Fenretinide: A Novel Treatment for Endometrial Cancer

<div><p>Resistance to progestin treatment is a major hurdle in the treatment of advanced and reoccurring endometrial cancer. Fenretinide is a synthetic retinoid that has been evaluated in clinical trials as a cancer therapeutic and chemo-preventive agent. Fenretinide has been established to be cytotoxic to many kinds of cancer cells. In the present study, we demonstrate that fenretinide decreased cell viability and induced apoptosis in Ishikawa cells, which are an endometrial cancer cell line, in dose dependent manner <i>in-vitro</i>. This effect <i>was found to be</i> independent of retinoic acid nuclear receptor signaling pathway. <i>Further</i>, we have shown that this induction of apoptosis by fenretinide may be caused by increased retinol uptake via STRA6. Silencing of STRA6 was shown to decrease apoptosis which was inhibited by knockdown of STRA6 expression in Ishikawa cells. Results of an <i>in-vivo</i> study demonstrated that intraperitoneal injections of fenretinide in endometrial cancer tumors (created using Ishikawa cells) in mice inhibited tumor growth effectively. Immunohistochemistry of mice tumors showed a decrease in Ki67 expression and an increase in cleaved caspase-3 staining after fenretinide treatment when compared to vehicle treated mice. Collectively, our results are the first to establish the efficacy of fenretinide as an antitumor agent for endometrial cancer both <i>in-vitro</i> and <i>in-vivo</i>, providing a valuable rationale for initiating more preclinical studies and clinical trials using fenretinide for the treatment of endometrial cancer.</p></div

Effects of megace on retinol uptake genes in endometrial cancer Ishikawa cells (A–C).

<p>Total RNA was isolated from Ishikawa cells treated with DMSO (Vehicle), megace, fenretinide and a combination of megace and fenretinide both. Relative mRNA expressions of (<b>A</b>) STRA6, (<b>B</b>) CRBP1 and (<b>C</b>) CYP26A1 were analyzed with RT- qPCR. Data are presented as fold change in relative expressions of mRNA from DMSO treated samples and represent the mean ±SEM of 5 independent experiments. An asterisk indicates a significant (*<i>P</i><0.05) increase in mRNA expression of these genes in Ishikawa cells treated with fenretinide or megace + fenretinide cells as compared to DMSO treated cells.</p

Inhibition of cell proliferation and increase in apoptosis following fenretinide treatment.

<p>Excised tumors were fixed in formalin, paraffin embedded and sectioned. (<b>A</b>) H & E staining and immunohistochemical staining with anti-Ki67 antibody (proliferation marker) and anti-cleaved caspase -3 (apoptosis marker) of whole sections of tumor xenografts. (<b>B</b>) The images are at 100<i>μ</i>m.</p

Effects of Fenretinide on Ishikawa cell viability and apoptosis (A–E).

<p>Ishikawa cells were treated with DMSO (Veh), (<b>A</b>) 2–20μM fenretinide, (<b>B</b>) 0.1–10μM megace or (<b>C</b>) a combination of megace + fenretinide for 24 h and cell viability was measured using the Prestoblue reagent. Data are presented as fold change in relative fluorescence units from DMSO treated samples and represent the mean ±SEM of 4–5 independent experiments. An asterisk indicates a significant (*<i>P</i><0.05) decrease in cell viability in Ishikawa cells treated with fenretinide as compared to the DMSO treated group. (<b>D</b>) Ishikawa cells were treated with DMSO (Veh) or 1–10μM fenretinide which induced apoptosis in a dose dependent manner within 24 h of treatment as demonstrated by increases in cleaved caspase-9 and cleaved-PARP protein levels by western blotting. (<b>E</b>) The treatment of Ishikawa cells with megace did not have any effect on these markers of apoptosis. Actin was used as loading control. Blots are representative of 3 independent experiments.</p

Inhibition of fenretinide induced apoptosis by knockdown of STRA6 gene expression.

<p>Ishikawa cells were transfected with 30 nM siCTL (nontargeting negative control siRNA) or siSTRA6. (<b>A</b>) Total RNA was isolated from untreated Ishikawa cells at 3 consecutive days of post transfection and the expression of STRA6 gene was measured by RT-qPCR. Data are presented as fold change in relative mRNA expression of STRA6 in siSTRA6 transfected cells and siCTL transfected cells and represent the mean ±SEM of 5 independent experiments. An asterisk indicates a significant (*<i>P</i><0.05) decrease in mRNA expression of STRA6. (<b>B</b>) Transfected Ishikawa cells were treated with DMSO (Veh) or 6μM fenretinide for 24 h after and immunoblotting for apoptotic markers was performed. STRA6 gene expression knockdown inhibited fenretinide induced apoptosis in Ishikawa cells as demonstrated by decreases in protein levels of cleaved caspase-9 and cleaved-PARP. Blots are representative of 3 independent experiments.</p

Effect of fenretinide on endometrial tumor progression in an Ishikawa xenograft mouse model (A–C).

<p>(<b>A</b>) Ovariectomized nude CD-1 female mice were injected with Ishikawa cells in both the flanks and estrogen pellets were implanted. Tumors were allowed to grow for 2 weeks. After 2 weeks of tumor establishment, the mice were treated with vehicle (20 mice), megace (19 mice), fenretinide (21 mice) or megace + fenretinide (20 mice) and tumors were harvested after 3 weeks of treatment. (<b>B</b>) Images of representative excised tumors from each group. (<b>C</b>) The fold change in tumor volume (from day 1 treatment to day of sacrifice) of vehicle, fenretinde, meagce or both fenretinide + megace treated mice was plotted after 3 weeks of treatment. Data are shown as means ± min to max of two different experiments. The P values indicate a significant inhibition by fenretinide, megace or megace + fenretinide both on tumor growth (*P<0.05). The largest decrease in tumor size was observed in the mice treated with fenretinide alone.</p