Time series transcriptomes resolve metabolic pathways underlying crocin's anti-cancer activity: Control FASTQ sequencing reads

Liver cancer can be treated with natural products, including saffron. The exact mechanism for the components of saffron, including crocin, to eliminate malignancy in HCC cells is not known. We used response screening from time series transcriptomic datasets to decipher the underlying mechanisms responsible for crocin’s anti-cancer activity. We treated HCC cells with 1 and 2 mM crocin for 2, 6, 12, and 24 hours and examined transcriptomic profiles at each timepoint compared to controls. The effect size was strongest at two hours and 1 mM crocin; later timepoints and the 2 mM dose showed similar responses, but the overall effect was diminished after two hours post-treatment. We observed the upregulation of transcripts for fatty acid oxidation enzymes, mitochondrial carnitine transport systems, bile acid synthesis, and cholesterol metabolism to varying degrees at all timepoints tested after crocin treatment. Pathway enrichment analyses showed that pathways for DNA damage checkpoint activation and senescence induction were upregulated while a variety of pro-proliferation pathways were downregulated. We saw 52 genes involved in non-alcoholic fatty liver disease down-regulated after 24 hours post-treatment (enrichment FDR p = 8 x 108), indicating a reversal of many pathways promoting carcinogenesis after crocin treatment. Expression of genes involved in senescence and autophagy were commonly dysregulated in all timepoints, with autophagy being apparently suppressed by crocin treatment. All time points tested showed the significant down-regulation of spliceosomal machinery genes. Interestingly, the cell fate-protecting PAX transcription factor was significantly upregulated while ELK oncogenes were downregulated after crocin treatment. Overall, the data outline an intricate cellular response leading to HCC spliceosome repression and induced senescence after crocin treatment. Our findings help to explain the cancer-fighting effects of saffron and outline an example of antioxidant ‘swamping’ in cancer cells by natural products.