Dose Response Curves Can be Used to Assess Sensitivity of Busulfan (Bu) in Neuroblastoma (NB) Cell Lines

Bu is an alkylating agent for conditioning regimens in transplantation. The purpose of this study was to evaluate apoptosis and cell death in- vitro in four NB cell lines (NGP, IMR-5, SK-N-SH and SKN-DOX) and two primary tumors cell lines (Patient A and B) treated with Bu. Cells were exposed in-vitro to Bu with various concentrations (0.001, 0.005, 0.01, 0.05, and 0.1 mg/ml). Cell viability was assessed using trypan blue, apoptosis and cell death by flow cytometry via Annexin V and Propidium Iodide. Samples were assessed at 6, 24, 48, and 72 hours. Untreated samples served as control. All experimentations were in triplicate and mean values were used. Table 1 illustrates the median percent at 24 hours for the comparison of the lowest (0.001 mg/ml) to highest (0.1 mg/ml) Bu dosage.  Cell lines with a high percentage (> 50%) in combination of apoptosis and cell death regardless of Bu concentrations were considered sensitive (SK-N_SH, SK-N-DOX, Patients A and B) while those with decreased percentage (<20%) of both were considered resistant (NGP, IMR-5) to Bu. This methodology can be used to assess prospective patients with NB and to determine if Bu should be included in their conditioning regimens for autologous hematopoietic stem cell transplants.

Table 1.

	Control			0.001 mg/ml			0.1 mg/ml
	Viability	Apoptosis	Death	Viability	Apoptosis	Death	Viability	Apoptosis	Death
NGP	91.5%	7%	1.5 %	72%	25%	3%	80%	15%	5%
IMR-5	63%	15%	22%	63%	12%	21%	56%	21%	23%
SK-N-SH	71%	13%	13%	29%	33%	38%	20%	27%	56%
SK-N-DOX	92%	3%	5%	31%	16%	41%	28%	17%	57%
Patient A	40%	28%	16%	35%	42%	23%	42%	42%	24%
Patient B	57%	14%	30%	29%	26%	41%	21%	22%	57%