Methionine depletion with recombinant methioninase: in vitro and in vivo efficacy against neuroblastoma and its synergism with chemotherapeutic drugs

Abstract

Methionine starvation can modulate gene methylation, cell cycle transition and pathways related to survival following DNA damage. Methionine depletion by recombinant methioninase (rMETase) may have in vitro and in vivo efficacy against neuroblastoma (NB), especially when combined with chemotherapeutic drugs. rMETase from Pseudomonas putida was produced in Escherichia coli and purified by ion-exchange chromatography. rMETase alone inhibited the proliferation of 15/15 NB cell lines in vitro. Among these 15 cell lines, only 66N demonstrated rMETase-induced apoptosis. rMETase alone suppressed LAN-1 and NMB-7 xenografts (p < 0.01) and no toxicities were noted other than reversible weight loss. In vitro efficacy experiments combining rMETase and chemotherapeutic agents were carried out using SK-N-LD and SK-N-BE (1)N established at diagnosis, as well as LAN-1, SK-N-BE (2)C and NMB-7 established at relapse. Microtubule depolymerization agents including vincristine, vinorelbine, vinblatine and mebendazole showed synergism when tested in combination with rMETase in all 5 cell lines. Among DNA damaging agents, synergy with rMETase was observed only in cell lines established at diagnosis and not at relapse. Cell cycle analysis showed that rMETase arrested G2 phase and not M phase. In vivo efficacy experiments using LAN-1 and NMB-7 xenografts showed that rMETase rendered vincristine more effective than vincristine alone in tumor growth suppression (p < 0.001). In conclusion, methionine depletion inhibited NB proliferation and arrested tumor cells at G2 phase. rMETase synergized with microtubule depolymerization agents. Moreover, synergism between rMETase and DNA damaging agents was dependent on whether cell lines were established at diagnosis or at relapse.

Figure 1. Molecular size and purity of rMETase

A: SDS-PAGE. Lane 1: Purified rMETase; Lane 2: Supernatant from whole cell extract; Lane 3: Whole cell crude extract, Lane 4: Marker. B: HPLC size exclusion profile of purified rMETase. The major peak at 15.547 min was rMETase.

Figure 2. Effect of vincristine with or without rMETase on NB cell proliferation of SK-N-BE(1)N and SK-N-LD

Open circle: vincristine alone; Solid triangle: rMETase + vincristine combined treatment.

Figure 3. Effect of rMETase with or without vincristine on cell cycle arrest of SK-N-BE(2)C

The flow cytometry results were presented in density plots, where the quadrants were set to include live cells. The lower left quadrant contained G1 phase population; the lower right quadrant contained G2 phase population; the upper right quadrant contained M phase population.

Figure 4. Efficacy of in vivo rMETase in NB xenografts

Open square: Control (N=10 for LAN-1 and N=26 for NMB-7); Solid circle: rMETase treatment (N=10 for LAN-1 and N=34 for NMB-7); ** p<0.01 when AUC of rMETase group was compared to the control group.

Figure 5. Efficacy of in vivo combination treatment with rMETase and vincristine

Open square: Control (N=9 for LAN-1 and NMB-7); Solid circle: rMETase alone (N=14 for LAN-1 and N=7 for NMB-7); Open circle: vincristine alone (N=8 for LAN-1 and N=7 for NMB-7); Solid triangle: rMETase + vincristine combined treatment (n=14 for LAN-1 and n=13 for NMB-7); ** p<0.001 when AUC of combination group was compared to other 3 groups, respectively.