A G-quadruplex ligand 3,3'-diethyloxadicarbocyanine iodide induces mitochondrion-mediated apoptosis but not decrease
Chung-Pin Li1, Jen-Hsin Huang, Ai-Chi Chang
1Cooperative Laboratory, Cancer Research Division, National Health Research Institutes, Taipei, Taiwan, ROC.
Purpose:
The G-quadruplex ligand 3,3'-diethyloxadicarbocyanine iodide (DODC) was reported to enhance the apoptotic potency of pheochromocytoma PC-12 and leukemia HL-60 cells through the inhibition of telomerase activity. In this study, a mitochondrion-mediated apoptotic pathway was demonstrated as another cytotoxic mechanism for DODC action.
Methods:
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and DNA laddering assays were performed to exhibit the cytotoxicity and apoptosis-inducing activity of DODC. Telomeric repeat amplification protocol (TRAP) assay was used to evaluate the effect of DODC on cellular telomerase. The mitochondrial uptake of probe 3,3'-dihexyloxacarbocyanine iodide was measured by flow cytometry. The mitochondrial proteomes were analyzed by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Western blot analyses were adopted to demonstrate the change of the distribution of mitochondrial proteins.
Results:
DODC alone was able to induce apoptotic cell death but not decrease of telomerase activity in nasopharyngeal carcinoma NPC-TW01 cells. Instead, we found evidence that DODC significantly affected cellular mitochondria. DODC inhibited the uptake of another mitochondrial probe 3,3'-dihexyloxacarbocyanine iodide. By proteomic comparative analysis, we found that DODC induced the increase of prohibitin level in the mitochondria, indicating the occurrence of mitochondrial perturbation. Moreover, DODC was found to induce the levels of p53 and an 18-kDa truncated Bax on mitochondria, which in turn potentiated the release of cytochrome c for activation of caspases.
Conclusions:
DODC induces NPC-TW01 cell apoptosis via a mitochondrion-mediated mechanism. This paper demonstrates another cytotoxic mechanism of DODC other than inhibition of telomerase.
Insights
The G-quadruplex ligand DODC induces apoptosis in NPC-TW01 cells by targeting mitochondria, not telomerase. This study reveals a novel mitochondrion-mediated cytotoxic pathway for DODC action.
Area of Science:
- Cell Biology
- Molecular Biology
- Cancer Research
Background:
- The G-quadruplex ligand 3,3'-diethyloxadicarbocyanine iodide (DODC) was previously shown to induce apoptosis in cancer cells by inhibiting telomerase.
- However, alternative cytotoxic mechanisms of DODC action require further investigation.
Purpose of the Study:
- To investigate the cytotoxic mechanisms of DODC in nasopharyngeal carcinoma NPC-TW01 cells.
- To determine if DODC induces apoptosis through a mitochondrion-mediated pathway.
Main Methods:
- Cytotoxicity and apoptosis were assessed using MTT assays and DNA laddering.
- Telomerase activity was evaluated using the Telomeric Repeat Amplification Protocol (TRAP) assay.
- Mitochondrial function and proteome changes were analyzed via flow cytometry, 2D gel electrophoresis, MALDI-TOF MS, and Western blotting.
Main Results:
- DODC induced apoptosis in NPC-TW01 cells without decreasing telomerase activity.
- DODC inhibited mitochondrial probe uptake and increased prohibitin levels, indicating mitochondrial perturbation.
- DODC upregulated p53 and truncated Bax in mitochondria, leading to cytochrome c release and caspase activation.
Conclusions:
- DODC induces apoptosis in NPC-TW01 cells through a mitochondrion-mediated pathway.
- This study identifies a novel cytotoxic mechanism for DODC involving mitochondrial dysfunction, distinct from telomerase inhibition.
