Purdue scientists hunt for 'secret X' to treat liver cancer

December 13, 2004

WEST LAFAYETTE, Ind. - Identifying the link between chronic hepatitis B infection and liver cancer may one day help cancer patients sidestep the poison of chemotherapy, a Purdue University study suggests.

The research group of Ourania M. Andrisani (oo-RAHN-ee-ah an-dri-SAH-nee) has shown that a protein the hepatitis virus instructs chronically infected liver cells to produce - known as the X protein - under certain conditions instructs precancerous infected liver cells to die. The discovery of how the X protein influences liver cell behavior could be harnessed as an anticancer therapy, turning the X protein's presence in the liver to patients' advantage.

"Instead of chemotherapy, drugs that influence the X protein's behavior might become an alternative cancer therapy," said Andrisani, a professor of basic medical sciences in Purdue's School of Veterinary Medicine. "Rather than give a patient chemicals that damage every cell in the body, therapy based on the X protein could potentially target only cancerous cells, slowing their growth."

The research, which Andrisani conducted with first author Wen Horng Wang, and Gérald Gregori and Ronald L. Hullinger, all of Purdue, appears in the current issue of Molecular and Cellular Biology.

Viruses like the hepatitis B virus incorporate their genes into a healthy cell's genetic material. This way, many viruses not only instruct the cell to make proteins necessary to assemble more virus particles, but they also change or deregulate the normal production of proteins by the healthy cell. One of the genes hepatitis B introduces into liver cells is called the X gene, a short sequence of DNA that "expresses," or creates, the X protein - a building block essential for creating hepatitis B. Scientists have suspected the gene and its corresponding protein to be accomplices in cancer development as well.

"The X protein is present in the livers of all chronic hepatitis B patients, and several past studies have implicated it in liver cancer development," Andrisani said. "We decided to look at the effect of the X protein on liver cells in isolation to find out what it was doing to the cells' life cycle."

As a molecular biologist, Andrisani studies how cells receive chemical messages from the body that instruct them to grow, differentiate and die at proper times - actions that are necessary for health in highly specialized bodies like our own. Andrisani's team theorized that the X protein was the chemical messenger that caused the liver cells to behave erratically, putting them out of step with healthy liver tissue.

"To find out what instructions the X protein was feeding the cells, we worked with samples of mouse liver tissue in the lab," she said. "We first took the X gene from hepatitis B and inserted it into liver cell nuclei. Then, after the gene started producing the X protein in the cells, we watched the behavior of these mouse liver samples to see whether they conformed to healthy liver cell life cycles."

To ease observation, they used samples that were only a single layer of cells thick. Observing these modified cells, the team found that the effects of the X gene were a bit complicated.

"The gene does different things to cells at different stages of their lives," Andrisani said. "We found, for example, that liver cells in the beginning of their life cycle will grow vigorously when the X protein is around, but under conditions of stress, it encourages them to die. We have other data, as yet unpublished, that shows the X protein can essentially 'rescue' cells from dying at the appropriate time. Of course, if you see cells growing uncontrollably and refusing to die, then you are looking at the mechanisms of cancer development."

Andrisani likens the X gene to a driver going downhill who doesn't use the car's brakes, allowing the vehicle to accelerate to breakneck speed. But before cells with the X gene can begin proliferating at such an unhealthy rate, it could be possible to counter this behavior with drugs. Andrisani said that it is currently within medicine's power to devise treatments that can eliminate precancerous X-expressing cells, based on their increased tendency to die. Researchers at other institutions also are optimistic about this aspect of the work's implications.

"In fact, some drugs already exist that could help slow the effects of the X protein within the livers of cancer patients," said Spiro Hiotis, a surgeon at New York University who is not affiliated with the study. "While a great deal of further research still needs to be done, Andrisani's team has shown that work with the X gene could someday lead to liver cancer treatments, and managing the gene's effects with drugs could be a viable approach."

This approach could mean that liver cancer could be treated at least in part without chemotherapy, Andrisani said, making for far less overall stress on a patient's body.

"This is what we in the business would call a mechanism-based treatment," she said. "Rather than kill all rapidly-dividing cells in the body - the healthy ones along with the cancerous ones - we would like to target the cancer cells alone. We are taking the next steps toward that goal now."

That next step involves work with live animals instead of tissue samples, she said, and work is now in progress in the labs of Marie-Annick Buendia and Pierre Tiollais of the Louis Pasteur Institute in France.

"In our lab, we are continuing with liver tissue culturing as well," Andrisani added. "We are now trying to work with 3-D cultures of many cell layers, which more closely resemble a normal liver. We hope that with a few years' effort, our work will pay off in the clinic."
-end-
Funding for this work was provided in part by the National Institutes of Health.

Andrisani is associated with the Purdue Cancer Center. One of just eight National Cancer Institute-designated basic-research facilities in the United States, the center attempts to help cancer patients by identifying new molecular targets and designing future agents and drugs for effectively detecting and treating cancer.

Writer: Chad Boutin, 765-494-2081, cboutin@purdue.edu

Sources: Ourania M. Andrisani, 765-494-8131, andrisao@purdue.edu
Spiro Hiotis, hiotis01@popmail.med.nyu.edu
Marie-Annick Buendia, mbuendia@pasteur.fr

Related Web sites: Purdue Cancer Center: http://www.cancer.purdue.edu

ABSTRACT

Combined and sustained activation of p38 and JNK pathways by Hepatitis B Virus X protein effects apoptosis via induction of Fas/FasL expression

Wen Horng Wang, Gérald Gregori, Ronald L. Hullinger and Ourania M. Andrisani

Activation of the cellular stress pathways (JNK, p38) by environmental stresses is linked to apoptosis. However, the role of their combined activation in mediating apoptosis remains unresolved. The Hepatitis B virus X protein activates both p38 and JNK pathways, and in response to weak apoptotic signals, sensitizes hepatocytes to apoptosis. Employing tetracyclineregulated, pX-expressing hepatocyte cell lines, we investigated the mechanism of the combined p38 and JNK pathway activation in apoptosis. Specific inhibitors for p38MAPK and JNK demonstrate that initiation of pX-mediated apoptosis is rescued by 80 percent via inhibition of the p38 pathway, whereas subsequent apoptotic events involve both pathways. The combined and sustained activation by pX of p38 and JNK pathways induces transcription of death-receptor family genes, Fas/FasL, TNFR1/ TNF-£, and p53-regulated genes Bax and Noxa. The pXdependent expression of Fas/FasL and TNFR1/ TNF-£ mediates caspase 8 activation, resulting in Bid cleavage. In turn, activated Bid, acting with the pX- induced Bax and Noxa, mediates mitochondrial release of cytochrome c, resulting in caspase 9 activation and apoptosis. Importantly, antibody neutralization of FasL reduces initiation of pX-mediated apoptosis. These results support the importance of the combined activation of p38 and JNK pathways in pXmediated apoptosis, and suggest this mechanism of apoptosis occurs in vivo, in response to weak apoptotic signals.

Purdue University

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