Sensitivity to sodium arsenite in human melanoma cells depends upon susceptibility to arsenite-induced mitotic arrest.

McNeely SC, Belshoff AC, Taylor BF, Fan TW, McCabe MJ Jr, Pinhas AR, States JC, 2008. Sensitivity to sodium arsenite in human melanoma cells depends upon susceptibility to arsenite-induced mitotic arrest. Toxicol Appl Pharmacol. 2008 Jun 1;229(2):252-61.

Summary

McNeely, et al. sought to investigate the efficacy and mechanism of sodium arsenite as a chemotherapeutic agent against human melanoma. Melanoma is a serious but less common form of skin cancer that is often deadly after metastasis. The authors used four different human melanoma cell lines as a model and exposed them to varying concentrations of sodium arsenite, which is an FDA-approved chemotherapy against certain types of leukemia. It is known that arsenite acts as a chemotherapeutic by inducing apoptosis in cancerous cells, but the mechanism for this induction has not been definitively determined.

Using AlamarBlue fluorescence as a measure of viability, the investigators found that two of the four human melanoma cell lines (A375 and SK-Mel-2) were sensitive to concentrations of arsenite that could be realistically and safely achieved in practical application [See Figure 1] The authors confirmed that cell death was due to apoptosis by visually observing membrane blebbing and more rigorously by the application of Western Blot. Western Blot showed cleavage of capase 3 and PARP in the two sensitive cell lines, which are two key proteins in the apoptotic pathway. This was sufficient to validate apoptosis as the method of cell death caused by arsenite. Furthermore, the investigators found that the two resistant cell lines required higher concentrations of arsenite to yield the same effect.

Figure 1

Next, the authors worked to uncover the nature of the arsenite resistance in the two resistant cell lines (SK-Mel-3 and SK-Mel-28). One major finding of the paper was evidence showing no significant difference in intracellular arsenite concentration among all 4 cell lines after 24 hours of arsenite treatment. This was surprising because the authors assumed that arsenite resistance would be the result of a greater ability to transport arsenite out of the cell and reduce the intracellular concentration. They used inductively coupled plasma mass spectrometry (ICP-MS) to determine intracellular arsenite concentrations. It was also shown that inhibiting the arsenite trafficking pathway significantly lowered the resistance of the Mel-3 and Mel-28 cell lines. This was done by treatment of the resistant cells with buthionine sulfoximine (BSO), which selectively inhibits proteins involved in transporting arsenite out of the cell. The authors concluded that BSO’s mechanism of reducing arsenite resistance is due to an increase in intracellular concentration of arsenite.

The authors went on to investigate mitotic arrest associated with the induction of apoptosis. The mitotic index is a measure of the percentage of cells engaged in mitosis at a given time. It was found that the sensitive cell lines have a significantly higher mitotic index at any given concentration of arsenite treatment. [See Figure 6B] Flow cytometry was performed for a more detailed measure of cells in specific phases of the cell cycle. [See Figure 6A] The sensitive cell lines were found to accumulate in the G2/M cycle compartment, while the resistant cell lines were found to accumulate in G1 and S compartments. The investigators concluded that in A375 and SK-Mel-2, arsenite is arresting cells at mitosis, which then causes the initiation of apoptosis. In resistant cells, arsenite does not arrest cells at mitosis and thus cannot induce apoptosis at the same concentrations.

Figure 6

Finally, McNeely et al. tested their hypothesis that resistance to mitotic arrest is due to differences in the function of the spindle checkpoint. The spindle checkpoint arrests the progression of mitosis in normal cells to prevent abnormal chromosome segregation prior to anaphase. To test this hypothesis, the investigators employed Paclitaxel, which is a compound that triggers the spindle checkpoint. By treating cells with Paclitaxel, they found that the arsenite-sensitive cells had a functional spindle checkpoint, while the arsenite-resistant cells had a malfunctioning one. [See Figure 7A & 7B] The authors investigated further by using Western Blot to show that one of the key proteins in the spindle checkpoint pathway (BUBR1) has reduced expression in the arsenite-resistant cells. The authors conclude that these observed differences in the spindle checkpoint can explain differences in arsenite-sensitivity of these four cell lines.

Figure 7

Critique

Certain aspects of the experiments performed by the authors may have been less than ideal. Several of the experiments were performed on just 2 of the 4 cell lines, and the results were generalized to all arsenite-sensitive or arsenite-resistant cells. This may have been done to save on costs and time, but it may not give a full picture of the effects of arsenite on all types of melanoma. Secondly, when the authors performed flow cytometry, multiple variables were varied rather than controlling all but one. This can be seen in Figure 2A of the paper. It would be ideal for the authors to control all but one variable in these experiments. In addition, there is a somewhat questionable interpretation of data for some of the authors’ experiments. For example, in the experiments involving BUBR1, the authors refer to a “slight increase in M index from 2% to 13%,” which is actually a 6x increase.

Significance

Melanoma is the most common form of skin cancer in young people. It is not the most common form of skin cancer in general, but its mortality rate is 50x higher than that of other forms. If caught early, melanoma can often be excised effectively, but if not, melanoma quickly metastasizes to other organs. At that point, the overall potential for treatment success is limited. Because of that, new chemotherapies for melanoma are extremely important, because they are some of the few options available to target cancer after metastasis. Furthermore, the findings of McNeely et al. support a new mechanism for the induction of apoptosis, and a new understanding of why certain cells may be resistant. Increasing our knowledge of the biology involved increases our potential ability to manipulate these cells. A greater knowledge of the behavior of mammalian cell lines also potentiates our mastery of these cells and their potential applications in tissue engineering.