Induction of differentiation and apoptosis in cancer cells through ligands of nuclear hormone receptors (NHRs) is a novel and promising approach to cancer therapy. All-trans-retinoic acid (ATRA), an RA receptor-specific NHR ligand, is now used for selective cancers.

We have synthesized and studied the ability of a series of seven novel 1 alpha,25(OH)2 vitamin D3 analogues to inhibit clonal growth of prostate cancer cells (LNCaP, PC-3 and DU-145). Addition of double and triple bonds to the C/D ring (C-16) and side chain (C-22 and C-23) as well as lengthening of the side chain were important for enhanced activity against LNCaP and PC-3. Reorientation of the side chain in the 20-epi configuration resulted in analogues that were extremely potent only against LNCaP (ED50 approximately 5 x 10(-11) M). Compounds with six fluorines on the end of the side chain were very active against both PC-3 and LNCaP (ED50 approximately 2 x 10(-8) M). DU-145 cells were relatively resistant to compounds with all of these modifications, but removal of C-19 (e.g. 1,25(OH)2-16-ene-23-yne-26,27-F6-19-nor-D3) resulted in an analogue that was inhibitory against all three prostate cell lines. Further analysis showed that pulse exposure (3 days, 10(-7) M) to this analogue was enough to inhibit clonal growth of PC-3 cells by 50%. The same exposure also induced cell cycle arrest of all three cell lines, accompanied by upregulated protein expression of the cyclin-dependent kinase inhibitor (CDKI) known as p21waf1 in all three cell lines, and the CDKI known as p27kip1 in LNCaP cells. Associated with upregulation of these CDKIs, partial differentiation occurred as measured by increased expression of both prostate-specific antigen by LNCaP cells and E-cadherin, a cell adhesion protein that may act as a putative tumour suppressor (LNCaP and PC-3 cells). In summary, this is the first report of a potent series of 19-nor-vitamin D3 analogues with the ability to inhibit proliferation of LNCaP, PC-3 and DU-145 prostate cancer cell lines. These compounds may mediate their potent anti-proliferative activities through a cell cycle arrest pathway.

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 gamma (PPARgamma), is a ligand-activated transcriptional factor that regulates cell proliferation, differentiation and apoptosis in both normal and cancer cells. Recent data indicate that PPARgamma 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 PPARgamma 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 PPARgamma ligands pioglitazone (PGZ), rosiglitazone (RGZ), GW7845 or its natural ligand 15-deoxy-delta 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 PPARgamma 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 PPARgamma 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 PPARgamma ligands may have therapeutic value for the treatment of highly invasive breast cancer by targeting its invasive behavior.

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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 PPARgamma (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, ERalpha, 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 x 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, ERalpha, HER-2/neu, bag-1, and BRCA1. Therefore, the combination of ligands for PPARgamma and retinoid receptors may have a therapeutic role for 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)-gamma. PPAR-gamma signalling is involved in a number of other disease conditions including cancer. In breast cancer cells, PPAR-gamma ligands inhibit proliferation and induce apoptosis both in vitro and in vivo. PPAR-gamma ligands also inhibit tumour angiogenesis and invasion. The only published clinical trial using a PPAR-gamma 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-gamma-independent effects. A better understanding of these mechanisms will be needed before PPAR-gamma ligands may be useful in the treatment of breast cancer patients.

Treatment of adult Philadelphia chromosome-positive lymphocytic leukemia is rarely successful. We report here the effects of TZD18, a novel dual ligand specific for peroxisome proliferatoractivated receptor ␣ and ␥ (PPAR␣/␥) on Ph ؉ lymphocytic leukemia cell lines BV173, SD1, and SupB-15. Exposure of these cells to TZD18 resulted in growth inhibition in a dose-and time-dependent manner that was associated with G 1 cell cycle arrest. This effect was much stronger than that mediated by the PPAR␥ ligand pioglitazone (PGZ), which also belongs to the thiazolidinediones (TZD) class of ligands. However, it may not be mediated through PPAR␥ or PPAR␣ activation because antagonists of PPAR␥ and PPAR␣ cannot reverse it. Study of the key regulators of cell cycle progression by Western blot analysis showed that the expression of the cyclin-dependent kinase inhibitor (CDKI) p27 kip1 , but not that of p21 cip1 , was enhanced, whereas that of c-Myc, cyclin E, cyclin D2, and cyclindependent kinases 2 and 4 (CDK-2 and CDK-4) was decreased when these cells were treated with TZD18 (10 or 20 M). Therefore, the up-regulation of p27 kip1 and the down-regulation of CDK-2 and CDK-4 may, at least in part, account for the G 1 cell cycle arrest. Furthermore, a remarkable induction of apoptosis was observed in the cells treated with this dual ligand.No obvious alteration of bcl-2 protein level occurred, but bax was up-regulated in these TZD18-treated cells. Activation of caspase 8 and caspase 9 by TZD18 was also observed. Importantly, NF-B DNAbinding activity was markedly decreased by the TZD18 treatment. In addition, TZD18 enhanced the growth inhibitory effect of imatinib, a specific tyrosine kinase inhibitor therapeutically used in the treatment of Ph ؉ leukemia. Overall, our findings strongly suggest that TZD18 may offer a new therapeutic approach to aid in the treatment of Ph ؉ lymphocytic leukemia. IntroductionAlthough significant progress has been made in the treatment of acute lymphocytic leukemia (ALL), the prognosis for patients with Philadelphia chromosome-positive (Ph ϩ )/Bcr-Abl ϩ adult ALL is still very poor. After first remission, allogeneic stem cell transplantation is the treatment of choice. However, most patients are not eligible for this therapy because of advanced age or lack of a suitable stem cell donor (for reviews, see Redaelli et al 1 and Bassan et al 2 ). Imatinib (Gleevec, previously known as STI571 and CGP57148), the selective tyrosine kinase inhibitor, displayed pronounced antileukemic activity in Ph ϩ chronic myeloid leukemia (CML) and ALL. It is used after allogeneic stem cell transplantation and is recommended for relapsed or refractory Ph ϩ ALL as salvage therapy to facilitate subsequent transplantation. 3 However, quick emergence of resistance to this agent is a major problem in the treatment of patients with Ph ϩ leukemia. Another major obstacle to imatinib-based therapies is the persistence of Ph ϩ cells despite the application of imatinib. Based on these arguments, the development of novel therapeutic a...