The Induction Of Differentiation Of Leukamenin E, An Ent-kaurane Diterpene, In Human Promyelocytic Leukemia Cells HL-60and A Brief Study Of Action Mechanisms

Isodon plants, owing approximately150species, are widely distributed inworldwide, and there are about90kinds, including more than30species as a folkherbal medicine for anti-inflammatory and anti-tumor in China. Ent-kaurane diterpeneare the main natural products in this genus from which more than400have beenextracted. Oridonin, a kind of ent-kaurene diterpenoids, has shown great potential inthe clinical treatment of leukemia. It could cleave AML1-ETO into a truncatedversion and induce apoptosis selectively in leukemia. The exciting research result hasbeing used in clinical treatments, which strongly suggest the important value existingin the research on antileukemia of those compounds. Nevertheless, of the thousands ofent-kaurane diterpenoids identified, little research has been conducted in applying thenovel compounds to inducting differentiation of leukemia. Leukamenin E, an ent-kaurane diterpene, which is separated from (Isodon excisoides (Sun ex C.H.Hu) C.Y_Wu et H.w.Li) in Gansu Province, has strong cytotoxicity and could induceapoptosis in various cancer cells. However, the research about induction ofdifferentiation of Leukamenin E on leukemia has not been reported. Humanpromyelocytic leukemia HL-60cells are tested cell line in this paper for the first timeto study effects of Leukamenin E on induction of differentiation of HL-60cells, andwe conducted a brief study of its action mechanisms. The main results as follows:1. The results of trypan blue staining discovered that0.3μmol·L-1,0.6μmol·L-1,0.9μmol·L-1,1.2μmol·L-1and1.5μmol·L-1Leukamenin E significantly inhibit HL-60cells proliferation in dose-and time dependent manners; PI staining assays by flowcytometry displayed that Leukamenin E could induce strong G1phase arrest in dosedependent manner. AO/EB double staining demonstrated that0.3μmol·L-1,0.6μmol·L-1,0.9μmol·L-1,1.2μmol·L-1Leukamenin E does not induce apoptosis ofHL-60cells.2. The results of Giemsa staining, investigation of phagocytosis, NBT reductionand expression of CD11b showed that HL-60cells treated with0.3μmol·L-1,0.6μmol·L-1,0.9μmol·L-1and1.2μmol·L-1Leukamenin E after24h~72h，in which the rate of renal nucleation and seg nucleation increased; nuclear-cytoplasmic ratiodecreased; NBT reduction increased; phagocytosis enhanced, expression of CD11bincreased in a concentration dependent manner. All above suggest that Leukamenin Ecould induce HL-60cells differentiation into maturity.3. We investigated the induction of Leukamenin E on microtubules and vimentinskeleton rearrangement of the HL-60cells exposed Leukamenin E for24h by indirectimmunofluorescence technique/fluorescent tags dyeing technology research combinedwith fluorescence microscope and flow cytometry. With the increase of concentration,microtubules and micro fibrous bundle in cytoplasm significantly increased; thecontent of vimentin and the bright spot in HL-60cells increased; and the content ofmicrofilament in HL-60cells increased and gathered towards interior of the cell.4. DCFH-DA staining displayed that the ROS levels in HL-60cells advanced forthe treatment of Leukamenin E after6h in a dose dependent manner.In conclusion, Leukamenin E, an ent-kaurane diterpenoid, could evoke strongG1phase arrest causing growth inhibition, during which the cystoskeleton in the HL-60cells rearranged and the nuclear morphometry changed go along with the advent ofthe renal nucleation and seg nucleation, and induce HL-60cells differentiation intomaturity. Furthermore, we speculated that reactive oxygen species probably mediatedthose events caused by Leukamenin E.