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Lysosomal Stress and Mitochondrial Dysfunction Define the Cellular Response to Photoactivated 1,9-Dimethyl-Methylene Blue

ABSTRACT: Photodynamic therapy (PDT) using 1,9-dimethyl methylene blue (DMMB) induces coordinated mitochondrial and lysosomal damage and results in strong cellular death induction. However, the underlying transcriptional regulation in response to DMMB remains elusive. We compared the transcriptome response of photoactivated DMMB to the gene signature triggered by autophagy-modulating agents: rapamycin (an autophagy activator) and bafilomycin A1 (a lysosomal acidification inhibitor). Transcriptome analysis revealed a pronounced transcriptomic response to photoactivated DMMB, with 884 differentially expressed genes (DEGs), compared to 291 for bafilomycin and 154 for rapamycin. DMMB treatment upregulated genes associated with autophagy, mitochondrial stress responses, proteostasis, and inflammation, while downregulating genes involved in differentiation, miRNA processing, and lipid catabolism. Rapamycin treatment downregulated amino acid metabolism and biosynthesis pathways, while upregulated processes associated with triglyceride metabolism and nutrient starvation. Conversely, bafilomycin upregulated genes related to lipid metabolism, cholesterol biosynthesis, and inflammatory signaling, while suppressing developmental and cytoskeletal programs. Interestingly, transcriptomic comparisons revealed a striking overlap between DMMB and bafilomycin signatures (95% concordant DEGs), suggesting a common transcriptome regulation. Among the several biological processes affected by DMMB, mitochondrial-related processes were strongly enriched. To determine whether the acute transcriptome changes caused by DMMB led to persistent functional effects, we stimulated cells with DMMB and assessed mitochondrial respiration after a recovery period. Photoactivated DMMB reduced basal respiration, ATP production, proton leak, and maximal respiration, indicating sustained mitochondrial dysfunction. These effects were not further altered by bafilomycin co-treatment but were markedly exacerbated by rapamycin. Collectively, we show that photoactivated DMMB leads to an extensive transcriptome rewiring, closely resembling autophagy inhibition with a sustained mitochondrial dysfunction. Our findings provide a valuable resource to understand the interplay between DMMB-induced lysosomal stress, transcriptional regulation, and PDT.

ORGANISM(S): Homo sapiens

PROVIDER: GSE305949 | GEO | 2026/08/17

REPOSITORIES: GEO

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Project description:Although malignant gliomas frequently show aberrant activation of the mammalian target of rapamycin (mTOR), mTOR inhibitors have performed poorly in clinical trials. Besides regulating cell growth and translation, mTOR controls the initiation of autophagy. By recycling cellular components, autophagy can mobilize energy resources, and has thus been attributed cancer-promoting effects. Here, we asked whether the activation of autophagy represents an escape mechanism to pharmacological mTOR inhibition, and explored co-treatment with mTOR and autophagy inhibitors as a therapeutic strategy. Mimicking conditions of the glioma microenvironment, glioma cells were exposed to nutrient starvation and hypoxia. Following treatment with mTOR inhibitor torin2 or rapamycin, and autophagy inhibitors bafilomycin A1 or MRT68921, we analyzed autophagic activity, cell growth, viability and oxygen consumption. Changes in global proteome were quantified by mass spectrometry. In the context of hypoxia and starvation, autophagy was strongly induced in glioma cells, and further increased by mTOR inhibition. While torin2 enhanced glioma cell survival, co-treatment with torin2 and bafilomycin A1 failed to promote cell death. Importantly, treatment with bafilomycin A1 alone also protected glioma cells from cell death. Mechanistically, both compounds significantly reduced glioma growth and oxygen consumption. Mass spectrometry analysis showed that bafilomycin A1 induced broad changes in the cellular proteome. More specifically, proteins downregulated by bafilomycin A1 were associated with the mitochondrial respiratory chain and ATP synthesis. Taken together, our results show that activation of autophagy does not account for the cytoprotective effects of mTOR inhibition in our in vitro model of the glioma microenvironment. Our proteomic findings suggest that the pharmacological inhibition of autophagy induces extensive changes in the cellular proteome that can support glioma cell survival under nutrient-deplete and hypoxic conditions. These findings provide a novel perspective on the complex role of autophagy in gliomas and may have implications for the design of future trials.

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Lysosomal Stress and Mitochondrial Dysfunction Define the Cellular Response to Photoactivated 1,9-Dimethyl-Methylene Blue

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