Development and Improvement of Leukemia Treatment Methods

Much progress has been made in the past few decades in the development and improvement of treatments for leukemia. However, many of the drugs currently in use have wide ranging and sometimes very severe side effects. Therefore, despite this advancement, it is still important to develop new anti-leukemia treatments.

One group of antileukemia compounds that shows some promise are molecules derived from Acridine, an organic compound that is a nitrogen heterocycle, and Acridone, which is Acridine with a carbonyl group. Acridine and acridone are chemicals that were originally used and pigments and dyes, but have since come to be used to form derivatives that have a wide range of medical applications. In the past few decades they have come to be used for treatments against bacteria, viruses, and even as treatments for psoriasis or malaria. Most importantly in reference for this article, many acridine derivatives have come into use as chemotherapy drugs for the treatment of cancer. In fact, some have begun the process of clinical trials to test their viability as cancer treatments, such as Amsacrine, which is used in combination with anti-cancer drugs to treat leukemia. This article was focused around a derivative of acridone, N-(2-(dimethylamino)ethyl)-1-((3-methoxybenzyl)amino)-5-nitro-9-oxo-9,10-dihydro-acridine-4-carboxamide, which they refer to as “8q”, that was synthesized by their laboratory had been previously demonstrated to display potent antitumor effects against CCRF-CEM cells, which are tumor forming cells associated with the development of T-cell acute lymphoblastic leukemia.

While the anti-leukemia effects had already been demonstrated in previous laboratory tests, the mechanism of action by which 8q suppressed these tumor cells was unknown. Many of the acridine/acridone derivatives used as anti-cancer drugs are known to target DNA or DNA related enzymes, such as DNA topoisomerases, however this is not the case for all of them, and researchers have found other mechanisms of actions, such as inducing apoptosis through inhibition of Poly (ADP-ribose) polymerase or by promoting oxidative stress.

Due to the promising nature of 8q as an effective antitumor compound, the authors of this paper make the case that it is necessary to determine the mechanism of action, because it is important for the sake of structural optimization and screening of lead compounds. Therefore, the purpose of this study was to attempt to determine the mechanism of action of 8q in CCRF-CEM cells.The authors of this paper did not put forward a formal prediction for what the mechanism of action of 8q is. They instead approached it utilizing a combination of metabolomics with bioanalysis methods in order to identify the mechanism action. As a result, there isn’t a well-defined expectation or hypothesis. At most, they only predicted that they would find a mechanism of action, they did not put forward a prediction for what it would be.

For this experiment, the research group synthesized 8q and utilized CCRF-CEM cells that were Human T Cell lymphoblast-like cell lines from various lineages. In addition to the assays that were later used for the determination of mechanism of action, the research group also performed an in silico utilizing different software packages in order to examine the possible affinities for various targets that 8q might display, as well as potential toxic effects and undesirable interactions. The leukemia cells were cultured in various mediums that varied based on different types of those cells, with antibiotic supplements added to those mediums. After the culture of the cells over 12-16 hours, the cell cultures were treated with different concentrations for 24 or 48 hours. After these treatments, a combination of many biological assays and analysis software packages were performed in order to try and narrow down the specific mechanism of action.

The first of these was a measure of cell viability after treatment. MTT assay, a technique which relies on the reduction of a special dye to measure metabolic activity, was used to analyze the viability of the cells. The researchers calculated a percent viability as a ratio between the cells that received the treatment drug, and those in the control group. The researchers also utilized a software program to measure the half maximal inhibitory concentrations. Also, in order to separate and analyze the samples for the purpose of metabolomics analysis, the researchers utilized a technique called “ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry”, which provided that that could be processed by a software program, different metabolic markers extracted from that data were identified by comparison to existing data in various databases, and changes in metabolic pathways were analyzed using special software tools. Many commonly utilized assay techniques were performed on these test cell cultures in order to bring in a wide range of data. Flow cytometry was used to measure apoptosis, and western blot analysis utilized gel electrophoresis in order to separate and analyze the proteins. In order to measure the levels of mitochondrial membrane potential, the treated leukemia cells were exposed to Rh123, a fluorescent dye commonly utilized for this purpose, and the subsequent fluorescent intensity was measured by the use of a fluorescent spectrophotometer. An ATP assay was performed that utilized an established method that utilizes luciferase. A different fluorescent assay, one utilizing DCFH DA, was used for the detection of Reactive Oxygen Species, and was also measured by the use of fluorescent spectrophotometer. The values gathered were subject to statistical analysis in the form of a t-test in order to generate a p value, and the authors of this paper chose as their standard that p values of less than 0.05 were significant. The analysis of the metabolites was subject to a more involved technique, that used a false discovery rate controlling approach, which gave a corrected p-value, or Q-value.