NOXA as critical mediator for drug combinations in polychemotherapy

Abstract

During polychemotherapy, cytotoxic drugs are given in combinations to enhance their anti-tumor effectiveness. For most drug combinations, underlying signaling mechanisms responsible for positive drug–drug interactions remain elusive. Here, we prove a decisive role for the Bcl-2 family member NOXA to mediate cell death by certain drug combinations, even if drugs were combined which acted independently from NOXA, when given alone. In proof-of-principle studies, betulinic acid, doxorubicin and vincristine induced cell death in a p53- and NOXA-independent pathway involving mitochondrial pore formation, release of cytochrome c and caspase activation. In contrast, when betulinic acid was combined with either doxorubicine or vincristine, cell death signaling changed considerably; the drug combinations clearly depended on both p53 and NOXA. Similarly and of high clinical relevance, in patient-derived childhood acute leukemia samples the drug combinations, but not the single drugs depended on p53 and NOXA, as shown by RNA interference studies in patient-derived cells. Our data emphasize that NOXA represents an important target molecule for combinations of drugs that alone do not target NOXA. NOXA might have a special role in regulating apoptosis sensitivity in the complex interplay of polychemotherapy. Deciphering the differences in signaling of single drugs and drug combinations might enable designing highly effective novel polychemotherapy regimens.

Impact of the p53 status of tumor cells on extrinsic and intrinsic apoptosis signaling

“…Another advantage of the employed experimental setting is that xenograft cells better resemble the in vivo situation as they do not feature non-physiologic alterations or comprise a selection of mutations that do not resemble the patient situation. We studied the impact of p53 on TRAIL sensitivity in xenografted ALL cells using the recently described experimental setting of RNA interference after amplification of primary childhood ALL cells in NOD/SCID mice [3,4,7,21-24]. RNA interference against p53 markedly reduced the p53 expression as described before (Additional file 1: Figure S4 and data not shown) [21].…”

“…Five different mutant forms of p53 were introduced by mutagenesis which are known to be associated with reduced protein stability (V143A), defect of DNA binding (R248W, R273H) or structural instability (R175H, R249S) (Figure 2 and Additional file 1: Figure S2) [16]. For the second approach, the previously described technique of RNA interference against p53 using a lentiviral system was used [2-4] to generate an efficient knockdown of p53 (Figure 3A and Additional file 1: Figure S3). For the last approach, access to pairs of tumor cells either expressing p53 or with somatic knock-out of p53 was obtained (Figures 1, 2 and 3A, Additional file 1: Figure S3B).…”

“…In contrast to the many ongoing studies using mostly biochemical modulations, the specific impact of p53 gene alterations was investigated in a limited number of studies with respect to the general impact on intrinsic cell death induction [5,7,10-12]. Within the research activities of the extrinsic cell death induction by death inducing ligands like TRAIL, the focus was exclusively drawn to the promising approaches of p53 activation by the addition of or pre-incubation with chemotherapeutic drugs or targeted stimuli [3,4,13]. …”

Platinum(IV) complex LA-12 exerts higher ability than cisplatin to enhance TRAIL-induced cancer cell apoptosis via stimulation of mitochondrial pathway

“…Therefore, we examined if the Noxa upregulation could contribute to the apoptosis-sensitizing action of the LA-12 and TRAIL combination. Although Noxa was suggested to play a decisive role in mediating efficient apoptosis induction for certain drug combinations [38], the siRNA-mediated individual silencing of Noxa was not sufficient to significantly affect the cell sensitivity to the apoptosis induced by LA-12/cisplatin and TRAIL in HCT116 cells. In addition to Noxa, we investigated whether LA-12/cisplatin-mediated stimulation of another important BH3-only protein, Bim, could contribute to potentiation of apoptosis triggered by TRAIL.…”

In Vivo Imaging Enables High Resolution Preclinical Trials on Patients’ Leukemia Cells Growing in Mice

“…Patient-derived leukemia cells were freshly isolated from mouse spleens, purified and cultured in RPMI medium supplemented with 20% FCS, 1% penicillin/streptomycin, 1% gentamycin, 6 µl/ml mixture of insulin, transferrin and selenium (Invitrogen, Carlsbad, CA), 1 mM sodium pyruvate and 50 µM 1-thioglycerole (Sigma-Aldrich, St. Louis, MO) in the absence of further cytokines as described [16], [17]. Cells were transduced overnight with GLuc virus in the additional presence of 3 µg/ml polybrene (Sigma, Hamburg, Germany).…”