p.s. DMSO versus tylophorine treatments, (?1) and (?2)p values
AGI-6780 synchronizing agents unambiguously revealed that tylophorine interferes with the cell cycle through a dominant G1 arrest and retardation of the S-phase progress in HepG2, HONE-1, and NUGC-3 carcinoma cells. The retardation in the S-phase progression was delayed for 48 h or longer (Fig. 3A) and thus masked the G1 arrest in asynchronizing cells (Fig. 2A), as was found by others [8] and [13]. In addition, the tylophorine inhibited DNA synthesis (data not shown) is suggested to be associated with the S-phase retardation. The tylophorine-induced G1 phase arrest was difficult to pass through, as was strongly evident by the increased proportion of cells in the G1 phase and slightly decreased proportion in the S and G2/M phase with time after release from G2/M arrest by thimidine-nacodazole (Fig. 3B). Thus, the dominant inhibitory effect of tylophorine for carcinoma cell growth is the arrested G1 phase rather than the retarded S-phase progression (Fig. 3A and B).Cyclin A2 is a cell cycle regulatory protein which gene expression is repressed during the G1 phase and induced at S-phase entry in non-transformed cells [23]. Ectopically expressed cyclin A2 overcomes G1 arrest in confluent cultures [24] and the induced expression of cyclin A2 via JunD releases the cell contact inhibition (G1 arrest) upon the TCDD treatment in rat liver oval cells [24]. Our results suggest that downregulated cyclin A2 is involved in the tylophorine-induced G1 arrest in carcinoma cells since overexpressed cyclin A2 was able to overcome the tylophorine-induced G1 arrest and facilitated the S-phase entry. Moreover, overexpressed cyclin A2 did not play a significant role for overcoming the S-phase retardation by tylophorine treatment, since the incremental trend of S-phase proportion remained the same in cells with basal or overexpressed cyclin A2 (Table 1). These results reflect the role of cyclin A2 in entry from G1 to S-phase [23].Collectively, phenanthroindolizidines (e.g. tylophorine, antofine, and DCB3503) exhibit anti-cancer growth activity through interfering
cell cycle progress but the detail effective cell cycle points and underlying mechanisms are suggested to be differentiated. In conclusion, we have clarified the effective points of tylophorine in carcinoma cell cycle progress and thus warrant further studies on the detail mechanisms of action. This study lays the foundation for future development of tylophorine analogues for anti-cancer therapeutic agents.AcknowledgmentsThis work was supported by National Health Research Institutes, Taiwan, ROC [Grants BP-094-PP-04; BP-095-PP-04; BP-096-PP-05; BP-097-PP-05] and the National Science Council of Taiwan, ROC [Grant 95-320-B-400-010-MY3]. We appreciate support from the flow cytometry core facility at National Health Research Institutes. Mr. Cheng-Wei Yang is currently a Ph.D. student of the Graduate Program of Biotechnology in Medicine sponsored by the National Tsing Hua University and the National Health Research Institutes.
