1. What is the problem and what is known about it so far?
The research article is focused on determining the possible role of 5’- methylthioadenosine (MTA) in inducing cell death or apoptosis in cancer cells. MTA is a sulfur-containing that is found in mammalian tissues and is generated through the polyamine biosynthetic pathway. The gene responsible for the production of MTA is found on the short arm of chromosome 9. It is known that MTA has the ability to stop specific reactions that are crucial for the normal functioning of cell, including cell proliferation, maturation, gene expression and apoptosis. Such inhibitory capability of MTA has thus rendered this molecule as a prospective agent for cancer therapeutics and tissue regeneration. It has also been earlier established that 5’- methylthioadenosine
Phosphorylase (MTAP) has the inherent capability to regulate the concentration of MTA inside a cell, through a positive feedback mechanism. Such capability is strongly associated to the capacity of cells to further divide and multiply, wherein the presence of MTAP inhibitors directly results in the decrease in the concentration of MTAP, which in turn results in the increase in the concentration of MTA, which consequently results in apoptosis. The chemical structure of MTAP has earlier been determined by kinetic isotope assays and quantum calculations. Other earlier investigations have generated analogues of MTAP which have similar inhibitory effects, including methylthio-DADMe-immucillin-A (MT-DADMe-ImmA).
2. Why did the researchers do this particular study?
The investigators of this research article conducted this particular study to determine the specific effects of the synthetic MTAP inhibitor MT-DADMe-ImmA in two head and neck squamous cell carcinoma cell lines, FaDu and Cal27. The study aims to determine whether MT-DADMe-ImmA confers a preferential toxic effect to specific types of cancer cells, of which is determined by alterations in methylation patterns of genes that consequently result in different protein expressions in cells.
Other research studies have established that deletions of the MTAP gene are frequently observed in different types of cancers, yet tumor progression still continues amidst such absence of MTAP activity. Interestingly, it has also been earlier determined that the deletion of the MTAP gene in the particularly affect tumor cells results in a different metabolic effect that when the MTAP gene is deleted in the entire organism. Hence, this particular study also aims to determine whether there are difference in metabolism if the MTAP gene is absent in the tumor cells and in the entire organism.
3. Who was studied?
Specific carcinoma cell lines were used in this particular study. Head and neck (FaDu and Cal27), breast (MCF7) and glioblastoma (U87) cancer cell lines were grown in vitro. In addition, a normal fibroblast cell lines, CRL2522 and GM02037, were also employed as control cell lines. These cultured cells were exposed to either 20 μM MTA or 1 μM MT-DADMe-ImmA at single, separate exposures or in combination. The response of the treated cells was compared to that of untreated cells.
4. How was the study done?
The response of the treated cells was quantified using several cellular and biochemical assays. The cytotoxic effect of MTA and MT-DADMe-ImmA were determined using the Alamar Blue assay and cellular apoptosis was tested through fluorescence-activated cell sorting (FACS). The treated cells were also analysed for MTAP, caspases 3, 7 and 9, PARP and actin using immunoblot analyses. The activity level of MTAP was also assessed using synthesized radiolabelled MTA that was set to react with the cells are assay using high-performance liquid chromatography (HPLC). The amount of polyamines and MTA in the treated cells was quantified by cation exchange chromatography and HPLC/fluorescence. The degree of methylation in the treated cells was evaluated by microarray profiling of CpG islands which are common DNA targets of methylation in cells.
5. What did the researchers find?
The researchers observed that FaDu cancer cells and CRL2522 fibroblast cells that were exposed to 1 μM MT-DADMe-ImmA and 20 μM MTA for 24 hr showed a 94% – 96% decrease in MTAP activity, suggesting that the synthetic MTAP analogue has the ability to specifically enter FaDu and CRL2522 cells. They also observed that such exposure results in a heightened level of putrescine, which is a polyamine that is an intermediate molecule in the polyamine biosynthetic pathway of cells. The heightened level of putrescine proves that the polyamine biosynthetic pathway has been blocked because it has accumulated in the treated cells and was not further processed into another molecule. Further testing of the treated cells with DFMO, an inhibitor of carboxylation, showed that the amount of polyamines in the treated cells was decreased but did not result in the apoptosis of FaDu cells, suggesting that the polyamine pathway is not directly responsible to inducing cell death in this particular type of cancer cells.
The study also revealed that MTAP is expressed only in selected cancer and normal cells such as FaDu, Cal27, CRL2522 and is not expressed in MCF7, A549 and U87. They also observed that MT-DADMe-ImmA and MTA did not decrease the growth rate and did not induce cell death in the cultured cells. Only the FaDu cancer cells were inhibited from growing, but only depending on the presence and concentration of both agents and not by exposure to a single chemical, suggesting the MTA may play a role in preventing cell growth and promoting cell death.
They observed that MTA specifically affected cells at the G2/M stage of interphase upon 2 days of treatment, as indicated in flow cytometry data. The other cancer cell lines reacted to the treatment only after 8 days of exposure to the MTAP inhibitor. An increase in the expression of caspases 3, 7 and 9 was also observed in FaDu cells, suggesting that cell death was induced by the introduction of the MTAP inhibitor. Microarray analysis of CpG islands showed that there was a significant decrease in the level of methylation in FaDu cells, suggesting that gene expression was inhibited by exposure to the MTAP inhibitor.
6. What were the limitations of the study?
The study only indirectly evaluated the gene expression levels in the treated cells by analyzing the degree of methylation of the CpG islands. It would be more informative if the study evaluated the global expression of gene in the entire cells so that they could present a more comprehensive description of the effect of the MTAP inhibitors. It would also be interesting to do this same evaluation in normal cells. In addition, the study only employed two concentrations of MTAP inhibitors, which is very limited in terms of information the study can generate. It would have been better if the investigators used approximately 5 different concentrations of MTAP inhibitors are treatment variables in their study. The study only described acute exposure to MTAP inhibitors, which is described as less than one week exposure. It would be interesting to know the effects of chronic exposure to the inhibitors, as well as intermittent exposures.
7. What are the implications of the study?
The study provides a better understanding of the mechanism of selectively inhibiting cell growth and inducing cell death or apoptosis in cells that are cultured in vitro. Such information may be instrumental to the design of further molecular treatments of different types of cancer. The use of MTAP inhibitor over general anticancer drugs seems to be more beneficial because it does not affect all types of cells but only specific type. Anticancer drugs are commonly very risky to use because these drugs attack all highly proliferative cells, including those of the gastric lining, scalp and blood, that the cancer patient experiences severe side-effects during chemotherapy. This study, until further testing and analysis, may provide an alternative mode for cancer treatment.