Analysis of gene expression identifies PLAB as a mediator of the apoptotic activity of fenretinide in human ovarian cancer cells.

Fenretinide (4-HPR) is a synthetic retinoid with antitumor activity, which induces apoptosis in cancer cell lines of different histotypes. To identify genes contributing to its apoptotic activity in ovarian cancer cells, we monitored, by cDNA arrays, gene expression changes after 4-HPR exposure in A2780, a human ovarian carcinoma cell line sensitive to the retinoid. Among the differentially expressed transcripts, PLAcental Bone morphogenetic protein (PLAB), a proapoptotic gene, was the most highly induced. In a panel of ovarian carcinoma cell lines with different 4-HPR sensitivities, PLAB upregulation was associated with cellular response to 4-HPR, its overexpression increased basal apoptosis and its silencing by small interfering RNA decreased the ability of 4-HPR to induce apoptosis. PLAB induction by 4-HPR was p53- and EGR-1 independent and was regulated, at least in part, by increased stability of PLAB mRNA. PLAB up-modulation by 4-HPR also occurred in vivo: in ascitic cells collected from patients with ovarian cancer before and after 4-HPR treatment, PLAB was upmodulated in 2/4 patients. Our results in certain ovarian cancer cell lines indicate a role for PLAB as a mediator of 4-HPR-induced apoptosis. The correlation of increased PLAB in vivo with antitumor activity remains to be established.

Involvement of c-Fos in fenretinide-induced apoptosis in human ovarian carcinoma cells.

Fenretinide (HPR), a synthetic retinoid that exhibits lower toxicity than other retinoids, has shown preventive and therapeutic activity against ovarian tumors. Although the growth inhibitory effects of HPR have been ascribed to its ability to induce apoptosis, little is known about the molecular mechanisms involved. Since the proto-oncogene c-Fos has been implicated in apoptosis induction, we analyzed its role in mediating HPR response in a human ovarian carcinoma cell line (A2780) sensitive to HPR apoptotic effect. In these cells, HPR treatment caused induction of c-Fos expression, whereas such an effect was not observed in cells made resistant to HPR-induced apoptosis (A2780/HPR). Moreover, in a panel of other human ovarian carcinoma cell lines, c-Fos inducibility and HPR sensitivity were closely associated. Ceramide, which is involved in HPR-induced apoptosis, was also involved in c-Fos induction because its upregulation by HPR was reduced by fumonisin B(1), a ceramide synthase inhibitor. The causal relationship between c-Fos induction and apoptosis was established by the finding of an increased apoptotic rate in cells overexpressing c-Fos. Similarly to that observed for c-Fos expression, HPR treatment increased c-Jun expression in HPR-sensitive but not in HPR-resistant cells, suggesting the involvement of the transcription factor activating protein 1 (AP-1) in HPR-induced apoptosis. In gene reporter experiments, HPR stimulated AP-1 transcriptional activity and potentiated the AP-1 activity induced by 12-tetradecanoylphorbol 13-acetate. Furthermore, inhibition of AP-1 DNA binding, by transfecting A2780 cells with a dominant-negative Fos gene, caused decreased sensitivity to HPR apoptotic effects. Overall, the results indicate that c-Fos plays a role in mediating HPR-induced growth inhibition and apoptosis in ovarian cancer cells and suggest that c-Fos regulates these processes as a member of the AP-1 transcription factor.

Long-term effects of fenretinide, a retinoic acid derivative, on the insulin-like growth factor system in women with early breast cancer.

High insulin-like growth factor-I (IGF-I) levels are associated with an increased risk of breast cancer in premenopausal women. Because the synthetic retinoid fenretinide showed a beneficial effect on second breast cancers in premenopausal women in a Phase III trial, we studied its long-term effects on IGF-I levels. We measured, at yearly intervals for up to 5 years, the circulating levels of IGF-I, IGF binding protein (BP)-3, and their molar ratio in 60 subjects < or = 50 years of age and 60 subjects > 50 years of age allocated either to fenretinide or no treatment. In women < or = 50 years of age, measurements of IGF-II, IGFBP-1, and IGFBP-2 were also performed. The associations between biomarkers and drug or metabolite plasma concentrations were also investigated. All biomarkers were relatively stable over 5 years in the control group. Compared with controls and after adjustment for baseline, treatment with fenretinide for 1 year induced the following changes: IGF-I, -13% [95% confidence interval (CI), -25 to 1%] in women < or = 50 years of age and -3% (95% CI, -16 to 13%) in women > 50 years of age; IGFBP-3, -4% (95% CI, -12 to 6%) in both age groups; IGF-I:IGFBP-3 molar ratio, -11% (95% CI, -22 to 1%) in women < or = 50 years of age and 1% (95% CI, -11 to 16%) in women > 50 years of age. These effects were apparently maintained for up to 5 years, although fewer samples were available as time progressed. No change in other IGF components was observed. Drug and metabolite concentrations were negatively correlated with IGF-I and IGF-I:IGFBP-3 molar ratio in women < or = 50 years of age. Fenretinide induces a moderate decline of IGF-I levels in women < or = 50 years of age. The association between IGF-I change and the reduction of second breast cancers in premenopausal women warrants further study.

Decrease in drug accumulation and in tumour aggressiveness marker expression in a fenretinide-induced resistant ovarian tumour cell line.

We investigated whether the efficacy of fenretinide (HPR) against ovarian tumours may be limited by induction of resistance. The human ovarian carcinoma cell line A2780, which is sensitive to a pharmacologically achievable HPR concentration (IC(50)= 1 microM), became 10-fold more resistant after exposure to increasing HPR concentrations. The cells (A2780/HPR) did not show cross-resistance to the synthetic retinoid 6-[3-adamantyl-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437) and were not sensitive, similarly to the parent line, to all-trans-retinoic acid, 13-cis-retinoic acid or N-(4-methoxyphenyl)retinamide. A2780/HPR cells showed, compared to parental cells, a 3-fold reduction in colony-forming ability in agar. The development of HPR resistance was associated with a marked increase in retinoic acid receptor beta (RARbeta) mRNA and protein levels, which decreased, together with drug resistance, after drug removal. The expression of cell surface molecules associated with tumour progression including HER-2, laminin receptor and beta1 integrin was markedly reduced. The increase in the levels of reactive oxygen species is not involved in HPR-resistance because it was similar in parental and resistant cells. Conversely differences in pharmacokinetics may account for resistance because, in A2780/HPR cells, intracellular peak drug levels were 2 times lower than in A2780 cells and an as yet unidentified polar metabolite was present. These data suggest that acquired resistance to HPR is associated with changes in marker expression, suggestive of a more differentiated status and may be explained, at least in part, by reduced drug accumulation and increased metabolism.

Role of retinoic acid receptor overexpression in sensitivity to fenretinide and tumorigenicity of human ovarian carcinoma cells.

The role of retinoic acid receptor (RAR) expression in sensitivity to N-(4-hydroxyphenyl)retinamide (4HPR or fenretinide) as well as on the tumorigenicity of human ovarian carcinoma cells was examined. Two human ovarian cancer cell lines, A2780 and IGROV-1, with a 10-fold difference in sensitivity to 4HPR were chosen to study RAR involvement in the response to 4HPR. To determine which RAR was effective, RARalpha, beta and gamma were individually overexpressed in A2780 cells, which are the most sensitive to 4HPR. Sensitivity to 4HPR was increased in RARbeta-overexpressing clones, whereas it was slightly decreased in RARalpha transfectants (which had diminished RARbeta expression) and was unchanged in clones transfected with RARgamma. IGROV-1 cells, which are RARbeta negative, were transfected with RARbeta. Surprisingly, none of the obtained IGROV-1 RARbeta transfectants expressed RARbeta protein, in spite of RARbeta mRNA transcription. All clones were similar to the parental IGROV-1 cells in their sensitivity to 4HPR. Treatment with a pharmacologically achievable concentration of 4HPR (1 microM) led to a rapid 2-fold increase in RARbeta mRNA levels in A2780 cells, but it did not induce RARbeta expression in IGROV-1 cells. Analysis of the tumorigenicity of A2780-transfected clones revealed that overexpression of RARalpha was associated with a significant reduction in tumor takes (50% and 67%, respectively, vs. 96% for the parent line) and with a reduced growth rate. Oncogenicity was clearly decreased in only 1 of the 2 RARbeta-overexpressing clones (33% takes) and was unchanged in the 2 clones with increased RARgamma expression. Our results demonstrate that basal expression and 4HPR inducibility of RARbeta play a role in mediating 4HPR response in ovarian cancer cells. The findings of reduced oncogenicity of clones overexpressing RARalpha and of one clone overexpressing RARbeta indicate that RARalpha and RARbeta might have a tumor-suppressive effect in ovarian tumors.

Pharmacokinetics and effects on plasma retinol concentrations of 13-cis-retinoic acid in melanoma patients.

The pharmacokinetics of 13-cis-retinoic acid (13cisRA) and its effects on retinol plasma levels were investigated after the first and the last doses in melanoma patients, who participated in a study run to assess tolerance over a long period of a treatment schedule of 13cisRA associated with recombinant interferon alpha2a (rIFN-alpha2a). Melanoma patients with regional node metastases after radical surgery were randomized to be treated for 3 months with rIFN-alpha2a, 3 x 10(6) IU s.c. every other day, associated with oral 13cisRA at doses of 20 mg day(-1) (five patients) or 40 mg every other day (seven patients). Maximum 13cisRA blood concentrations usually occurred 4 h after drug administration, with average values of 406 and 633 ng ml(-1) (i.e. 1.3 and 2.1 microM) after the 20 and 40 mg dose respectively. The average half-life (t(1/2)) was approximately 30 h. The maximum concentration, the t(1/2) and the area under the concentration-time curves from 0 to 48 h (AUC(0-48)) of 13cisRA did not change after multiple dosing, whereas the AUC(0-48) of its major blood metabolite, 4-oxo-13-cis-retinoic acid, increased. Immediately after 13cisRA treatment, retinol plasma levels started to decline and they reached the lowest values (approximately 20% reduction) shortly after the time of maximum 13cisRA concentrations (i.e. 4-12 h after drug intake). Afterwards, values returned to baseline. The amount of retinol reduction in time was correlated with 13cisRA maximum concentrations.