Cellular and molecular parameters of sanguinarine induced bimodal cell death

Weerasinghe, Priya (2002) Cellular and molecular parameters of sanguinarine induced bimodal cell death. Doctoral (PhD) thesis, Memorial University of Newfoundland.

[English] PDF (Migrated (PDF/A Conversion) from original format: (application/pdf)) - Accepted Version Available under License - The author retains copyright ownership and moral rights in this thesis. Neither the thesis nor substantial extracts from it may be printed or otherwise reproduced without the author's permission. Download (20MB) [English] PDF - Accepted Version Available under License - The author retains copyright ownership and moral rights in this thesis. Neither the thesis nor substantial extracts from it may be printed or otherwise reproduced without the author's permission. (Original Version)

Abstract

Sanguinarine is a potential anti-cancer drug belonging to the benzophenanthridine alkaloids. K562 erythroleukemia cells treated with sanguinarine at concentrations of 1.5 (ig/ml for 2 hours displayed the morphology of apoptosis or programmed cell death (PCD) and at 12.5 |ig/ml, displayed the morphology of blister formation or blister cell death (BCD). This dual modality of cell death processes was coined "bimodal cell death" (BMCD). The present study is devoted to the cellular and molecular characterization of bimodal cell death. -- Sanguinarine-induced PCD in the Bcl-2 (anti-apoptotic) low-expressing K562 erythroleukemia cells was found to have increased expression of the pro-apoptotic Bax protein as well as caspase-3 activation, whereas BCD was found to have neither. cDNA expression array analysis of sanguinarine induced PCD in K562 cells failed to reveal the presence of Bax at the gene transcript level, which suggests the relevance of postradiational modification of proteins as opposed to its de novo synthesis. On the other hand, high Bcl-2 protein expressing JM1 pre-B lymphoblastic cells, when treated with sanguinarine, failed to undergo the morphology of either PCD or BCD. Western blotting analysis of JM1 cells treated with sanguinarine showed an increase in Bcl-2 protein, over and above its high endogenous levels while there was no detectable Bax expression or caspase-3 activation. Bcl-2 appeared to have played the role of being anti-PCD as well as anti-BCD in JM1 cells during sanguinarine treatment. The role of several other members ofthe Bcl-2 family: Bad, Bak, Bcl-xL? Bcl-xs, Hrk, NBK, Bik, Bid as well as the tumour suppressor protein p53 was found to be negligible. -- A comparative analysis of PCD and BCD in K562 and JM1 cells treated with sanguinarine was also done at the cellular level using the following methods: light and electron microscopy; terminal deoxynucleotidyl transferase (TdT) end labeling; 51Cr release; trypan blue exclusion; propidium iodide exclusion and annexin-V-binding. These studies further characterized PCD and BCD at the cellular level in K562 cells, and at the same time highlighted the anti-PCD and anti-BCD roles played by Bcl-2 in sanguinarine- resistant JM1 cells. -- Immunofluorescence-flow cytometry studies of protein expression in K562 cells treated with sanguinarine showed a high percentage of Bax positive cells with low levels ofthe nuclear transcription factor NF-kB in PCD, and a high percentage of Bax negative cells with high levels of NF-kB in BCD. JM1 cells showed elevated levels of Bcl-2 in response to sanguinarine treatment. This flow cytometry study also helped confirm the findings of Western blot analysis. Thus, the interaction between Bcl-2, Bax and NF-kB might be crucial to understand sanguinarine-induced PCD and BCD. -- Sanguinarine was also found to overcome the P-glycoprotein-mediated drug efflux pump, widely believed to be associated with the classical multidrug resistant (MDR) phenomenon, in the vinblastin resistant CEM-VLB leukemia cells. Studies pertaining to the cellular and molecular parameters of sanguinarine induced PCD and BCD in the MDR CEM-VLB cells and in their wild type counterparts CEM-T4 (P-gp negative), showed that the bimodal cell death pattern in both cell lines was qualitatively similar. Moreover, flowcytometric studies in these cells showed an increased presence of sanguinarine in cells during BCD but not PCD. Thus, the phenomenon of BCD, associated with elevated intracellular levels of sanguinarine, may have had a role to play in overcoming the P-gp mediated multidrug resistance. -- Understanding different cell death mechanisms of individual anti-cancer agents may lead to their effective administration, alone or in combination with other established therapies. Accordingly, we have attempted to characterize the novel form of cell death- BCD, described in literature as the "blebbing phenomenon", and propose it as a form of cell death, distinct from apoptosis.

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