Basis of Action Found for Anti-inflammatories’ Anticancer Role

Aspirin, Other NSAIDs Block Cell Cycle, Boost Cell Death

It is over 100 years since acetyl salicylic acid, better known as aspirin, was first synthesized and marketed as an analgesic, but scientists are still finding new uses for it. Currently prescribed as an anti-inflammatory, an anti-coagulant, and a pain killer, could aspirin, or another nonsteroidal anti-inflammatory drug (NSAID), be the next treatment for cancer? Though not exactly the first drugs that come to mind when one thinks of cancer therapy, epidemiological evidence suggests that NSAIDs can help prevent a variety of tumors, including colon, breast, and ovarian cancers—observations that have left some researchers scratching their heads for an explanation. Now, in the Dec. 15 Cancer Research, Towia Libermann, HMS associate professor of medicine at Beth Israel Deaconess Medical Center and director of the BID Genomics Center, and colleagues report that many NSAIDs deal cancer cells a double blow, putting a halt to the cell cycle and inducing an apoptotic spiral of cell death, all at the behest of the cytokine interleukin 24 (IL-24). “The finding will help in the identification and development of more potent anticancer treatments,” said Libermann.

Photo by Graham Ramsay

Though combination therapy is becoming an important option in oncology, choosing the right mix of drugs can be challenging. By identifying the way nonsteroidal anti-inflammatory drugs retard the growth of cancer cells, Towia Libermann (right), Luiz Zerbini, and colleagues have provided a rationale for their use in cancer treatment.

The Interleukin Connection
That some NSAIDs induce apoptosis in cancer cells is not new, but exactly how they promote programmed cell death or arrest cell growth was unclear. Libermann and colleagues found the link with IL-24, also known as melanoma differentiation associated gene-7 (MDA-7), when they treated a variety of cancer cells with the NSAID sulindac sulfide, one of two active metabolites of the cyclooxygenase-2 (COX-2) inhibitor sulindac sulfoxide. Joint lead authors Luiz Zerbini, an HMS instructor in medicine and researcher in the Genomics Center at BID, and research fellow Akos Czibere used DNA microarrays to obtain a transcriptional profile of cancer cell lines treated with the drug. “What we found was both dramatic and quite surprising,” said Libermann.

Not only did the profiling reveal that sulindac sulfide alters expression of genes involved in apoptosis and the cell cycle, but it showed that IL-24 was, by far, the most upregulated gene in two prostate cancer cell lines, increased by 140-fold in PC-3 cells and 722-fold in DU145 cells. And the effect was not limited to prostate cancer cells or to this specific anti-inflammatory. Using quantitative RNA amplification to measure IL-24, the researchers found that it is also upregulated by 10- to 20-fold in a variety of kidney, breast, stomach, and other prostate cancer cells in response to sulindac sulfide, while a range of different NSAIDs, including aspirin, flurbiprofen, and diclofenac—in fact all anti-inflammatories that induce apoptosis in cancer cells—also increased expression of IL-24. Though these drugs have widely different structures and modes of action, their effects all seem to converge to upregulate this one cytokine. In contrast, NSAIDs with no apparent apoptotic effect failed to induce the interleukin.

Double Shot
IL-24 is best known for its role in immune regulation, but in 2002 Paul Fisher, a co-author on the paper, and colleagues at Columbia University Medical Center in New York City, found that overexpression of the cytokine induces apoptosis in human melanoma cells. It appears to do this by upping expression of two other proteins called growth arrest and DNA damage inducible 45-alpha (GADD45-alpha) and GADD45-gamma, which are checkpoint proteins that put a halt to the cell cycle. The same signaling pathway seems to be driven by the apoptotic NSAIDs because Libermann and colleagues also found increased expression of GADD45-alpha and gamma in cancer cells treated with sulindac sulfide. This induction, and apoptosis itself, could be almost completely blocked in prostate cancer cells treated with RNA interference (RNAi) to knock down IL-24. NSAIDs also failed to arrest the cell cycle in the absence of the interleukin.

Two for one. By inducing expression of IL-24 and subsequently GADD45-alpha and GADD45-gamma, nonsteroidal anti-inflammatory drugs (NSAIDs) modulate two distinct pathways that retard cancer cells. JNK activation promotes apoptosis, or programmed cell death, while repression of checkpoint protein Cdc2-cyclin B puts a stop to the cell cycle and retards cell growth.

Diagram courtesy of Towia Libermann

The involvement of GADD45 proteins in blocking cell growth makes perfect sense, because they inhibit the Cdc2-cyclin B complex that is essential for transition from the G2 to the M phase of the cell cycle. In fact, Zerbini and Czibere found that Cdc2 activity was substantially decreased in PC-3 cells 24 hours after treatment with sulindac sulfide. It is harder to see how the GADD45 proteins might spur programmed cell death and yet, when the team used RNAi to knock down these checkpoint proteins, it prevented NSAID-driven apoptosis. Could these proteins also affect the apoptotic machinery?

Libermann’s and other research groups previously found that GADD45-alpha and gamma interact with the mitogen activated protein kinase (MAPK) signaling pathway, which can spur activation of c-Jun NH2-terminal kinase (JNK), an enzyme that can induce apoptosis in cancer cells. Specifically, GADD45-alpha and gamma can activate MAPK kinase kinase 4. To see if this pathway might be activated by NSAIDs, Zerbini and Czibere tested cancer cells for JNK activation. While active JNK is absent from PC-3 and DU145 prostate cancer cells under normal culture conditions, addition of sulindac sulfide caused a robust increase in JNK activity, an induction that was attenuated in PC-3 cells by about 60 and 80 percent, respectively, when GADD45-alpha and GADD45-gamma were knocked down by RNAi. All told, the data show that by activating IL-24, and consequently the two GADD45 proteins, NSAIDs can trigger separate signaling pathways that stop the cell cycle and turn on apoptosis (see figure).

“These interesting findings suggest an alternate means of inducing and exploiting MDA-7/IL-24 to promote cancer-specific cell killing,” said David Curiel, director of the Division of Human Gene Therapy and the Gene Therapy Center at the University of Alabama at Birmingham and an expert on viral vector gene delivery for cancer treatment, who was not an author on the paper. “Moreover, this strategy represents a novel way of enhancing the therapeutic benefit of MDA-7/IL-24 with genuine potential to develop small molecules that can promote anticancer activity by exploiting induction of this therapeutic cytokine, rather than by direct gene delivery.” Adenoviral delivery of MDA-7/IL-24 is currently being evaluated in phase I/II clinical trials for advanced carcinomas.

Whether the same NSAID effects can be achieved in vivo in human cancers remains to be seen, but the researchers found that sulindac sulfide reduces the average tumor weight in a mouse model of prostate cancer by almost 40 percent. That might not seem like much, but as Libermann emphasized, the main clinical application of NSAIDs in cancer will almost definitely be in combination with other drugs. “One thing is becoming very clear,” he said. “While everyone is busy looking for new cancer drugs, there are already many approved drugs available, both prescription and over-the-counter, that have anticancer activity.” The key is using them in the right combination, and “right now it is not always easy to decide what to combine with what.” Now that his lab has uncovered some of the anticancer secrets of NSAIDs, finding that right combination may be a lot easier.

—Tom Fagan

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