Am80, a RARα Specific Synthetic Retinoid Induced a Strong Growth Inhibition in Hematologic Malignant Cells Including Myeloid and Lymphoid Cell Lines.
Authors: Jimi Shiro , Mashima Kota , Sakabe Mitsukuni , Ushijima Emiko , Suzumiya Junji , Hara Shuji , Tamura Kazuo 
Published: October 2019

Abstract
Acute promyelocytic leukemia (APL) is an objective disease for treatment with retinoids including ATRA, however adverse effects appear due to non-specific binding to different retinoic acid receptors (RARs). To overcome this disadvantage a targeting drug namely Am-80, Tamibarotene, has been developed, which can react specifically to RARα. On the other hand, recent studies addressed that other therapeutic applications with retinoids can be use against malignant tumors other than APL. Therefore the mechanisms of selective action of Am-80 on tumor cells were still obscure. In the present study, we studied effects of Am-80 regarding cell death and growth inhibition using myeloid and lymphoid malignant cultured cells, i.e., HL60, HL60R, K-562, Kasumi-1, MEG01, Raji, U266B1, and U937. Expression of RARs was analyzed by a FACS-based GUAVA PCA method using 3 sets of cell samples [1st antibody (−)/2nd PE-antibody (−), (−/+), (+/+)], then median value of fluorescence intensity was obtained and calculated a relative expression value with following formula: ((+/+)-((−/+)-(−/−)))/(−/−), by which each tumor cell with different background level can be standardized. In normal growing condition, RARα in HL60 was significantly greater (p<0.006) than in not only HL60R but also all of other cells. Effects of Am-80 were examined on cell growth during 9 days of incubation with 0.5% ethanol and 10−7~−5 M Am-80 in ethanol. In this culture we always kept cells in an exponential growth by adjusting cell number to 5x105/10 ml/flask at the time of subculture in each 3-day-interval. All of the time points viable and dead cell number was monitored by FACS-based GUAVA ViaCount assay. HL-60 was found to be only a cell type sensitive to Am-80, by which viable cell density was reduced more than 95 % after 9 days of incubation. Dead/total cell density gradually increased after 3 days of incubation, and reached about 40 % after 9 days of incubation. Mode of cell death was examined by assessments for annexin V, intracellular dissociated-caspases and TUNEL, showing that most of dead cells were apoptotic cells, but necrotic cells appeared at a minimum level. Expression of RARα in HL60 with Am-80 decreased strongly during 9 days of incubation, and reached a level lower than that in HL60R. While, no change was found in HL60R during incubation with Am-80. Next, Am-80-inducing growth inhibition was examined. Growth inhibition was time- and dose-dependently occurred in all of the cells, and reached 40–70 % by 10−5 M Am-80 after 9 days of incubation. The growth inhibition value was negatively correlated with RARα expression value (r=−0.534, p=0.04). When TGFβ in cultures with different concentrations of Am-80 was quantified by an ELISA method, TGFβ was released dose-dependently, and its reactivity was negatively correlated with RARα expression value. These results indicated that Am-80-inducing strong growth inhibition was mediated by TGFβ in an autocrine manner, in which RARα pool size may be a regulatory factor.