Abstracts Division 3

70. The senescence-associated secretory phenotype (SASP) from colorectal cancer cells induce neurotoxicity: potential involvement of CD38-NAD+ pathways

Wu W.B.1,2, Hageman G.J. 1,2, van Schooten F.J.1,2

1
Department of Pharmacology and Toxicology, Maastricht University, Maastricht, The Netherlands
2 School of Nutrition and Translational Research in Metabolism, Faculty of Health, Medicine and Life Sciences, Maastricht University, Maastricht, The Netherlands

Background
About 70% of colorectal cancer survivors who undergo chemotherapy often have com-plaints, such as fatigue, depression and cognitive decline. Even though the underlying mechanisms remain elusive, these complaints are highly related to neurotoxicity and disruption of the normal neuronal functions. Anti-neoplastic therapy can induce (cancer) cell senescence, with concomitant senescence-associated secretory phenotype (SASP) known to secrete several inflammatory cytokines, proteases and chemokines. These cytokines can by-pass the blood-brain-barrier and induce inflammation by activating CD38. CD38 uses NAD+ as its substrate and has been identified as the major regulator of cellular NAD+ levels. NAD+ decline was also reported to be related to ageing and neurodegenerative diseases. This study investigated if the mentioned pathway can lead to neurotoxicity, which may provide a biological explanation towards neuropathy and fatigue syndrome observed in cancer survivors. 

Methods
Colorectal cancer lines HCT116 and HT29 were exposed with three senescence inductors hydrogen peroxide, 5-fluorouracil and doxorubicin for 24h and continued culturing with growth media for 7 days. The cellular senescent phenotype was validated by increased beta-galactosidase activity, and significantly increased mRNA levels of p16, p21, IL-6, TNF-a, IL-1b and VEGF. Conditioned medium (CM) from the senescent HT29 and HCT116 cells was used to treat human micro-glial cells (HMC3 cell line) and neuronal cells (SHY5Y cell line) in a mono and a co-culture. Neuronal cells were also exposed to CM from microglial cells (CM-HMC3) treated for 2 h and 24h with CM with/ without CD38 inhibitor 78c. ATP production, mRNA levels on kynurenine pathway, inflammation markers, NAD+ status, and mitochondrial stress test by seahorse assay were preformed after exposure with CM in both differentiation and undifferentiating SHY5Y cells.    

Results
Significantly higher IL-6, TNF-a, IL-1b, TGF-a mRNA levels and increased CD38 activity was found in microglial cells treated with CM for 24h. Exposure of the human neuronal cells to CM-HMC3 for 2h and 24h, respectively, significantly decreased cell via-bility, NAD+ and ATP production in both differentiation and undifferentiating SHY5Y cells. In a co-culture system, when exposed with CM from colorectal cancer lines for 24h, a significantly decreased cell viability and ATP production was found in SHY5Y cells; When co-exposed to CM with 50 nM CD38 inhibitor 78c for 24h, the cell viability and ATP production was restored in SHY5Y cells. 

Conclusion
Our data suggest that SASP released by senescent HT29 and HT116 cells can induce decreased energy production and disruption of cellular NAD+ homeostasis in SHY5Y cells mediated by increased CD38 activity.

Keywords
cancer chemotherapy, neuropathy, senescence, NAD+, CD38

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