GZ17-6.02 interacts with carboplatin and etoposide to kill neuroblastoma cells
Michael R Booth1, Laurence Booth1, Jane L Roberts1
1Department of Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, Virginia.
Abstract:
The biology of GZ17-6.02 alone and more so in combination with either of the standard-of-care agents etoposide or carboplatin killed MYCN overexpressing neuroblastoma (NB) cells is unknown. The methods involved in this study are in-cell immunoblotting, trypan blue exclusion, plasmid and siRNA transfection, assessment of autophagy using a plasmid expressing LC3-GFP-RFP. GZ17-6.02 (602) comprises, by mass, a ratio of curcumin (1.0), harmine (1.3), and isovanillin (7.7). In tumors dosed with 602, the ratio becomes curcumin (1.0), harmine (16), and isovanillin (6.1) (602NR). GZ17-6.02 activated ATM, AMPK, ULK1, ATG13, and PERK and inactivated ERBB1, ERBB2, ERBB3, ERBB4, AKT, mTORC1, mTORC2, SRC, NFκB, YAP, and eIF2α. 602 enhanced autophagosome formation and autophagic flux that was amplified when it was combined with etoposide or carboplatin. Compared with 602, 602NR caused significantly greater autophagosome formation that was also amplified when in combination with chemotherapy and which was reduced ~40% by knockdown of ATM or AMPKα and abolished by knockdown of Beclin1 or ATG5. Knockdown of ATM or AMPKα significantly reduced tumor cell death caused by 602 of 602NR, whereas endoplasmic reticulum stress (eIF2α) and macroautophagy (Beclin1, ATG5) were more effective at maintaining tumor cell survival. Combined knockdown of Beclin1 and the death receptor CD95 almost abolished the antitumor actions of 602 and 602NR. 602, and more so 602NR, kills MYCN NB cells and interacts with standard-of-care chemotherapeutics to cause further killing via autophagy and death receptor signaling.
Insights
GZ17-6.02 and its modified form 602NR kill MYCN neuroblastoma cells by activating autophagy and death receptor pathways. Combining these agents with chemotherapy enhances cell killing, offering new therapeutic strategies.
Area of Science:
- Oncology
- Cell Biology
- Pharmacology
Background:
- Neuroblastoma (NB) is a pediatric cancer often overexpressing the MYCN gene.
- The anti-cancer potential of GZ17-6.02, a combination of curcumin, harmine, and isovanillin, against MYCN-overexpressing NB cells is largely unexplored.
- Understanding the molecular mechanisms of GZ17-6.02, alone and with standard chemotherapeutics, is crucial for therapeutic development.
Purpose of the Study:
- To investigate the anti-cancer effects of GZ17-6.02 and a modified ratio (602NR) on MYCN-overexpressing neuroblastoma cells.
- To elucidate the cellular pathways, including autophagy and death receptor signaling, involved in the anti-tumor activity of GZ17-6.02 and 602NR.
- To evaluate the synergistic effects of GZ17-6.02/602NR in combination with etoposide or carboplatin.
Main Methods:
- In-cell immunoblotting, trypan blue exclusion assay, plasmid/siRNA transfection.
- Assessment of autophagy using LC3-GFP-RFP expressing plasmids.
- Analysis of key signaling pathways including ATM, AMPK, mTOR, ER stress, and death receptors.
Main Results:
- GZ17-6.02 and 602NR activated pro-autophagy and pro-apoptotic signaling proteins while inactivating survival pathways.
- Both agents enhanced autophagosome formation and autophagic flux, effects amplified by etoposide or carboplatin.
- 602NR demonstrated enhanced autophagosome formation compared to GZ17-6.02, and its anti-tumor effects were dependent on ATM, AMPK, Beclin1, ATG5, and CD95 signaling.
Conclusions:
- GZ17-6.02 and 602NR exhibit potent anti-neuroblastoma activity against MYCN-overexpressing cells.
- The anti-tumor effects are mediated through the induction of autophagy and activation of death receptor pathways.
- Combination therapy with GZ17-6.02/602NR and standard chemotherapeutics offers a promising strategy for neuroblastoma treatment.
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