Proteasome Inhibitors Can Increase Efficacy of Topoisomerase Poisons in NSCLC

CLEVELAND—Tinkering with NFkB has little effect on the activity of topoisomerase poisons in non-small cell-lung cancer (NSCLC), but proteasome inhibitors can increase the efficacy of drugs such as irinotecan and etoposide. Information on proteasome inhibitors was presented at the Vanderbilt University Symposium by Ram Ganapathi, MD, staff scientist at the Cleveland Clinic’s Taussig Cancer Center.

"There is a need for more information about what actually happens after DNA damage, including questions about apoptosis and about other mechanisms of cell death," Dr. Ganapathi said. "In a variety of tumor cells, treatment with topoisomerase poisons produces increased DNA binding activity of NFkB. We became interested in whether the activation of NFkB is required for producing apoptosis and cytotoxicity."

Possible Influences

A number of possible factors influence the progression from the DNA cleavable complex to cell death in tumor cells treated with topoisomerase poisons. Studies were done using two human non-small-cell lung cancer (NSCLC) cell lines. One (NSCLC-3) is a large-cell undifferentiated carcinoma from the lower lobe of the left lung. The other (NSCLC-5) is a poorly differentiated carcinoma with stage IV metastatic brain lesions. Dr. Ganapathi said the cell-death pathway was explored using dominant-negative mutants for NFkB and testing the effects of treatment with SN-38 (the active metabolite of irinotecan), and etoposide, which is a topoisomerase II poison.

"Both etoposide and SN-38 activate NFkB," Dr. Ganapathi said. "Paclitaxel (Taxol) and cisplatin (Platinol) do not." Maximum activation was at 2 to 3 hours. Dr. Ganapathi pointed out that in NSCLC, NFkB may not be required for apoptosis, since it can be induced by drugs such as paclitaxel that do not induce NFkB.

Role of Proteasomes

The role of proteasomes was explored using the proteasome inhibitor MG-132. "Treatment with MG-132 followed by etoposide or SN-38 dramatically decreases upregulation of NFkB," Dr. Ganapathi said. Shutting down NFkB inhibited apoptotic pathways in the short-term, but apoptosis had largely resumed by 24 hours after treatment.

Ultimately, Dr. Ganapathi is trying to determine what signals drive cells to apoptosis.

"Transfection of NSCLC cells with a dominant negative mutant for NFkB inhibited the transcription and DNA binding activity of NFkB usually induced by SN-38 or by etoposide, but this did not alter drug-induced apoptosis," Dr. Ganapathi explained. "Regulation of apoptosis by mitochondrial release of cytochrome C and activation of pro-caspase-9 followed by cleavage of poly-ADP-ribose polymerase by effector caspase-3 and caspase-7 was similar in control cells and in the mutant cells treated with SN-38 or etoposide.

In contrast to pretreatment with the proteasome inhibitor MG-132, exposure to MG-132 following SN-38 or etoposide enhanced apoptosis compared to drug alone," he continued (Figure 1). "We conclude from this that apoptosis induced by topoisomerase poisons in human NSCLC is not mediated by NFkB but can be manipulated by proteasome inhibitors."