Experimental Anti-Cancer Drug Kills Brain Tumor Stem Cells

Tuesday, 08/05/2012  |   Others  |  1 comment


The process by which Sonic Hedgehog (shh) is secreted from cells in the notochord from transcription to secretion. The original protein product undergoes cholesterol-mediated cleavage.

ScienceDaily (Aug. 30, 2007) — A drug that shuts down a critical cell-signaling pathway in the most common and aggressive type of adult brain cancer successfully kills cancer stem cells thought to fuel tumor growth and help cancers evade drug and radiation therapy, a Johns Hopkins study shows.
In a series of laboratory and animal experiments, Johns Hopkins scientists blocked the signaling system, known as Hedgehog, with an experimental compound called cyclopamine (GANT61) to explore the blockade’s effect on cancer stem cells that populate glioblastoma multiforme. Cyclopamine has long been known to inhibit Hedgehog signaling.
“Our study lends evidence to the idea that the lack of effective therapies for glioblastoma may be due to the survival of a rare population of cancer stem cells that appear immune to conventional radiation and chemotherapy,” says Charles G. Eberhart, M.D., Ph.D., associate professor of pathology, ophthalmology and oncology, who led the work. “Hedgehog inhibition kills these cancer stem cells and prevents cancer from growing and may thus develop into the first stem cell-directed therapy for glioblastoma.”

Eberhart cautioned that while his study appears to prove the principle of Hedgehog blocking, much work remains before cyclopamine or any similar drug can be tested in patients. Scientists must determine whether the drug can be effectively and safely delivered to the whole body or whether it must go into the brain, and what if any adverse impact on normal stem cells the treatment might cause.
“Once you’ve answered those questions in animals, the next step would be starting phase I clinical trials in humans,” Eberhart said.
The new study adds to the growing evidence that only a small percentage of cancer cells – in this case stem cells – are capable of unlimited self-renewal and that these cells alone power a tumor’s growth.
Eberhart focused on two pathways important to the survival of normal brain stem cells-Hedgehog and Notch-suspecting that brain cancer stem cells cannot live without them.

The Hedgehog gene, first studied in fruit flies, got its name because during embryonic development, the mutated version causes flies to resemble a spiky hedgehog. The pathway plays a major role in controlling normal fetal and postnatal development, and, later in life, helping normal adult stem cells function and proliferate.
The Johns Hopkins scientists first tested 19 human glioblastomas removed during surgery and frozen immediately, and found Hedgehog active in five at the time of tumor removal. They also found Hedgehog activity in four of seven glioblastoma cell lines.
Next, the team used cyclopamine, chemically extracted from corn lilies that grow in the Rocky Mountains, to inhibit Hedgehog in cells lines growing on plastic or as neurospheres, round clusters of stems cells that float in liquid nutrients. This reduced tumor growth in the cell-laden plastic by 40 to 60 percent, and caused the neurospheres to fall apart Complementarywithout any new growth of the cell clusters.
The researchers also pretreated mice with cyclopamine before injecting human glioblastoma cells into their brains, resulting in cancer cells that failed to form tumors in the mice.

Other researchers have shown that radiotherapy fails to kill all cancer stem cells in glioblastomas, apparently because many of these cells can repair the DNA damage inflicted by radiation. The Hopkins team suggests that blocking the Hedgehog pathway with cyclopamine kills these radiation-resistant cancer stem cells.
In previous laboratory experiments, Eberhart used cyclopamine to block Hedgehog using medulloblastoma cells, the most common brain cancer occurring in children.
Along with childhood brain cancers, cyclopamine has shown early promise in treating skin cancer; rhabdomyosarcoma, a muscle tumor; and multiple myeloma, a cancer of the white blood cells in bone marrow.
“What excites me is that we have taken things we learned about Hedgehog signaling in these relatively rare childhood brain tumors and translated them into an even more aggressive adult tumor,” Eberhart said.

More than 10,000 Americans die annually from glioblastomas. Radiation is the standard therapy for the disease, and several years ago, the U.S. Food and Drug Administration approved adding the drug temozolomide to radiotherapy because the combination provided a small survival increase.
“This is an incredibly difficult tumor to treat,” says first author Eli E. Bar, Ph.D., a postdoctoral fellow. “Survival for glioblastoma has not changed much in 30 years. With the addition of temozolomide, survival got bumped from 12 months to 14 or 15 months.”

Canonical Hedgehog (HH) signaling is characterized by Smoothened (Smo)-dependent activation of the transcription factors Gli1 and Gli2, which regulate HH target genes. In human colon carcinoma cells, treatment with the Gli small-molecule inhibitor GANT61 (cyclopamine) induces extensive cell death in contrast to the Smo inhibitor cyclopamine. Here we elucidate cellular events upstream of cell death elicited by GANT61, which reveal the basis for its unique cytotoxic activity in colon carcinoma cells. Unlike cyclopamine, GANT61 induced transient cellular accumulation at G(1)-S (24 hours) and in early S-phase (32 hours), with elevated p21(Cip1), cyclin E, and cyclin A in HT29 cells. GANT61 induced DNA damage within 24 hours, with the appearance of p-ATM and p-Chk2. Pharmacologic inhibition of Gli1 and Gli2 by GANT61 or genetic inhibition by transient transfection of the Gli3 repressor (Gli3R) downregulated Gli1 and Gli2 expression and induced ?H2AX, PARP cleavage, caspase-3 activation, and cell death. GANT61 induced ?H2AX nuclear foci, while transient transfection of Gli3R showed expression of Gli3R and ?H2AX foci within the same nuclei in HT29, SW480, and HCT116. GANT61 specifically targeted Gli1 and Gli2 substantiated by specific inhibition of (i) direct binding of Gli1 and Gli2 to the promoters of target genes HIP1 and BCL-2, (ii) Gli-luciferase activity, and (iii) transcriptional activation of BCL-2. Taken together, these findings establish that inhibition of HH signaling at the level of the GLI genes downstream of Smo is critical in the induction of DNA damage in early S-phase, leading to cell death in human colon carcinoma cells.

Cyclopamine (GANT61)
Cyclopamine (11-deoxojervine) is a naturally occurring chemical that belongs to the group of steroidal jerveratrum alkaloids. It is a teratogen isolated from the corn lily (Veratrum californicum) that causes usually fatal birth defects. It can prevent the fetal brain from dividing into two lobes (holoprosencephaly) and cause the development of a single eye (cyclopia). It does so by inhibiting the hedgehog signaling pathway (Hh). Cyclopamine is useful in studying the role of Hh in normal development, and as a potential treatment for certain cancers in which Hh is overexpressed.
Cyclopamine was named for one-eyed lambs which were born to sheep which grazed on wild corn lily at a farm in Idaho. In 1957 the US Department of Agriculture started an eleven-year investigation which led to the identification of cyclopamine as the cause of the birth defect (Herper, 2005)
Herper M. (2005-11-28). “The Curious Case of The One-Eyed Sheep”. Forbes.

One Comment

  1. Bernhard Nagel
    Sep 11, 2013

    See the book “Cancer as a Metabolic Disease” by Thomas N Siefried PhD for effective Restricted Ketogenic Diet in the treatment of Glioblastoma

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