Dicentrine Analogue-Induced G2/M Arrest and Apoptosis through Inhibition of Topoisomerase II Activity in Human Cancer Cells

Abstract

Lindera megaphylla has been traditionally used as an antineoplastic and wound healing remedy. We previously demonstrated the antitumor effects of D-dicentrine, a natural aporphine alkaloid from the root of L. megaphylla. To generate analogues, series of phenanthrene alkaloids from D-dicentrine were synthesized by degradation with ethyl chloroformate in pyridine, base hydrolysis, and N-alkylation. In this study, we demonstrated that one of the synthesized D-dicentrine analogues (here after designated as analogue 1) exhibited more potent cytotoxic effects than D-dicentrine in colon adenocarcinoma, hepatoma, leukemia, and epidermoid carcinoma cells. We performed cell cycle and apoptotic analysis by flow cytometry, an apoptotic DNA detection ELISA assay, and topoisomerase II activity by the kinetoplast DNA concatenation assay for studying their cytotoxic mechanisms. We found that both D-dicentrine and analogue 1 induced apoptosis and G₂/M arrest in HL-60 leukemia cells. The percentage of apoptotic cells induced by analogue 1 was 4.5-fold higher than that induced by D-dicentrine as evident from measuring the amount of histone-bound DNA fragments. Moreover, we found that analogue 1 was 28-fold more potent than D-dicentrine for inhibition of topoisomerase II activity by the kinetoplast DNA concatenation assay. Our findings indicate that D-dicentrine analogue 1 is very promising as a potential antitumor agent for future study.

Key words

Lindera megaphylla - Lauraceae - D-dicentrine - dicentrine analogues - cytotoxicity - apoptosis - topoisomerase II inhibitor - cell cycle arrest

^* These authors contributed equally to this work.

• References

• 1 Liu LF. DNA topoisomerase poisons as antitumor drugs. Annu Rev Biochem 1989; 58: 351-375
• 2 Nitiss JL. Targeting DNA topoisomerase II in cancer chemotherapy. Nat Rev Cancer 2009; 9: 338-350
• 3 Oppegard LM, Ougolkov AV, Luchini DN, Schoon RA, Goodell JR, Kaur H, Billadeau DD, Ferguson DM, Hiasa H. Novel acridine-based compounds that exhibit an anti-pancreatic cancer activity are catalytic inhibitors of human topoisomerase II. Eur J Pharmacol 2009; 602: 223-229
• 4 Huang RL, Chen CC, Huang YL, Ou JC, Hu CP, Chen CF, Chang C. Anti-tumor effects of d-dicentrine from the root of Lindera megaphylla . Planta Med 1998; 64: 212-215
• 5 Shen CC, Huang RL, Lin HF, Liao JF, Chen CC. Synthesis and cytotoxic activity of dicentrine analogues. Chin Pharmaceut J 2000; 52: 353-360
• 6 Huang HL, Yeh WC, Lai MZ, Mirtsos C, Chau H, Chou CH, Benchimol S. Impaired TNFalpha-induced A20 expression in E1A/Ras-transformed cells. Br J Cancer 2009; 101: 1555-1564
• 7 Chau H, Mirtsos C, Huang HL. Regulation of death complexes formation in tumor necrosis factor receptor signaling. Exp Cell Res 2011; 317: 1841-1850
• 8 Tsai TJ, Lin RH, Chang CC, Chen YM, Chen CF, Ko FN, Teng CM. Vasodilator agents modulate rat glomerular mesangial cell growth and collagen synthesis. Nephron 1995; 70: 91-99
• 9 Montrucchio DP, Cordova MM, Santos AR. Plant derived aporphinic alkaloid S-(+)-dicentrine induces antinociceptive effect in both acute and chronic inflammatory pain models: evidence for a role of TRPA1 channels. PLoS One 2013; 8: e67730
• 10 Teng CM, Yu SM, Ko FN, Chen CC, Huang YL, Huang TF. Dicentrine, a natural vascular alpha 1-adrenoceptor antagonist, isolated from Lindera megaphylla . Br J Pharmacol 1991; 104: 651-656
• 11 Chen CC, Huang YL, Ou JC, Su MJ, Yu SM, Teng CM. Bioactive principles from the roots of Lindera megaphylla . Planta Med 1991; 57: 406-408
• 12 Yu SM, Chen CC, Ko FN, Huang YL, Huang TF, Teng CM. Dicentrine, a novel antiplatelet agent inhibiting thromboxane formation and increasing the cyclic AMP level of rabbit platelets. Biochem Pharmacol 1992; 43: 323-329
• 13 Woo SH, Sun NJ, Cassady JM, Snapka RM. Topoisomerase II inhibition by aporphine alkaloids. Biochem Pharmacol 1999; 57: 1141-1145
• 14 Wong N, Yeo W, Wong WL, Wong NL, Chan KY, Mo FK, Koh J, Chan SL, Chan AT, Lai PB, Ching AK, Tong JH, Ng HK, Johnson PJ, To KF. TOP2A overexpression in hepatocellular carcinoma correlates with early age onset, shorter patients survival and chemoresistance. Int J Cancer 2009; 124: 644-652
• 15 Perrin D, van Hille B, Barret JM, Kruczynski A, Etievant C, Imbert T, Hill BT. F 11782, a novel epipodophylloid non-intercalating dual catalytic inhibitor of topoisomerases I and II with an original mechanism of action. Biochem Pharmacol 2000; 59: 807-819
• 16 Etievant C, Kruczynski A, Barret JM, Perrin D, van Hille B, Guminski Y, Hill BT. F 11782, a dual inhibitor of topoisomerases I and II with an original mechanism of action in vitro, and markedly superior in vivo antitumour activity, relative to three other dual topoisomerase inhibitors, intoplicin, aclarubicin and TAS-103. Cancer Chemother Pharmacol 2000; 46: 101-113
• 17 Huang RL, Chen CC, Huang YL, Hsieh DJ, Hu CP, Chen CF, Chang C. Osthole increases glycosylation of hepatitis B surface antigen and suppresses the secretion of hepatitis B virus in vitro . Hepatology 1996; 24: 508-515
• 18 Carmichael J, DeGraff WG, Gazdar AF, Minna JD, Mitchell JB. Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing. Cancer Res 1987; 47: 936-942
• 19 Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, Riccardi C. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Methods 1991; 139: 271-279
• 20 Huang HL, Chen CC, Yeh CY, Huang RL. Reactive oxygen species mediation of baizhu-induced apoptosis in human leukemia cells. J Ethnopharmacol 2005; 97: 21-29
• 21 Galluzzi L, Aaronson SA, Abrams J, Alnemri ES, Andrews DW, Baehrecke EH, Bazan NG, Blagosklonny MV, Blomgren K, Borner C, Bredesen DE, Brenner C, Castedo M, Cidlowski JA, Ciechanover A, Cohen GM, De Laurenzi V, De Maria R, Deshmukh M, Dynlacht BD, El-Deiry WS, Flavell RA, Fulda S, Garrido C, Golstein P, Gougeon ML, Green DR, Gronemeyer H, Hajnoczky G, Hardwick JM, Hengartner MO, Ichijo H, Jaattela M, Kepp O, Kimchi A, Klionsky DJ, Knight RA, Kornbluth S, Kumar S, Levine B, Lipton SA, Lugli E, Madeo F, Malomi W, Marine JC, Martin SJ, Medema JP, Mehlen P, Melino G, Moll UM, Morselli E, Nagata S, Nicholson DW, Nicotera P, Nunez G, Oren M, Penninger J, Pervaiz S, Peter ME, Piacentini M, Prehn JH, Puthalakath H, Rabinovich GA, Rizzuto R, Rodrigues CM, Rubinsztein DC, Rudel T, Scorrano L, Simon HU, Steller H, Tschopp J, Tsujimoto Y, Vandenabeele P, Vitale I, Vousden KH, Youle RJ, Yuan J, Zhivotovsky B, Kroemer G. Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes. Cell Death Differ 2009; 16: 1093-1107
• 22 Larsen AK, Grondard L, Couprie J, Desoize B, Comoe L, Jardillier JC, Riou JF. The antileukemic alkaloid fagaronine is an inhibitor of DNA topoisomerases I and II. Biochem Pharmacol 1993; 46: 1403-1412