Exploring the Protective Role of Caffeine against Taraxacum-Induced Ribotoxic Stress: Insights into Autophagy and Mitochondrial Depolarization

Background: The ribotoxic stress response (RSR) is a pathway that gets activated when ribosomes get impaired, leading to disruptions in protein synthesis, increased inflammatory signaling, and cell death if left unresolved. Taraxacum can induce apoptosis-associated ribosomal RNA (rRNA) cleavage, however, the exact working mechanism of Taraxacum-induced rRNA cleavage remains unclear. Methods: The RNA integrity (RIN) value, 28S/18S ratio, mRNAs, and proteins were for the drug screening for Taraxacum-induced rRNA cleavage process. Flowcytometry analysis was to examine the effects on necrosis, apoptosis, ROS, and mitochondrial membrane potential of Taraxacum-induced cells. Results: We first used the RIN value and 28S/18S ratio to confirm the integrity of experiments. Our RNA sequencing data showed that T. formosanum upregulated 893 genes and downregulated 509 genes and triggered hallmark genes of spliceosomes, TNF-α signaling via NF-κB, inflammatory response, and IL6-JAK-STAT3 signaling. Additionally, T. formosanum imbalanced the levels of ribosomal proteins of the large and small subunits. We found that caffeine was the only screening agent that could rescue the cleavage of 28S and 18S rRNA induced by T. formosanum. However, caffeine failed to rescue T. formosanum-targeted mRNAs when the RIN values were relatively lower. T. formosanum induced the N-terminal clipping of histone H3, which was observed not only in human HeLa cervical cancer cells but also in human Huh6 and HepG2 liver cancer cells. Conclusion: Our study revealed that caffeine could reverse the effects of T. formosanum on the reduction of autophagy and the disruption of mitochondrial membrane potential. However, caffeine could only change the populations of necrotic and apoptotic cells but not T. formosanum-induced cell death. In this study, we utilized RNA sequencing analysis to examine the effects of T. formosanum extract on gene expression in HeLa cells. HeLa cells were exposed to either 7 mg/ml of T. formosanum extract alone or in combination with 10 mM caffeine.