Glioblastoma multiforme (GBM) is the most common and lethal brain tumor in adults. Full removal of the tumor by surgery is impossible, because it is highly invasive with finger-like tentacles. The poor prognosis and high recurrence rate attract tremendous attention for developing drugs to treat GBM. Currently, most chemotherapy agents aim to induce apoptosis in tumor cells. However, GBM cells harbor gene mutations that promote drug-resistance to apoptosis-inducing agents. Another challenge associated with the treatment of GBM is that the blood brain barrier (BBB) limits entry of drugs into intracranial tumors.
Methuosis is a novel non-apoptotic cell death pathway associated with rapid cellular vacuolization from macropinosomes, late endosomes and autophagosomes, followed by massive cell death. Caspase inhibitors or suppression of autophagy do not prevent methuosis. Our lab has synthesized a series of indole chalcone compounds to induce methuosis in both parental and drug-resistant human GBM U251 cells. MOMIPP (3-(5-methoxy-2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one) is the most potent one that induces cellular vacuolization at 1 h and cell death after 24 h. Our previous mechanistic studies have established a role of MOMIPP in inducing oxidative stress and perturbations of trafficking pathways. In this dissertation, I continued studying stress-related MAPK signaling pathways, aiming to elucidate the exact working mechanism of methuosis. I also performed in vivo studies with a human GBM xenograft mouse model to evaluate anti-tumor efficacy of MOMIPP. In a separate study, I examined the effects of MOPIPP, a non-lethal MOMIPP derivative, on exosome production.
1) I found that MOMIPP decreased cell viability in different types of cancer cell lines, including melanoma, lung cancer, colorectal cancer, pancreatic cancer, cervical cancer, and osteosarcoma. Methuosis is distinguishable from necroptosis, as a necroptosis inhibitor (Necrostatin-1) did not prevent cell death induced by MOMIPP. The mechanistic studies of stress-activated MAPK signaling pathways have established that activation of JNK 1/2 kinase, but not p38, is required for MOMIPP to induce cell death. JNK 1/2 substrates, c-Jun and anti-apoptotic Bcl-2 family proteins (Bcl-2 and Bcl-xL), were phosphorylated by MOMIPP. The expression of pro-apoptotic proteins, BAX and BID, was not affected during methuosis. Inhibition of c-Jun by induction of c-Jun dominant-negative expression failed to restore viability in cells treated with MOMIPP, suggesting that post-translational inactivation of Bcl-2 and Bcl-xL, via JNK 1/2-mediated phosphorylation, may take precedence over c-Jun-mediated transcriptional events, to cause mitochondrial damage and cell rupture in methuosis. My studies also showed that mitochondrial membrane potential was decreased with MOMIPP treatment, suggesting mitochondrial membrane depolarization. PIKfyve has been found to be a potential target of MOMIPP. However, comparison of the effects of MOMIPP with known PIKfyve inhibitors (Vacuolin-1 and YM201636) on U251 cells indicates that MOMIPP is much more potent in activating the JNK 1/2 signaling pathway.
2) I performed in vivo studies to evaluate the pharmacokinetic properties of MOMIPP in normal mice and assessed the anti-tumor efficacy of the compound in an orthotopic GBM xenograft model. MOMIPP was shown to successfully cross the BBB and maintain a therapeutic level in mouse brain for approximately 12 h. With MOMIPP administered by I.P. injection (q.d.) at 80 mg/kg for 5 days, the drug concentration in brain was above the expected therapeutic concentration when measured 8 h after dosing on each day. In a separate efficacy study, bioluminescence imaging of tumors engineered to express luciferase indicated that MOMIPP effectively reached the tumors and significantly inhibited tumor growth. However, when assessed by measurements of tumor cross-sectional area in histological sections, the suppression of tumor progression by MOMIPP did not reach statistical significance. Treatment with MOMIPP for 15 days was not associated with weight loss, and blood chemistry profiles did not reveal any systemic toxicity. Overall, the results suggest that MOMIPP can effectively penetrate the BBB. While efficacy studies with intracranial xenografts revealed a promising trend toward tumor growth inhibition, further studies will be needed to verify this effect through the use of new formulations that can sustain therapeutic drug levels for longer periods of time.
3) I assessed effects of non-toxic vacuole-inducing compounds, MOPIPP and Vacuolin-1, on exosome production. During our study of methuosis, we found accumulated vacuoles derived from late endosomes in cells treated with non-cytotoxic indole-based chalcones, with MOPIPP being the prime example. The intralumenal vesicles (ILVs) within multivesicular late endosomes (MVEs) are the origin of exosomes. MOPIPP only induces vacuolization without cytotoxicity, which is beneficial for dissecting the relationship between late endosome accumulation and exosome production. In vacuolated cells treated with MOPIPP or Vacuolin-1, the yield of exosomes was substantially increased. The exosomes from the treated cells exhibited morphologies similar to controls. The profiles of selected miRNA cargoes carried by the exosomes were generally unaltered in cells treated with the vacuole-inducing compounds.