Molecular Mechanisms of Paraptosis Induction: Implications for a Non-Genetically Modified Tumor Vaccine

Abstract

Paraptosis is the programmed cell death pathway that leads to cellular necrosis. Previously, rodent and human monocytes/macrophages killed glioma cells bearing the membrane macrophage colony stimulating factor (mM-CSF) through paraptosis, but the molecular mechanism of this killing process was never identified. We have demonstrated that paraptosis of rat T9 glioma cells can be initiated through a large potassium channel (BK)-dependent process initiated by reactive oxygen species. Macrophage mediated cytotoxicity upon the mM-CSF expressing T9-C2 cells was not prevented by the addition of the caspase inhibitor, zVAD-fmk. By a combination of fluorescent confocal and electron microscopy, flow cytometry, electrophysiology, pharmacology, and genetic knock-down approaches, we demonstrated that these ion channels control cellular swelling and vacuolization of rat T9 glioma cells. Cell lysis is preceded by a depletion of intracellular ATP. Six-hour exposure to BK channel activation caused T9 cells to over express heat shock proteins (Hsp 60, 70, 90 and gp96). This same treatment forced HMGB1 translocation from the nuclear region to the periphery. These last molecules are “danger signals” that can stimulate immune responses. Similar inductions of mitochondrial swelling and increased Hsp70 and 90 expressions by BK channel activation were observed with the non-immunogenic F98 glioma cells. Rats injected with T9 cells which were killed by prolonged BK channel activation developed immunity against the T9 cells, while the injection of x-irradiated apoptotic T9 cells failed to produce the vaccinating effect. These results are the first to show that glioma cellular death induced by prolonged BK channel activation improves tumor immunogenicity; this treatment reproduces the vaccinating effects of mM-CSF transduced cells. Elucidation of strategies as described in this study may prove quite valuable in the development of clinical immunotherapy against cancer.

Histomorphological changes in the cornea of the rat following monocular eyelid closure

“…[14] It is characterized by cytoplasmic reorganization, enlargement of the endoplasmic reticulum and mitochondria, swelling of the cell and formation of extensive cytoplasmic vacuoles. [15] In the eye, paraptosis has been portrayed in retinal ganglion cells following glaucoma induced retinal ischemia/reperfusion injury, a major cause of vision loss in the human. [16,17] In addition, paraptosis in the corneal epithelium may occur in pathological conditions such as corneal dystrophies.…”

Glioma Big Potassium Channel Expression in Human Cancers and Possible T Cell Epitopes for Their Immunotherapy

“…Earlier, we showed that prolonged activation of BK channels of T9 or U251 gliomas in vitro causes cell swelling and eventually leads to paraptosis (7,8). When paraptotic BK channel-activated/killed T9 cells were used as the immunogen, T9-specific tumor immunity toward the T9 glioma cells was generated within naive rats (8). BK channel activators were created and successfully prevented cardiac reperfusion injury in animals (37,38).…”

“…Both chloride channel-1 and aquaporins are also overexpressed in glioma (41)(42)(43). BK channel activation leads to cell swelling, whereas CLIC1 activation leads to cell shrinkage (7,8,44). Both of these functions, swelling and shrinking, are needed for cell volume regulation and motility.…”

“…This programmed cell death pathway leads to necrosis (5,6) and is characterized by swollen mitochondria and endoplasmic reticulum (ER). Human U251 (7) and rat T9 gliomas (8) immediately swelled upon activation of the big potassium (BK) channels using a BK ionophore; paraptosis ultimately occurred within 18-24 h. Functional membrane BK channels were detected on the human and rat glioma cells by using patch-clamping techniques. Additionally, BK channels were found on the ER and the mitochondria (7,8), providing a plausible rationale for why these organelles are specifically targeted in paraptosis.…”

“…Human U251 (7) and rat T9 gliomas (8) immediately swelled upon activation of the big potassium (BK) channels using a BK ionophore; paraptosis ultimately occurred within 18-24 h. Functional membrane BK channels were detected on the human and rat glioma cells by using patch-clamping techniques. Additionally, BK channels were found on the ER and the mitochondria (7,8), providing a plausible rationale for why these organelles are specifically targeted in paraptosis. These ion channels are also called Maxi-K, hSlo, mSlo, KCNMA1, calcium-dependent, large conductance-, or voltage-activated channels (9)(10)(11)(12)(13)(14)(15)(16)(17).…”