In-Vitro Study on the Cytotoxic Effects and Mechanisms of Action of Arsenic Trioxide on Human Neuroblastoma Cells

This dissertation, "In-vitro Study on the Cytotoxic Effects and Mechanisms of Action of Arsenic Trioxide on Human Neuroblastoma Cells" by On-lit, Yeung, 楊安烈, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled IN-VITRO STUDY ON THE CYTOTOXIC EFFECTS AND MECHANISMS OF ACTION OF ARSENIC TRIOXIDE ON HUMAN NEUROBLASTOMA CELLS. Submitted by YEUNG ON LIT for the Degree of Master of Philosophy at the University of Hong Kong December 2005 Recent in vitro and in vivo studies showed that arsenic trioxide (As O ) could induce 2 3 apoptosis in neuroblastoma cells through either intrinsic or extrinsic pathways with various degrees of response. We investigated the variation of response to As O in 2 3 neuroblastoma by stratifying fifteen neuroblastoma cell lines according to their response (IC ) to As O We then correlated their chemo-responsiveness to known genetic 50 2 3. (MYCN, 1p-) or epigenetic (hyper-methylation of caspase 8) prognostic markers. Two previously un-explored cytotoxic mechanisms of As O on neuroblastoma were 2 3 explored. They are the cell cycle control and the change in the expression level of survivin. We investigated these 2 aspects in seven selected cell lines with different chemo-responsiveness to As O . To explore further on the effects of over-expression of 2 3 MYCN, we tested the MYCN over-expressed or MYCN repressed TET-2/N cells with a panel of common chemotherapeutic drugs including As O . Finally, the cytotoxic effects 2 3 of a selected arsenic related drugs, mainly bismuth (Bi) and antimony (Sb) compounds were examined. We found that As O could induce apoptosis in most of the cell lines tested (12/15) with 2 3 their IC within the dosage range of 2-5 μM The As O sensitivity did not correlate 50 . 2 3 with their known MYCN and caspase 8 expression statuses. For the possible cytotoxic mechanisms involved, 4/7 selected cell lines showed cell cycle arrest at the G2/M phase and all these 4 cell lines had activated caspase 3. Apoptosis was confirmed by the increase of sub-G1 phase peak using flow cytometry. Down-regulation of survivin was observed in all cell lines tested, even in those relative resistant cells. When compared with As O, a log higher concentration of Sb O and 2-log higher concentration of 2 3 2 3 Bi O, was required to induce cell death. Bi O could only induce cell death in the 2 3 2 3 As O sensitive cells under prolonged exposure (5 days). The two commonly used Bi 2 3 and Sb compounds (Colloidal bismuth subcitrate and Sodium stibogluconate) did not have any cytotoxic effect on neuroblastoma cells even with extremely high concentrations. The cytotoxic potential of As O may differ in neuroblstoma cells with 2 3 distinct biological characteristics. Cell cycle arrest at the G2/M phase might be an important arsenic induced apoptotic mechanism involved in some neuroblastoma cells. As O induced down-regulation of survivin disregarding of their As O sensitivities, 2 3 2 3 suggestive that As O can serve as a good adjunct with other chemotherapeutic agents in 2 3 overcoming the surviving related chemo-resistance. Over-expression of MYCN was paradoxically associated with increased chemo-sensitivity and development of secondary genetic and epigenetic events and it might be accounted for the emergence of chemo-resistance in surviving neuroblastoma cells with amplified MYCN. As O 2 3 remained to be the most potent cytotoxic agent within the group V compounds in the periodic table so no potential replacement could be identified yet. Our in