Imaging bone metastases in breast cancer: techniques and recommendations for diagnosis.

Bone is the most common site of distant metastases from breast carcinoma. The presence of bone metastases affects a patient's prognosis, quality of life, and the planning of their treatment. We discuss recent innovations in bone imaging and present algorithms, based on the strengths and weaknesses of each technique, to facilitate the most successful and cost-effective choice of imaging studies for the detection of osseous metastases. Skeletal scintigraphy (bone scan) is very sensitive in the detection of osseous metastases and is recommended as the first imaging study in patients who are asymptomatic. Radiographs are recommended for the assessment of abnormal radionuclide uptake or the risk of pathological fracture and as initial imaging studies in patients with bone pain. MRI or PET-CT can be considered for cases of abnormal radionuclide uptake that are not addressed by radiography. Osseous metastases can lead to emergent situations, such as spinal-cord compression or impending fracture of a weight-bearing bone, and imaging guidelines are essential for early detection and initiation of appropriate therapy. The imaging method used in non-emergent situations, such as assessment of the ribs, sternum, pelvis, hips, and joints, should be guided by the strengths and limitations of each technique.

Programmed cell death 4 inhibits breast cancer cell invasion by increasing tissue inhibitor of metalloproteinases-2 expression.

High levels of the cyclooxygenase-2 (COX-2) protein have been associated with invasion and metastasis of breast tumors. Both prostaglandin E(2) (PGE(2)) and interleukin-8 (IL-8) have been shown to mediate the invasive activity of COX-2 in breast cancer cells. Here we expand these studies to determine how COX-2 uses PGE(2) and IL-8 to induce breast cancer cell invasion. We demonstrated that PGE(2) and IL-8 decreased the expression of the tumor suppressor protein Programmed Cell Death 4 (PDCD4). We hypothesized that suppression of PDCD4 expression is vital to the invasive activity of PGE(2) and IL-8. In MCF-7 cells overexpressing PDCD4 (MCF-7/PDCD4), PGE(2) and IL-8 failed to induce invasion, in contrast to the parental MCF-7 cells, thus indicating that PDCD4 blocks breast cancer cell invasion. MCF-7/PDCD4 cells produced higher levels of the Tissue Inhibitor of Metalloproteinases-2 (TIMP-2) than the parental cells. Silencing TIMP-2 mRNA in MCF-7/PDCD4 cells reversed the anti-invasive effects of PDCD4, allowing PGE(2) and IL-8 to induce the invasion of these cells. Here we report the novel findings that suppression of PDCD4 expression is vital for the invasive activity of COX-2 mediated by PGE(2) and IL-8, and that PDCD4 increases TIMP-2 expression to inhibit breast cancer cell invasion.

TIMP-2 mediates the anti-invasive effects of the nitric oxide-releasing prodrug JS-K in breast cancer cells.

INTRODUCTION: Tumor invasion and metastasis remain a major cause of mortality in breast cancer patients. High concentrations of nitric oxide (NO) suppress tumor invasion and metastasis in vivo. NO prodrugs generate large amounts of NO upon metabolism by appropriate intracellular enzymes, and therefore could have potential in the prevention and therapy of metastatic breast cancer. METHODS: The present study was designed to determine the effects of the NO-releasing prodrug O2-(2,4-dinitrophenyl) 1- [(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K) on breast cancer invasion and the mechanisms involved. MDA-MB-231, MDA-MB-231/F10, and MCF-7/COX-2 were the three breast cancer cell lines tested. NO levels were determined spectrophotometrically using a NO assay kit. Invasion and the expression of matrix metalloproteinases (MMPs) and tissue inhibitor of MMPs were determined using Matrigel invasion assays, an MMP array kit and ELISAs. The activity and expression of extracellular signal-regulated kinase 1/2, p38, and c-Jun N-terminal kinase mitogen-activated protein kinases were determined using western blot analyses. RESULTS: Under conditions by which JS-K was not cytotoxic, JS-K significantly decreased (P < 0.05) the invasiveness of breast cancer cells across the Matrigel basement membrane, which was directly correlated with NO production. JS-43-126, a non-NO-releasing analog of JS-K, had no effect on NO levels or invasion. JS-K increased (P < 0.05) TIMP-2 production, and blocking TIMP-2 activity with a neutralizing antibody significantly increased (P < 0.05) the invasive activity of JS-K-treated cells across Matrigel. JS-K decreased p38 activity, whereas the activity and the expression of extracellular signal-regulated kinase 1/2 and c-Jun N-terminal kinase were unaffected. CONCLUSION: We report the novel findings that JS-K inhibits breast cancer invasion across the Matrigel basement membrane, and NO production is vital for this activity. Upregulation of TIMP-2 production is one mechanism by which JS-K mediates its anti-invasive effects. JS-K and other NO prodrugs may represent an innovative biological approach in the prevention and treatment of metastatic breast cancer.

Wilms' tumor 1 protein and focal adhesion kinase mediate keratinocyte growth factor signaling in breast cancer cells.

BACKGROUND: Keratinocyte growth factor (KGF) has been shown to induce breast cancer metastasis in animal models. cDNA microarrays have revealed that KGF increased Wilms tumor 1 (WT1) and focal adhesion kinase (FAK) expression in breast cancer cells. The role of WT1 and FAK in KGF signaling was investigated. MATERIALS AND METHODS: A cell culture wounding model was used to study the effects of WT1 and FAK down-regulation on KGF-induced proliferation and motility in breast cancer cells. RESULTS: WT1 down-regulation inhibited KGF-mediated proliferation and motility of breast cancer cells, while FAK down-regulation inhibited proliferation, but had no significant effect on cell motility. WT1 down-regulation, but not FAK down-regulation, led to Erk1,2 inactivation. CONCLUSION: KGF-mediated signaling employs WT1 and FAK to regulate breast cancer cell proliferation and motility and may represent therapeutic targets for the prevention of breast cancer progression.

Phosphorylated Pak1 level in the cytoplasm correlates with shorter survival time in patients with glioblastoma.

PURPOSE: Glioblastoma is the most common primary malignant tumor in the brain. It aggressively invades the surrounding parenchyma, often allowing the tumor to progress after surgery. Accumulating evidence has shown that phosphorylated p21-activated kinase 1 (Pak1), a mediator of small guanosine triphosphatases, plays a role in the proliferation, survival, and invasiveness of cancer cells. Thus, we examined patterns of Pak1 expression in glioblastoma and sought to determine whether the level of phosphorylated Pak1 in glioblastoma cells is associated with patient survival time. EXPERIMENTAL DESIGN: We carried out immunohistochemical staining for phosphorylated Pak1 in tumor specimens from 136 patients with glioblastoma; the tumors were classified according to Pak1 protein levels in the cytoplasm and nucleus. We compared the patients' overall survival times using Kaplan-Meier analysis and estimated the effects of levels of cytoplasmic or nuclear phosphorylated Pak1. We then down-regulated Pak1 by using small interfering RNA to knock down Pak1 in two glioblastoma cell lines to determine whether Pak1 contributed to cell viability and invasion. RESULTS: Median overall survival was significantly shorter in patients with tumors showing a moderate or high level of cytoplasmic phosphorylated Pak1 than in patients with tumors showing no cytoplasmic phosphorylated Pak1. The level of nuclear phosphorylated Pak1 was not related to survival time. Knockdown of Pak1 suppressed the invasion, but not the viability, of U87-MG and U373-MG cells. CONCLUSIONS: The presence of phosphorylated Pak1 in the cytoplasm of glioblastoma cells is associated with shorter survival, and Pak1 plays a role in the invasiveness of glioblastoma. These data suggest that Pak1 might be a potential target for the management of glioblastoma.

Adenovirus type 5 E1A-induced apoptosis in COX-2-overexpressing breast cancer cells.

INTRODUCTION: Suppression of Bcl-2 expression can overcome cellular resistance to apoptosis induced by the adenovirus type 5 gene E1A in models of ovarian and breast cancer. Celecoxib, a cyclooxygenase-2 (COX-2) inhibitor, is known to downregulate Bcl-2 expression. We hypothesized that celecoxib would enhance E1A-induced apoptosis by suppressing Bcl-2 through suppressing COX-2 expression. If successful, this strategy could represent a means of overcoming resistance to E1A gene therapy. METHODS: We first established the cytotoxicity of celecoxib in two COX-2-overexpressing E1A-transfected breast cancer cell lines (MDA-MB-231 and MDA-MB-435) and in two low-COX-2-expressing E1A-transfected cell lines (MCF-7 (breast cancer) and SKOV3.ip1 (ovarian cancer)). We next tested whether higher sensitivity to celecoxib among these cell lines resulted from increased apoptosis by flow cytometry and western blotting. We further investigated whether suppression of Bcl-2 by celecoxib was involved in the apoptosis resulting from celecoxib treatment, and we explored whether the celecoxib-induced apoptosis in these cells depends on a COX-2 downstream pathway. RESULTS: The two COX-2-overexpressing cell lines MDA-MB-231-E1A and MDA-MB-435-E1A were more sensitive to celecoxib than the corresponding control cells, but the two low-COX-2-expressing cell lines MCF-7-E1A and SKOV3.ip1-E1A were no more sensitive than control cells to celecoxib. Therefore, we used the MDA-MB-231-E1A and MDA-MB-435-E1A cells for all further experiments. In both cell lines, sub-G1 fraction was increased, or cleavage of PARP and caspase-9 were increased after 5 days of exposure to 40 microM celecoxib. However, Bcl-2 was suppressed only in the MDA-MB-435-E1A cells and not in the MDA-MB-231-E1A cells. Restoring Bcl-2 expression in the MDA-MB-435-E1A stable transfectants did not affect their sensitivity to celecoxib. However, adding prostaglandin E2 (PGE2) or PGF2alpha blunted the sensitivity to celecoxib of both E1A stable transfectants. CONCLUSION: We speculate that one mechanism by which celecoxib enhances E1A-induced apoptosis in cells that express high levels of COX-2 is through blocking PGE2 or PGF2alpha.

Cyclooxygenase-2 uses the protein kinase C/ interleukin-8/urokinase-type plasminogen activator pathway to increase the invasiveness of breast cancer cells.

Cyclooxygenase-2 (COX-2) increases breast cancer cell invasion. Expression of various pro-angiogenic and pro-invasive factors has been correlated with high expression of COX-2. However, whether these factors are essential to COX-2-mediated breast cancer invasion, and the mechanisms by which COX-2 increases the expression of these factors are unknown. Our microarray results indicate that higher COX-2 expression was associated with increased levels of interleukin-8 (IL-8), a key factor in breast cancer invasion and metastasis. COX-2 overexpressing cells (MCF-7/COX-2), generated by transfecting COX-2-encoding plasmids into the poorly invasive MCF-7 breast cancer cells, were more invasive and produced higher IL-8 levels than the parental cells. To investigate the role of IL-8 in COX-2-mediated invasion, MCF-7 parental cells were incubated with IL-8. Exogenous IL-8 increased the invasiveness of MCF-7 cells. IL-8 is one pathway by which COX-2 mediates breast cancer invasion. Protein kinase A (PKA) and protein kinase C (PKC) are activated by COX-2 and are involved in IL-8 regulation. Inhibition of PKC, not PKA, decreased IL-8 production and invasion in MCF-7/COX-2 cells. Activation of PKC, not PKA, increased IL-8 production and invasion in MCF-7 cells. Thus, the invasive effects of COX-2 are mediated by PKC, not PKA. Activity of the urokinase-type plasminogen activator (uPA) was increased in MCF-7 cells by COX-2 overexpression or by the addition of a PKC activator or by IL-8. Inhibition of PKC decreased uPA activity in MCF-7/COX-2 cells. Furthermore, inhibition of uPA activity decreased the invasiveness of MCF-7/COX-2 cells, indicating that uPA was essential to COX-2-mediated invasion. Herein we demonstrate for the first time a detailed mechanism by which COX-2 increases breast cancer invasion: the PKC/IL-8/uPA pathway.

Liposome-incorporated Grb2 antisense oligodeoxynucleotide increases the survival of mice bearing bcr-abl-positive leukemia xenografts.

We previously demonstrated that liposome-incorporated antisense oligodeoxynucleotide specific for the grb2 mRNA (L-Grb2) inhibited Grb2 protein expression and the proliferation of bcr-abl-positive leukemia cell lines. To determine whether L-Grb2 has the potential of being a therapeutic modality against bcr-abl-positive leukemia, we studied the tissue distribution of L-Grb2 in normal mice before studying its effects in mice bearing bcr-abl-positive leukemia xenografts. L-Grb2 was widely distributed in the body. The highest tissue concentrations of L-Grb2 were found in the spleen and liver, which are the organs where the tumor mass of bcr-abl-positive leukemia is mainly found. At 4 h post-injection, the amount of L-Grb2 detected per g of tissue was 64 microg in spleen and 50 microg in liver. Intravenous injection of bcr-abl-positive 32D mouse leukemia cells into radiated NOD/scid mice caused a lethal leukemia syndrome; we determined whether L-Grb2 could prolong the survival of mice bearing such xenografts. One day after leukemia cell inoculation, mice received twice weekly intravenous injections of L-Grb2. At an injection dose of 15 mg of L-Grb2 per kg of mouse body weight, 80% of mice treated with L-Grb2 survived to 48 days (end of study) whereas 0% of mice treated with the same dose of liposomal control oligonucleotide survived; the mean survival duration of these groups was 44 and 20 days, respectively. Our data indicate that L-Grb2 prolonged the survival of mice bearing bcr-abl-positive leukemia xenografts. L-Grb2 may be used as a novel cancer therapeutic modality.

HER2/neu increases the expression of Wilms' Tumor 1 (WT1) protein to stimulate S-phase proliferation and inhibit apoptosis in breast cancer cells.

High levels of the Wilms' Tumor 1 (WT1) protein and mRNA had been associated with aggressive phenotypes of breast tumors. Here we report that the HER2/neu oncogene increases WT1 expression. Approximately threefold higher levels of WT1 protein were observed in MCF-7 breast cancer cells transfected with the HER2/neu oncogene than in parental MCF-7 cells. Conversely, inhibition of HER2/neu with the anti-HER2/neu trastuzumab (Herceptintrade mark) antibody decreased WT1 protein levels in HER2/neu-overexpressing BT-474 and SKBr3 cells. We also found that HER2/neu engages Akt to regulate WT1 levels since inhibition of Akt reduced WT1 levels. Decreased expression of WT1 protein led to cell cycle arrest at the G1 phase and increased apoptosis in HER2/neu-overexpressing cells, which is correlated with decreased cyclin D1 and Bcl-2 levels. Our data indicate that HER2/neu engages Akt to increase WT1 expression, and that WT1 protein plays a vital role in regulating cell cycle progression and apoptosis in HER2/neu-overexpressing breast cancer cells.

Cyclooxygenase-2 is essential for HER2/neu to suppress N- (4-hydroxyphenyl)retinamide apoptotic effects in breast cancer cells.

We reported that HER2/neu reduces the sensitivity of breast cancer cells to N-(4-hydroxyphenyl)retinamide (4-HPR) by suppressing nitric oxide production. We show that HER2/neu uses Akt to induce cyclooxygenase-2 (COX-2) expression and that inhibition of Akt or COX-2 increases 4-HPR-induced apoptosis and nitric oxide production. Apoptosis induced by the 4-HPR and COX-2 inhibitor combination, although unaffected by an anti-HER2/neu antibody, was reversed by the COX-2 product prostaglandin E(2), indicating that COX-2 is a major mechanism by which HER2/neu suppresses 4-HPR apoptosis in breast cancer cells. Combining 4-HPR with COX-2 inhibitors may be a novel chemopreventive strategy against HER2/neu-overexpressing breast tumors.

Her2/neu induces all-trans retinoic acid (ATRA) resistance in breast cancer cells.

We observed that all-trans retinoic acid (ATRA) inhibited the growth of MCF-7 breast cancer cells, but not those transfected with HER2/NEU or its transactivating ligand HEREGULIN. This suggests that Her2/neu causes breast cancer cells to be resistant to the growth inhibitory effects of ATRA. To confirm this observation, MDA-MB-453 and BT-474 cells, which have high levels of Her2/neu and are resistant to ATRA, were incubated with the trastuzumab (Herceptin) antibody so that we could determine whether inhibition of the expression and function of Her2/neu would resensitize these cells to ATRA. Indeed, we found that MDA-MB-453 and BT-474 cells treated with trastuzumab were growth inhibitory by ATRA. We then determined whether Her2/neu uses Grb2 and Akt proteins to induce ATRA resistance. Liposome-incorporated Grb2 antisense oligonucleotides (L-Grb2) and a dominant negative (DN) AKT mutant were used to down-regulate Grb2 expression and inhibit Akt activity, respectively. When incubated with L-Grb2 or transfected with the DN AKT mutant, ATRA-resistant, Her2/neu-overexpressing cells became sensitive to ATRA. Our results indicate that Her2/neu utilizes Grb2 and Akt proteins to induce ATRA resistance in breast cancer cells. ATRA sensitivity was also correlated with RARalpha protein levels since higher RARalpha protein levels were observed in cells in which the Her2/neu pathway was inhibited.

Mechanism of action of a novel "combi-triazene" engineered to possess a polar functional group on the alkylating moiety: evidence for enhancement of potency.

Previous studies showed that SMA41, a 3-methyltriazene termed "combi-molecules" possessing a dual epidermal growth factor receptor (EGFR)/DNA targeting properties induced potent antiproliferative activity against alkylating-agent-resistant cells expressing EGFR in vitro. However, despite its marked potency, its antitumour activity in vivo was significantly hampered by its poor hydrosolubility and the moderate reactivity of its alkylating moiety. To circumvent this problem, we designed the quinazolinotriazene ZRBA1 to contain a N,N-dimethylaminoethyl group grafted to the 3-position of the triazene chain where it could serve both as a water soluble and a more potent alkylating moiety. ZRBA1 exhibited five-fold stronger EGFR tyrosine kinase (TK) inhibitory activity (IC(50)=37nM) than SMA41, decomposed into a 6-amino-quinazoline FD105 (IC(50)=200nM) and preferentially blocked EGF- over platelet-derived growth factor (PDGF)-or serum-induced cell growth. ZRBA1 induced DNA damage, concomitantly blocked EGF-stimulated EGFR phosphorylation by a partially irreversible mechanism in MDA-MB-468 breast cancer cells, and induced partially irreversible antiproliferative activity. It also prevented EGFR-mediated MAP kinase activation and, in contrast to FD105 and SMA41, induced high levels of apoptosis. Furthermore, ZRBA1 showed significantly greater antitumor activity (p<0.05) than SMA41 in the human MDA-MB-468 breast cancer xenograft model. The results in toto indicate that the appendage of N,N-dimethylaminoethyl to combi-triazenes may be an alternative to the reduced hydrosolubility and also to the lack of potency of monofunctional combi-triazenes against resistant tumours.

A novel mechanism by which N-(4-hydroxyphenyl)retinamide inhibits breast cancer cell growth: the production of nitric oxide.

N-(4-Hydroxyphenyl)retinamide (4-HPR) induces apoptosis in breast cancer cells; however, the molecular basis by which 4-HPR induces apoptosis is not well understood. In breast cancer cells, nitric oxide (NO) is predominantly an apoptotic inducer. Apoptotic agents, such as phorbol ester, tumor necrosis factor-alpha, and peptide hormones, have been shown to increase NO production in breast cancer cells. Therefore, we hypothesized that the production of No is vital for 4-HPR to induce apoptosis in breast cancer cells. We found that 4-HPR induced NO production in a dose-dependent manner in all of the breast cancer cell lines tested. The degree of growth inhibition and apoptotic induction by 4-HPR was directly correlated with the amount of NO produced. To prove that NO is essential for 4-HPR to induce apoptosis, breast cancer cells were coincubated with a competitive NO synthase (NOS) inhibitor, NG-monomethyl-L-arginine (L-NMMA), and 4-HPR, L-NMMA prevented 4-HPR from inducing inhibitory effects, indicating that NO is crucial for 4-HPR to induce its apoptotic effects in breast cancer cells. IFNs and tamoxifen (TAM) have been shown to potentiate 4-HPR effects in breast cancer cells. Both IFN-gamma and TAM enhanced the ability of 4-HPR to induce NO production in breast cancer cells, which was correlated with increased apoptosis. Alone, 4-HPR increased expression of both inducible NOS (NOSII) and endothelial NOS (NOSIII). When combined with 4-HPR, IFN-gamma and TAM enhanced NOSII expression. Thus, we have identified a novel mechanism by which 4-HPR induces apoptosis in breast cancer cells, i.e., by increasing NOS expression to induce NO production.

KGF-induced motility of breast cancer cells is dependent on Grb2 and Erk1,2.

Breast cancer metastasis is directly associated with breast cancer cell motility. Using a cell culture wounding model, we have demonstrated that keratinocyte growth factor (KGF) enhanced the motility of estrogen receptor-positive breast cancer cells. However, the mechanisms by which KGF enhanced motility of breast cancer cells are not known. In the present study, we report that KGF-induced motility requires intact tyrosine kinase signaling since genistein, a tyrosine kinase inhibitor, led to decreased motility of breast cancer cells mediated by KGF. Using cDNA microarrays, we previously found that KGF increased the expression of Grb2 mRNA by 2 3-fold. Since Grb2 plays an important role in tyrosine kinase signaling, we examined the involvement of Grb2 in KGF-induced motility. Down-regulation of Grb2 protein expression inhibited KGF-induced motility. Since Grb2 is known to regulate Erk1,2 and Akt kinase activities we determined whether these downstream proteins may be vital to KGF-induced motility. Inhibiting the activation of Erk1,2 by PD98059 suppressed KGF-induced motility whereas inhibiting the activation of Akt by wortmannin did not affect KGF-induced motility. In conclusion, these results indicate that KGF mediated signal transduction employs Grb2 to transduce the tyrosine kinase signals resulting in the activation of Erk1,2 and breast cancer cell motility.

Bcl-2-related antisense therapy.

High expression of the bcl-2 proto-oncogene is found in various human hematologic malignancies and solid tumors. Bcl-2 protein exerts its oncogenic role by preventing tumor cells from undergoing apoptosis induced by radiation, chemotherapy, and hormonal therapy. Antisense oligonucleotides directed toward the open reading frame of the bcl-2 gene have been used to inhibit Bcl-2 expression. Inhibition of Bcl-2 expression sensitizes lymphoma and leukemia cells to radiation and chemotherapy. However, it remains to be determined whether Bcl-2 antisense oligonucleotides will have a beneficial effect in solid tumors, such as breast cancer. Laboratory results indicate that Bcl-2 overexpression induces endocrine and chemoresistance in breast cancer cells. However, high levels of Bcl-2 have been associated with favorable prognostic factors, suggesting that Bcl-2 may not be an appropriate target in breast cancer. We will discuss the paradoxical role of Bcl-2 and the potential therapeutic application of Bcl-2 antisense oligonucleotides in breast cancer.

HER2/neu uses Akt to suppress retinoic acid response element binding activity in MDA-MB-453 breast cancer cells.

We previously demonstrated that HER2/neu prevents all trans-retinoic acid (ATRA) from inducing growth inhibition in MDA-MB-453 breast cancer cells. For ATRA to induce growth inhibition, it needs to bind to retinoic acid receptors and modulate gene transcription via retinoic acid response elements (RAREs). We hypothesized that HER2/neu suppresses RARE binding activity to prevent ATRA from inducing growth arrest in breast cancer cells. Electrophoretic mobility shift assays showed that when HER2/neu was inhibited by the trastuzumab antibody, RARE binding activity increased, indicating that HER2/neu suppresses RARE binding. Since trastuzumab also decreased Akt activity, we determined whether Akt regulates RARE binding activity. Compared to parental MDA-MB-453 cells, MDA-MB-453 cells transfected with a dominant negative Akt mutant (MDA-MB-453/DN-Akt) had higher RARE binding activity. However, trastuzumab did not further increase RARE binding activity in MDA-MB-453/DN-Akt cells. These data indicate that HER2/neu predominantly uses Akt to suppress RARE binding activity, which may be one mechanism by which HER2/neu induces ATRA resistance in breast cancer cells.

Nuclear retinoid receptors are involved in N-(4-hydroxyphenyl) retinamide (Fenretinide)-induced gene expression and growth inhibition in HL-60 acute myeloid leukemia cells.

N-(4-hydroxyphenyl) retinamide (Fenretinide, 4-HPR) inhibits cell growth by inducing apoptosis in numerous tumor cell types including all-trans-retinoic acid (ATRA)-resistant tumor cells. However, the mechanism(s) by which 4-HPR mediates its anti-proliferative effects remains unclear. Here, we determined whether 4-HPR induced growth inhibition and gene expression involve retinoid receptors in human acute myeloid leukemia (AML) cells (HL-60). We treated HL-60 and ATRA-resistant HL-60 (HL-60R) cells that express mutated RARalpha and very low levels of RARbeta, RARgamma and RXRalpha with 4-HPR (2 microM) for 3 days. 4-HPR showed significant anti-proliferative effects against both cell types and induced growth inhibition (92.7%) in HL-60 cells. However, at the same dose, 4-HPR induced only 53.4% growth inhibition in HL-60R cells. Growth inhibition by 4-HPR was significantly enhanced in HL-60R cells that were retroviraly transduced to express human RARalpha, RARbeta or RXRalpha (95.6%, 97.1%, and 75.6%, respectively), in comparison to HL-60R cells (P < 0.05), but not in HL-60R cells expressing RARgamma. Although ATRA and 4-HPR induced expression of CYP26, an ATRA-inducible gene encoding a cytochrome P450 enzyme, in HL-60 cells, both retinoids failed to induce CYP26 in HL-60R cells. However, induction of CYP26 mRNA by 4-HPR was restored in HL-60R cells expressing RARalpha and RARgamma, but not RARbeta and RXRalpha. In conclusion, our data suggest that nuclear retinoid receptors are involved in 4-HPR-induced growth inhibition and gene expression, and that 4-HPR can mediate its anti-proliferative effects through retinoid receptor-dependent mechanisms in HL-60 cells.

Cellular pharmacology of P-ethoxy antisense oligonucleotides targeted to Bcl-2 in a follicular lymphoma cell line.

A P-ethoxy oligonucleotide (oligo), 20 bases long and specific for the translation initiation site of human Bcl-2 mRNA, was incorporated into liposomes to increase its intracellular delivery. This oligo selectively inhibited Bcl-2 protein expression and induced growth inhibition in t(14;18)-positive transformed follicular lymphoma (FL) cell lines. We studied the inhibitory effects of shorter liposomal P-ethoxy oligos (7, 9, 11 or 15 mer) in order to determine the activity of different oligo chain lengths targeted to the same Bcl-2 mRNA. At 12 microM, all the oligos inhibited the growth of a FL cell line. We compared the 7-mer oligo with the 20-mer oligo. The two oligos inhibited Bcl-2 protein expression similarly: 66% and 60% for the 7- and 20-mer, respectively. The uptake and retention of both oligos were also very similar. Our results indicate that the Bcl-2 inhibitory activity is maintained with P-ethoxy antisense oligos ranging from 7 to 20 bases.

The HER2/Grb2/Akt pathway regulates the DNA binding activity of AP-1 in breast cancer cells.

We showed that the HER2/Grb2/Akt pathway induces all-trans retinoic acid (ATRA) resistance in breast cancer cells by suppressing the DNA binding activity of retinoic acid receptors (RAR). AP-1 activation was shown to induce ATRA resistance. Here, we determined whether AP-1 binding activity is correlated with ATRA resistance in HER2-overexpressing cells. Inhibition of HER2/Grb2/Akt decreased AP-1 binding activity in HER2-transfected cells, but increased AP-1 activity in cells that are naturally HER2-overexpressing. Since HER2/Grb2/Akt inhibition sensitized both cell types to ATRA, our results indicate that, unlike RAR, AP-1 binding activity is not correlated with ATRA sensitivity in HER2-overexpressing breast cancer cells.

Cyclooxygenase-2 utilizes Jun N-terminal kinases to induce invasion, but not tamoxifen resistance, in MCF-7 breast cancer cells.

Elevated cyclooxygenase-2 (COX-2) expression in breast tumors is associated with a lower survival rate in patients with estrogen receptor α (ERα)-positive tumors. We hypothesized that COX-2 reduces the survival rate of breast cancer patients with ERα-positive tumors since COX-2 increases the invasiveness of ERα-positive breast tumors and decreases tumor sensitivity to tamoxifen. Previously, we demonstrated that COX-2 stimulates the activity of protein kinase C (PKC) to increase the invasiveness of ERα-positive MCF-7 breast cancer cells and to decrease the sensitivity of MCF-7 cells to tamoxifen. High levels of COX-2 are associated with the activation of the mitogen-activated protein kinase (MAPK) family and the Akt kinase. However, it is not known whether these kinases mediate COX-2-induced invasive activity and tamoxifen resistance. In the present study, we report that COX-2 utilizes PKC to enhance the phosphorylation of Jun N-terminal kinases (JNKs), but not that of other MAPK family members or Akt. Inhibition aimed at JNKs reduced COX-2-induced invasion but not COX-2-induced tamoxifen resistance. We conclude that JNKs are essential for induced cell invasion by COX-2, but not tamoxifen resistance, in ERα-positive breast cancer cells.