The BH3 Mimetic Obatoclax Accumulates in Lysosomes and Causes Their Alkalinization

Abstract

Obatoclax belongs to a class of compounds known as BH3 mimetics which function as antagonists of Bcl-2 family apoptosis regulators. It has undergone extensive preclinical and clinical evaluation as a cancer therapeutic. Despite this, it is clear that obatoclax has additional pharmacological effects that contribute to its cytotoxic activity. It has been claimed that obatoclax, either alone or in combination with other molecularly targeted therapeutics, induces an autophagic form of cell death. In addition, obatoclax has been shown to inhibit lysosomal function, but the mechanism of this has not been elucidated. We have evaluated the mechanism of action of obatoclax in eight ovarian cancer cell lines. Consistent with its function as a BH3 mimetic, obatoclax induced apoptosis in three cell lines. However, in the remaining cell lines another form of cell death was evident because caspase activation and PARP cleavage were not observed. Obatoclax also failed to show synergy with carboplatin and paclitaxel, chemotherapeutic agents which we have previously shown to be synergistic with authentic Bcl-2 family antagonists. Obatoclax induced a profound accumulation of LC-3 but knockdown of Atg-5 or beclin had only minor effects on the activity of obatoclax in cell growth assays suggesting that the inhibition of lysosomal function rather than stimulation of autophagy may play a more prominent role in these cells. To evaluate how obatoclax inhibits lysosomal function, confocal microscopy studies were conducted which demonstrated that obatoclax, which contains two basic pyrrole groups, accumulates in lysosomes. Studies using pH sensitive dyes demonstrated that obatoclax induced lysosomal alkalinization. Furthermore, obatoclax was synergistic in cell growth/survival assays with bafilomycin and chloroquine, two other drugs which cause lysosomal alkalinization. These studies explain, for the first time, how obatoclax inhibits lysosomal function and suggest that lysosomal alkalinization contributes to the cytotoxic activity of obatoclax.

Obatoclax kills anaplastic thyroid cancer cells by inducing lysosome neutralization and necrosis

“…Our data, however, significantly extend these findings, providing a defined mechanistic framework for Obatoclax-induced lysosomal damage and cell necrosis. Stamelos and co-workers have recently found that Obatoclax accumulates into lysosomes of ovarian cancer cells, leading to their alkalization [50], providing an independent corroboration of our data. However, in contrast to our findings, apoptosis appeared to be the primary cause of cell death in several of the cell lines analyzed, and in general cell death occurred late, after 48 to 72 hours from Obatoclax treatment, while we show evidence of necrosis in thyroid cancer cells as early as six hours post-treatment.…”

Obatoclax Inhibits Alphavirus Membrane Fusion by Neutralizing the Acidic Environment of Endocytic Compartments

“…Indeed, three recent reports have provided independent corroboration of our findings and showed that OLX not only increases the lysosomal pH but also accumulates in the lysosomes and causes a loss of lysosomal function (37)(38)(39). The fact that OLX is a weak base and the likelihood that it is protonated and accumulates in lysosomes, similar to another lysomotrophic agent, chloroquine, suggest a possible mechanism for the rapid elevation in the lysosomal pH (38). Additionally, Champa et al have suggested that OLX could mediate the exchange of Cl Ϫ and HCO 3 Ϫ ions between the cytoplasm and the lysosome, which would also cause a rapid elevation in the lysosomal pH (37).…”

Preparation of Cyclic Prodiginines by Mutasynthesis in Pseudomonas putida KT2440

“…Modulation of autophagy is evaluated as a promising therapeutic approach for many diseases such as neurodegenerative disorders, metabolic disease, or cancer . Modulation of autophagy has been described for prodigiosin ( 2 ), for prodidiosin derivatives with different chain lengths, and for obatoclax mesylate (GX15‐070), a synthetic derivative investigated in several phase II clinical trials . The key cellular structures of autophagy are autophagosomes, double‐layer membrane vesicles that receive input from the endocytic pathway.…”