Buthionine Sulfoximine and Myeloablative Concentrations of Melphalan Overcome Resistance in a Melphalan-Resistant Neuroblastoma Cell Line

Abstract

Background 

Alkylator resistance contributes to treatment failure in high-risk neuroblastoma. Buthionine sulfoximine (BSO) can deplete glutathione and synergistically enhance in vitro sensitivity to the alkylating agent melphalan (L-PAM) for many neuroblastoma cell lines, but optimal use of this combination needs to be defined because clinical responses have been less frequent and not durable.

Patients and Methods 

The authors established and characterized a neuroblastoma cell line (CHLA-171) from a patient who died of progressive disease after treatment with BSO and low-dose L-PAM.

Results 

CHLA-171 lacks MYCN amplification, expresses PGP (P-glycoprotein) 9.5 RNA, and shows cell surface antigen expression (human leukocyte antigen class I weakly positive, but HSAN 1.2 (hybridoma, SAN 1.2) and anti-GD2 (anti-ganglioside GD2 antibody) strongly positive) characteristic of neuroblastoma cell lines. Twenty-four hours of BSO treatment (0–1,000 μmol/L) maximally depleted CHLA-171 glutathione to 36% of baseline. The cytotoxic response of CHLA-171 to BSO and L-PAM, alone and in combination, was measured by digital image microscopy (DIMSCAN) over a range of drug concentrations and compared with drug levels obtained in the patient during BSO/L-PAM therapy. As single agents, CHLA-171 was highly resistant to L-PAM (LD ₉₀ = 42 μmol/L; peak plasma concentration in the patient equals 3.9 μmol/L) and moderately resistant to BSO (LD ₉₀ = 509 μmol/L; steady-state concentration in the patient equals 397 μmol/L). Treatment with a 10:1 (BSO:L-PAM) fixed ratio combination synergistically overcame resistance (3–4 logs of cell kill, combination index <1) at clinically achievable levels of BSO (100–400 μmol/L) and levels of L-PAM (10–40 μmol/L) clinically achievable only with hematopoietic stem cell support.

Conclusions 

The in vitro results obtained for CHLA-171 suggest that BSO/L-PAM therapy may be optimally effective for drug-resistant neuroblastoma using myeloablative doses of L-PAM.