.

Anti-tumor effect of honokiol alone and in combination with other anti-cancer agents in breast cancer

Honokiol, an active component isolated and purified from Chinese traditional herb magnolia, was demonstrated to inhibit growth and induce apoptosis of different cancer cell lines such as human leukaemia, colon, and lung cancer cell lines; to attenuate the angiogenic activities of human endothelial cells in vitro; and to efficiently suppress the growth of angiosarcoma in nude mice. In this study, we have demonstrated that treatment of different human breast cancer cell lines with honokiol resulted in a time- and concentration-dependent growth inhibition in both estrogen receptor-positive and -negative breast cancer cell lines, as well as in drug-resistant breast cancer cell lines such as adriamycin-resistant and tamoxifen-resistant cell lines. The inhibition of growth was associated with a G1-phase cell cycle arrest and induction of caspase-dependent apoptosis. The effects of honokiol might be reversely related to the expression level of human epidermal growth receptor 2, (HER-2, also known as erbB2, c-erbB2) since knockdown of her-2 expression by siRNA significantly enhanced the sensitivity of the her-2 over-expressed BT-474 cells to the honokiol-induced apoptosis. Furthermore, inhibition of HER-2 signalling by specific human epidermal growth receptor 1/HER-2 (EGFR/HER-2) kinase inhibitor lapatinib synergistically enhanced the anti-cancer effects of honokiol in her-2 over-expressed breast cancer cells. Finally, we showed that honokiol was able to attenuate the PI3K/Akt/mTOR (Phosphoinositide 3-kinases/Akt/mammalian target of rapamycin) signalling by down-regulation of Akt phosphorylation and upregulation of PTEN (Phosphatase and Tensin homolog deleted on chromosome Ten) expression. Combination of honokiol with the mTOR inhibitor rapamycin presented synergistic effects on induction of apoptosis of breast cancer cells. In conclusion, honokiol, either alone or in combination with other therapeutics, could serve as a new, promising approach for breast cancer treatment.

Peroxisome proliferator-activated receptor-γ ligands for the treatment of breast cancer

Pioglitazone and rosiglitazone are thiazolidinediones used for the treatment of Type 2 diabetes mellitus. They modulate glucose and fat metabolism, mainly by binding to the nuclear hormone receptor peroxisome proliferator-activated receptor (PPAR)-γ. PPAR-γ signalling is involved in a number of other disease conditions including cancer. In breast cancer cells, PPAR-γ ligands inhibit proliferation and induce apoptosis both in vitro and in vivo. PPAR-γ ligands also inhibit tumour angiogenesis and invasion. The only published clinical trial using a PPAR-γ ligand in patients with metastatic breast cancer failed to show any clinical benefits. The mechanism of action of the thiazolidinediones in breast cancer cells is not fully understood but involves interactions with other nuclear hormone receptors, transcriptional co-activators and repressors as well as PPAR-γ-independent effects. A better understanding of these mechanisms will be needed before PPAR-γ ligands may be useful in the treatment of breast cancer patients.

Ligands for PPARγ and RAR Cause Induction of Growth Inhibition and Apoptosis in Human Glioblastomas

High-grade gliomas are characterized by a rapid proliferation rate, invasiveness and angiogenesis. Our previous data indicated that the combination of ligands for peroxisome proliferator-activated receptor γ (PPARγ) and retinoic acid receptor (RAR) induces apoptosis of breast cancer cells in vitro and in a murine model. In this study, we have shown that 11 glioblastoma cell lines and nine fresh glioblastoma tissue samples from patients expressed high-levels of PPARγ. In contrast, glia from nine healthy human brains expressed very low levels of PPARγ. No mutations or polymorphisms of the PPARγ gene were observed in these cell lines. The effect of the PPARγ ligand Pioglitazone (PGZ) either in the absence or in the presence of a RAR ligand [all-trans retinoic acid (ATRA)] on the proliferation and apoptosis of glioblastoma cells was examined using two glioblastoma cell lines (N39 and DBTRG05MG). PGZ and/or ATRA inhibited significantly the proliferation of both cell lines. Flow cytometry analysis showed that G1 cell cycle arrest was induced by these ligands. In addition, apoptosis occurred in both cell lines treated with either PGZ or ATRA, which was associated with a downregulation of bcl-2 and an upregulation of bax proteins. An enhanced effect was observed when PGZ and ATRA were combined. Furthermore, treatment of fresh glioblastoma tissue from patients with PGZ, either alone or in combination with ATRA, induced a significant level of tumor cell apoptosis together with a downregulation of bcl-2 protein level as compared with untreated control brain tissue. Taken together, our data demonstrated that PGZ, either alone or in combination with ATRA, induced apoptosis and inhibited proliferation of glioblastoma cells, and more interestingly, induced apoptosis of fresh glioblastoma cells from patients. Therefore, we conclude that these ligands may possess adjuvant therapeutic potential for patients with glioblastoma.

Ligands of peroxisome proliferator-activated receptor γ induce apoptosis in multiple myeloma

The activation of proliferator-activated receptor &ggr; (PPAR-&ggr;) by its natural and synthetic ligands induces apoptosis in several tumor cell lines, including malignant B-lineage cells. We investigated whether treatment with pioglitazone (PGZ), rosiglitazone (RGZ) or 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) inhibited tumor cell growth in five human multiple myeloma cell lines (LP-1, U-266, RPMI-8226-S, OPM-2 and IM-9) and human bone marrow myeloma cells expressing PPAR-&ggr; protein. MTT assays revealed growth arrest induced by the natural activator of PPAR-&ggr; 15d-PGJ2 and a lower antiproliferative effect with thiazolidinediones (PGZ and RGZ) in a dose-dependent manner. Induction of apoptosis was indicated by Annexin-V staining. At a dose of 50 μM, 15d-PGJ2 led to a high rate of apoptosis in all cell lines (60–92%). Furthermore, induction of apoptosis in sorted bone marrow plasma cells from myeloma patients was detected. Thiazolidinediones comprise anti-myeloma activity in vitro and should be explored further for the treatment of multiple myeloma.

Induction of endoplasmic reticulum stress response by TZD18, a novel dual ligand for peroxisome proliferator-activated receptor α/γ, in human breast cancer cells

Previously we reported that the peroxisome proliferator-activated receptor α/γ dual ligand TZD18 inhibited growth and induced apoptosis of leukemia and glioblastoma cells. Now we show that TZD18 also has the same effects against six human breast cancer cell lines. To obtain insights into the mechanism involved in TZD18-induced growth inhibition and apoptosis in breast cancer, the gene expression profiles of TZD18-treated and untreated MCF-7 and MDA-MB-231 cells were compared by microarray analysis. Results reveal that many genes implicated in endoplasmic reticulum stress signaling, such as CHOP (also known as DDIT3 or GADD153), GRP78 (HSPA5), and ATF4, are highly up-regulated, suggesting endoplasmic reticulum stress is induced. This is supported by our data that treatment of MCF-7 and MDA-MB-231 cells with TZD18 induces phosphorylation of PERK and the α subunit of eukaryotic initiation factor 2 (eIF2α), as well as an up-regulation of GRP78 and an activation of ATF6, all of which are specific markers for endoplasmic reticulum stress. Furthermore, this ligand increases the endoplasmic reticulum stress–related cell death–regulators such as CHOP, DR5, GADD34, Bax, and Bak in these cells. Importantly, knockdown of CHOP by small interference RNA antagonizes the TZD18-induced apoptosis, indicating a crucial role of CHOP in the apoptotic process triggered by TZD18. In addition, TZD18 also activates stress-sensitive mitogen-activated protein kinase (MAPK) pathways including p38, ERK, and JNK. The specific inhibitors of these MAPKs attenuated the TZD18-induced growth inhibition in these cells. These results clearly show that activation of these MAPKs is important for TZD18-induced growth inhibition. In summary, TZD18-treatment leads to the activation of endoplasmic reticulum stress response and, subsequently, growth arrest and apoptosis in breast cancer cells. [Mol Cancer Ther 2009;8(8):2296–307]

Sensitivity of CFU-GM from normal human bone marrow and leukaemic clonogenic cells (CFU-L) from blood of patients with myelogenous leukaemia to 3′-deoxy-3′-fluorothymidine in comparison to 3′-azido-3′-deoxythymidine

Summary3′-Deoxy-3′-fluorothymidine (FT), a thymidine analogue highly effective against HIV 1 in vitro was investigated as to its in vitro effect on normal human bone marrow CFU-GM (agar colony assay) and on human peripheral myeloid leukaemic clonogenic cells (CFU-L, colony assay in methylcellulose). For comparison, 3′-azido-3′-deoxythymidine (AZT), structurally related and used in AIDS treatment, was included in the study. Both compounds inhibit the formation of clusters and colonies from bone marrow stem cells with an [IC]50 between 10−6 and 10−5M. In concentrations only 5–10 times lower than the [IC]50, FT begins to stimulate cluster and colony formation. AZT and FT also inhibit the formation of clusters and colonies from CFU-L. Compared to CFU-GM, CFU-L were more sensitive to FT, and a stimulation was not seen. It is concluded that similar side effects on bone marrow could be expected for possible use of FT against AIDS as have been found for AZT and that both compounds are potential candidates for antileukaemic drugs.

Modification of MCF-10A Cells with Pioglitazone and Serum-Rich Growth Medium Increases Soluble Factors in the Conditioned Medium, Likely Reducing BT-474 Cell Growth

In the present study, we aimed to preincubate MCF-10A cells with pioglitazone and/or serum-rich growth media and to determine adhesive and non-adhesive interactions of the preincubated MCF-10A cells with BT-474 cells. For this purpose, the MCF-10A cells were preincubated with pioglitazone and/or serum-rich growth media, at appropriate concentrations, for 1 week. The MCF-10A cells preincubated with pioglitazone and/or serum-rich growth media were then co-cultured adhesively and non-adhesively with BT-474 cells for another week. Co-culture of BT-474 cells with the preincubated MCF-10A cells, both adhesively and non-adhesively, reduced the growth of the cancer cells. The inhibitory effect of the preincubated MCF-10A cells against the growth of BT-474 cells was likely produced by increasing levels of soluble factors secreted by the preincubated MCF-10A cells into the conditioned medium, as immunoassayed by ELISA. However, only an elevated level of a soluble factor distinguished the conditioned medium collected from the MCF-10A cells preincubated with pioglitazone and serum-rich growth medium than that with pioglitazone alone. This finding was further confirmed by the induction of the soluble factor transcript expression in the preincubated MCF-10A cells, as determined using real-time PCR, for the above phenomenon. Furthermore, modification of the MCF-10A cells through preincubation did not change the morphology of the cells, indicating that the preincubated cells may potentially be injected into mammary fat pads to reduce cancer growth in patients or to be used for others cell-mediated therapy.

Comparative analysis of the influences of IL-1, IL-3 and GM-CSF on the commitment of granulocyte-macrophage progenitors in vitro

SummaryOur experiments were directed towards the detection of the influence of interleukin-1 (IL-1); interleukin-3 (IL-3), and granulocyte-macrophage colonystimulating factor (GM-CSF) on the generation of granulocyte-macrophage progenitor cells. We also set out to examine whether this process is connected with changes within the early precursor cell compartment. Bone marrow suspension cultures (12 days) supplemented with these cytokines were tested for the presence of GM colony-forming cells (GM-CFC) in a colony-forming unit assay. The percentage of CD 34+ and HLA-DR+ as well as the number of blasts and promyelocytes were estimated cytofluorometrically and morphologically. The proliferative effect of GM-CSF was associated with a net increase of GM-CFC and HLA-DR+ myeloid cells and a decrease in the percentage of CD 34+ early precursor cells. IL-3 acted similarly and also caused an absolute decrease of CD 34+ cells in the cultures. IL-1 did not stimulate the generation of blasts or GM-CFC but elevated the number of CD 34− as well as HLA-DR-expressing cells in the cultures. These results imply that GM-CSF supported the maintenance of hematopoiesis in vitro. The transition from early precursor cells to committed myeloid progenitor cells (GM-CFC) and more mature precursor cells (G-CFC, M-CFC) may be supported by GM-CSF without affecting the self-renewing capacity of CD 34+ early precursors. In contrast, the blast-generating and proliferation-inducing action of IL-3 is associated with a drop in the total number of CD 34+ stem cells. An efficient renewal of this population obviously depends on the presence of IL-1.