Influence of p53 status on the response of Hodgkin lymphoma cells to etoposide and putative synergistic effect of resveratrol

In malignant cells mutations in the tumor suppressor gene p53 occur frequently, rendering cells less sensitive to chemotherapeutic agents. Hodgkin lymphoma are currently mainly treated by chemotherapy, with varying efficacy, depending e.g. on mutations in tumor suppressor- and proto-oncogenes.
This study investigates the mode of action of the chemotherapeutic agent etoposide as sole active substance as well as in combination resveratrol, a natural compound with diverse positive properties, in two Hodgkin lymphoma cell lines, differing in p53-status. Whereas cell line L 540 shows the wildtype of p53, L 428 harbors a mutation in its DNA-binding domain. Etoposide is a widely used cytostatic drug, leading to DNA strand breaks. As tumors are increasingly developing resistance to chemotherapeutic agents the development of new therapeutic strategies is indispensable. Resveratrol is a polyphenolic phytoalexin that occurs in many foods, especially in grapes. Its anti-oxidant, anti-inflammatory, anti-cancer properties make it an interesting compound for combination studies.
At several time points of a 24-hour etoposide treatment (with or without a 72-hours pre-treatment with resveratrol) gene expression analyses of selected apoptosis-related and other target genes were done by RT-qPCR and Western blot. Nuclear fragmentation was investigated in DAPI-stained cell preparations and apoptosis/necrosis was monitored via Annexin V staining.
Treatment duration, etoposide concentration and the type of cell line had a significant effect on the expression of most genes. Etoposide treatment led to strongest overexpression in the apoptosis-inducing gene GADD45 (more pronounced in the p53-mutant cell line, indicating p53-independent activation), the opposite effect was seen in p53. Strong downregulation of TNFα and HO1 in the mutant L-428 whereas upregulation in L-540 point out their opposing mechanisms of action. Nuclear fragmentation occurred in wildtype L-540 earlier and more pronounced (4 hr) than in L-428 (24 hr), as well as apoptosis was induced in L-540 already after 3 hours, necrosis setting in after 7 hours. Both mechanisms were not influenced in L-428, pointing out the crucial role of p53 in induction of cell death. The 72-hours pre-treatment with resveratrol did not show a distinct effect, with influence on the expression of some genes as well was on nuclear fragmentation of L-428, which was probably due to the low concentration used in the pretreatment.