A novel ProTide, NUC-7738, induces AMPK activation and cell death in both cell lines and ex vivo tissues of human renal cell carcinoma


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Mary Kudsy1,Mustafa Elshani1,Sarah Puthur1,Wen Chen1,In Hwa Um1,Grant D. Stewart2,Maeve Rahilly3,Alex Chapman3,Michelle Myers4,David J. Harrison1
1University of St Andrews,2University of Cambridge,3Victoria Hospital, NHS Fife,4Nucana

Abstract

Background

Cordycepin or 3'-deoxyadenosine inhibits the growth of cancer cells by several possible mechanisms. NUC-7738, the ProTide transformation of cordycepin, is designed to overcome cancer resistance mechanisms through direct release of 3’-deoxyadenosine monophosphate in cells. We hypothesised that it might activate AMP-activated protein kinase (AMPK), and thus disrupt metabolic homeostasis in clear cell renal cell carcinoma (ccRCC) which is considered as a metabolic disease.

Method

The expression of pAMPK and AMPK in ccRCC from 293 patients, and the expression of adenosine monophosphate deaminase 2 (AMPD2) and pAMPK in 10 ccRCC whole tissue sections were analysed by immunofluorescence, images were captured digitally and were analysed using QuPath software. The effect of NUC-7738 on the growth of nine renal cancer cell lines was assessed using SRB assay, under both 0.5% oxygen and normoxic conditions. Western blotting was used to assess changes in pAMPK caused by NUC-7738 and results read using LiCor Odyssey. Effect of NUC-7738 on AMPK activation and cleaved caspase-3 levels in ex vivo ccRCC tissue slices was analysed using immunofluorescence and QuPath software.

Results

Whereas AMPK was widely expressed in ccRCC, pAMPK was focal and very heterogeneous. Cell lines by contrast strongly expressed pAMPK, but this was reduced when culture conditions were altered to be more physiologically appropriate through reduction of oxygen tension and lowering glucose levels. An inverse relationship between AMPD2 and pAMPK levels was observed in ccRCC whole tissue sections. NUC-7738 inhibited the growth of renal cancer cell lines under both hypoxic and normoxic conditions, and increased pAMPK levels were noted after 1 hour, 6 hours, 24 hours, and 48 hours treatment. NUC-7738 also increased pAMPK and cleaved caspase-3 levels in ex vivo ccRCC tissue slices.

Conclusion

Activation of AMPK was generally low in both primary ccRCC tissue and cell lines grown under physiologically appropriate conditions. NUC-7738 activated AMPK in ex vivo ccRCC tissue slices and in cell lines. An induction of apoptosis in ex vivo tissues and inhibition of growth in cell lines were also caused by NUC-7738. This suggests that AMPK activation may be one mechanism through which NUC-7738 exerts its activity in clear cell renal cell carcinoma.