In this study, we identified the cardiac glycoside lanatoside C as a potent sensitizer of GBM cells to TRAIL-induced apoptosis. Furthermore, this drug on its own showed a significant anti-glioma effect in culture as well as tumor xenograft in mice by activating an alternative cell-death pathway. To our knowledge, this is one of the first examples of use of caspase-independent cell-death inducers to trigger tumor regression in vivo.
Recently, several studies suggested cardiac glycosides as potential therapies for different types of cancer,22
although few have made it to the clinic because of their therapeutic index and/or their low antitumor activity, as well as their cardiotoxicity.37
When combined with a low dose of TRAIL, a low dose of lanatoside C provides a high anti-glioblastoma activity, both in cultured cells and in GBM xenografts in vivo. These results could present one leap toward testing of this drug combination for glioma therapy because TRAIL is currently being tested for the treatment of different tumors in humans. Also, cardiac glycosides have been approved by the US Food and Drug Administration and have been used for decades for the treatment of congestive heart failure.
A previous study showed a synergistic effect of cardiac glycosides with TRAIL in culture caused by upregulation of the death receptors (DR) on non–small cell lung cancer cells while reporting no toxicity of cardiac glycoside treatment without TRAIL.23
Another recent study of malignant epithelial cells had suggested that cardiac glycosides act as anoikis sensitizers while causing no cytotoxicity on adherent cells.38
Although it seems that the activity of cardiac glycosides is cell-type specific, their mechanism of action on cancer cells is not fully elucidated, and several possible mechanism have been suggested.22
A chemical genetic screen for small molecules with neuroprotective effect in ischemic stroke revealed 4 cardiac glycosides (neriifolin, ouabain, digitoxin, and digoxin).39
Neriifolin, being the most potent of the 4 hits, demonstrated a neuroprotective effect in hypoxia/ischemia animal models as well. This provides additional evidence of the use of cardiac glycosides in brain treatment and the lack of toxicity of these drugs on normal brain cells. Another major advantage of cardiac glycosides in GBM therapy may be their ability to cross the blood-brain barrier.40
Therefore, we decided to investigate the mechanism of action of lanatoside C and inspect a possible cross-talk between this cardiac glycoside and the TRAIL pathway. We observed that treatment of GBM cells with lanatoside C upregulated the mRNA expression of several genes that are members of the TNF superfamily. We also observed an upregulation of DR5 protein expression on the cell surface after lanatoside C treatment, suggesting that the sensitization of glioblastoma cells to TRAIL might be due to the cardiac glycoside-induced expression of this death receptor on the cell surface. A drug screen for inhibitors of polyglutamine-dependent activation of caspase-3 on human embryonic kidney cells (HEK293) by Piccioni et al41
identified 6 cardiac glycosides as validated drug hits. In the absence of caspase activation, a necrotic cell death mechanism induced through the DR can supervene.42
On the other hand, ATP depletion can trigger autophagy,34
and more interestingly, it can favor necrosis over apoptosis,43,44
partly because of the role of ATP in apoptosome formation and the activation of caspases.45,46
One possible explanation for lanatoside C–induced cell death is that this drug might indirectly inhibit caspase activity through ATP depletion while activating the death receptors, thereby inducing programmed necrosis.
A major remaining challenge is to translate the combined therapeutic strategy for glioma therapy, because of the inefficiency of TRAIL in crossing the blood-brain barrier.47
On the other hand, TRAIL has been infused locally into the brain by convection-enhanced delivery, which showed wide distribution in the brain without cytoxicity to normal cells and no clinical symptoms by hepatotoxicity evaluation, with the exception of modest inflammation immediately adjacent to the needle tract.48
TRAIL has also been delivered to brain tumors with adeno-associated viral vectors, as well as neural precursor cells expressing secreted TRAIL; both resulted in glioma regression in different models.49
These delivery strategies could potentially be tested in combination with systemic injection of lanatoside C.
Evading apoptosis is a hallmark of cancer.50
The apoptosis resistance mechanisms are made possible through a myriad of genetic and epigenetic processes. One example of these apoptosis-resistance mechanisms is the enhanced expression of Bcl-2 anti-apoptotic proteins.51
Here, we showed that lanatoside C can overcome this resistance by activating an alternative cell-death mechanism. Chemotherapeutic agents that induce nonapoptotic cell-death pathways might be of great benefit and could outwit tumor resistance. Moreover, combining drugs that induce different cell-death mechanisms, as seems to occur with lanatoside C (caspase-independent cell death) and TRAIL (apoptotic cell death) on GBM cells, may help achieve a maximal therapeutic benefit. A deeper understanding of programmed necrosis pathways and identification of markers for this caspase-independent mechanism are still needed. Use of drugs such as lanatoside C combined with known therapeutics, such as TRAIL, may be a useful strategy to counter resistance of cancer cells to apoptosis and provide new opportunities for cancer treatment in general and for GBM in particular.