6,6'-((Methylazanedyl)bis(methylene))bis(2,4-dimethylphenol) Induces Autophagic Associated Cell Death through
Nicharat Sriratanasak1,2, Worawat Wattanathana3, Pithi Chanvorachote1,2
1Department of Pharmacology and Physiology, Faculty of Pharmaceutical Sciences, Bangkok 10330, Thailand.
Abstract:
Autophagy is the multistep mechanism for the elimination of damaged organelles and misfolded proteins. This mechanism is preceded and may induce other program cell deaths such as apoptosis. This study unraveled the potential pharmacological effect of 24MD in inducing the autophagy of lung cancer cells. Results showed that 24MD was concomitant with autophagy induction, indicating by autophagosome staining and the induction of ATG5, ATG7 and ubiquitinated protein, p62 expression after 12-h treatment. LC3-I was strongly conversed to LC3-II, and p62 was downregulated after 24-h treatment. The apoptosis-inducing activity was found after 48-h treatment as indicated by annexin V-FITC/propidium iodide staining and the activation of caspase-3. From a mechanistic perspective, 24-h treatment of 24MD at 60 μM substantially downregulated p-mTOR. Meanwhile, p-PI3K and p-Akt were also suppressed by 24MD at concentrations of 80 and 100 μM, respectively. We further confirmed m-TOR-mediated autophagic activity by comparing the effect of 24MD with rapamycin, a potent standard mTOR1 inhibitor through Western blot and immunofluorescence assays. Although 24MD could not suppress p-mTOR as much as rapamycin, the combination of rapamycin and 24MD could increase the mTOR suppressive activity and LC3 activation. Changing the substituent groups (R groups) from dimethylphenol to ethylphenol in EMD or changing methylazanedyl to cyclohexylazanedyl in 24CD could only induce apoptosis activity but not autophagic inducing activity. We identified 24MD as a novel compound targeting autophagic cell death by affecting mTOR-mediated autophagy.
Insights
The novel compound 24MD effectively induces autophagy and apoptosis in lung cancer cells by targeting the mTOR pathway. This compound shows potential as a therapeutic agent for lung cancer by promoting autophagic cell death.
Area of Science:
- Cell Biology
- Pharmacology
- Oncology
Background:
- Autophagy is a cellular degradation process crucial for eliminating damaged components.
- Dysregulation of autophagy is implicated in various diseases, including cancer.
- Understanding novel autophagy inducers is vital for developing new cancer therapies.
Purpose of the Study:
- To investigate the pharmacological effects of 24MD on lung cancer cells.
- To determine if 24MD can induce autophagy and/or apoptosis.
- To elucidate the underlying molecular mechanisms of 24MD's action.
Main Methods:
- Treatment of lung cancer cells with 24MD.
- Assessment of autophagy markers (autophagosome staining, ATG5, ATG7, LC3-I/II, p62).
- Evaluation of apoptosis markers (Annexin V-FITC/PI staining, caspase-3 activation).
- Mechanistic studies involving Western blotting and immunofluorescence to analyze signaling pathways (mTOR, PI3K, Akt).
- Comparison with rapamycin, an mTOR inhibitor.
Main Results:
- 24MD treatment led to significant autophagy induction, evidenced by increased autophagosomes and ATG proteins, and LC3-I to LC3-II conversion.
- p62 was downregulated, indicating autophagic flux.
- Apoptosis was induced after prolonged 24MD treatment, confirmed by Annexin V/PI staining and caspase-3 activation.
- 24MD suppressed mTOR signaling in a dose-dependent manner.
- Combination therapy with rapamycin enhanced 24MD's autophagy-inducing and mTOR-suppressing effects.
- Structural modifications of 24MD analogs (EMD, 24CD) resulted in apoptosis induction but not autophagy induction.
Conclusions:
- 24MD is a novel compound that effectively induces autophagy in lung cancer cells.
- The mechanism involves the suppression of the mTOR signaling pathway.
- 24MD also exhibits apoptosis-inducing activity, suggesting a dual role in cancer cell death.
- 24MD represents a promising therapeutic candidate targeting autophagic cell death in lung cancer.
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