Chuanbing Zang

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.

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.

Dual PPARalpha/gamma ligand TZD18 either alone or in combination with imatinib inhibits proliferation and induces apoptosis of human CML cell lines.

Despite progress in the treatment of early-stage chronic myeloid leukemia (CML), the accelerated and blastic phases of CML still remain a therapeutic challenge. Persistence of BCR-ABL-positive (bcr-abl+) cells or secondary resistance during imatinib therapy frequently occurs. In this study, we investigated the activity of a novel dual ligand specific for peroxisome proliferator-activated receptor α and γ (PPARα/γ) against CML blast crisis cell lines. Exposure of these cell lines (K562, KU812 and KCL22) to TZD18 resulted in a growth inhibition in a dose- and time-dependent manner. This effect may not be mediated through PPARγ and/or PPARα activation, since antagonists of PPARg and/or PPARa could not reverse this inhibition. Western blotting analysis showed that expression of the cyclin dependent kinase inhibitor (CDKI) p27kip1 was enhanced, whereas levels of cyclin E, cyclin D2 and cyclin dependent kinase 2 (CDK-2) were decreased when these cells were treated with TZD18. Most interestingly, TZD18 synergistically enhanced the antiproliferative and pro-apoptotic effects of imatinib. Overall, our findings strongly suggest that TZD18, either alone or in combination with imatinib may be beneficial for the treatment of CML in myeloid blast crisis.

Inhibition of pan-class I phosphatidyl-inositol-3-kinase by NVP-BKM120 effectively blocks proliferation and induces cell death in diffuse large B-cell lymphoma.

Abstract Diffuse large B-cell lymphoma (DLBCL) is the most frequent aggressive lymphoma, with a great demand for novel treatments for relapsing and refractory disease. Constitutive activation of the phosphatidyl-inositol-3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway is often detected in this lymphoma. Inhibition of this signaling cascade with the pan-class I PI3K inhibitor NVP-BKM120 decreased cell proliferation and increased apoptotic cell death. DLBCL proliferation was further decreased if NVP-BKM120-induced autophagy was blocked. Treatment with NVP-BKM120 was associated with an increase of the pro-apoptotic BH3-only proteins Puma and Bim and down-regulation of the anti-apoptotic Bcl-xL and Mcl-1. Translation of Bcl-xL and Mcl-1 is facilitated by cap-dependent mRNA translation, a process that was partially inhibited by NVP-BKM120. Overall, we demonstrated here the potential of NVP-BKM120 for the treatment of DLBCL.

Concurrent inhibition of PI3-Kinase and mTOR induces cell death in diffuse large B cell lymphomas, a mechanism involving down regulation of Mcl-1

Phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling is frequently dysregulated in diffuse large B cell lymphoma (DLBCL) including the favorable germinal centre B-cell (GCB) and the unfavorable activated B-cell (ABC) subtypes. mTOR promotes cap-dependent translation of proteins, like Mcl-1, through inhibitory phosphorylation of the eukaryotic translation initiation factor 4E binding protein 1 (4EBP1). Inhibition of mTOR by RAD001 reduces proliferation but fails to dephosphorylate 4EBP1 and to induce cell death in either DLBCL subtype. In contrast, concurrent inhibition of PI3K and mTOR with NVP-BEZ235 inhibits proliferation, dephosphorylates 4EBP1, and induces cells death, notably more pronounced in CGB cells. Small RNA interference identifies Mcl-1 as a crucial cell death mediator of both DLBCL subtypes. Inhibition of the PI3K/mTOR/4EBP1 by NVP-BEZ235 results in suppression of the cap-dependent translation initiation complex and concomitant downregulation of Mcl-1 in GCB cell lines. In ABC cell lines, this suppression is possibly compensated by NF-κB- or Pim kinase-mediated signaling.

The proteasome inhibitor bortezomib stimulates osteoblastic differentiation of human osteoblast precursors via upregulation of vitamin D receptor signalling

Interactions of myeloma cells with the bone marrow microenvironment lead to enhanced osteoclast recruitment and impaired osteoblast activity. Recent evidence revealed that the proteasome inhibitor bortezomib stimulates osteoblast differentiation, but the mechanisms are not fully elucidated. We hypothesised that bortezomib could influence osteoblastic differentiation via alteration of vitamin D signalling by blocking the proteasomal degradation of the vitamin D receptor (VDR). This is of clinical importance, as a high rate of vitamin D deficiency was reported in patients with myeloma. We performed cocultures of primary human mesenchymal stem cells (hMSCs) and human osteoblasts (hOBs) with myeloma cells, which resulted in an inhibition of the vitamin D‐dependent differentiation of osteoblast precursors. Treatment with bortezomib led to a moderate increase in osteoblastic differentiation markers in hMSCs and hOBs. Importantly, this effect could be strikingly increased when vitamin D was added. Bortezomib led to enhanced nuclear VDR protein levels in hMSCs. Primary hMSCs transfected with a VDR luciferase reporter construct showed a strong increase in VDR signalling with bortezomib. In summary, stimulation of VDR signalling is a mechanism for the bortezomib‐induced stimulation of osteoblastic differentiation. The data suggest that supplementation of vitamin D in patients with myeloma treated with bortezomib is crucial for optimal bone formation.

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]