Abstract
Among ribosomal proteins essential for protein synthesis, the functions of ribosomal protein L5 (RPL5) and RPL11 still remain unclear to date. Here, the roles of RPL5 and RPL11 were investigated in association with p53/p21 signaling in the antitumor effect of puromycin mainly in HCT116 and H1299 cancer cells. Cell proliferation assays using 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assays and colony formation assays, cell cycle analysis, Reverse transcription polymerase chain reaction (RT-PCR) and Western blotting were performed in cancer cells. Puromycin exerted cytotoxic and anti-proliferative effects in p53 wild-type HCT116 more than in p53 null H1299 cells. Consistently, puromycin increased sub-G1, cleaved Poly (ADP-ribose) polymerase (PARP), activated p53, p21, and Mouse double minute 2 homolog (MDM2), and attenuated expression of c-Myc in HCT116 cells. Notably, puromycin upregulated the expression of RPL5 and RPL11 to directly bind to MDM2 in HCT116 cells. Conversely, deletion of RPL5 and RPL11 blocked the activation of p53, p21, and MDM2 in HCT116 cells. Also, puromycin enhanced the antitumor effect with reactivating p53 and inducing tumor apoptosis (RITA) or doxorubicin in HCT116 cells. These findings suggest that puromycin induces p53-dependent apoptosis via upregulation of RPL5 or RPL11 for binding with MDM2, and so can be used more effectively in p53 wild-type cancers by combination with RITA or doxorubicin.
Highlights
Puromycin, an old antibiotic derived from Streptomyces alboniger as a structural analog of tyrosyl tRN 1 [1], is known to induce apoptosis in breast cancer cells by insulin-like growth factor 1 (IGF-I)and exert antitumor activity in MDA-MB-231 cells via the suppression of 45S pre-ribosomal RNA and upstream binding factor (UBF) [2,3], since it terminates the ribosomal protein synthesis process by causing the premature release of a polypeptide from the ribosome in malignant cells compared to normal cells [4]
In the present study, the roles of ribosomal protein L5 (RPL5) and RPL11 were elucidated in association with p53/Mouse double minute 2 homolog (MDM2) signaling in the puromycin-induced antitumor effect in p53 sensitive and deficient cancer cells
Puromycin significantly suppressed the viability of p53 wild-type HCT116 cells in a concentration and time-dependent fashion compared to SW620, HCT15, and H1299 cells using an MTT assay (Figure 1A,B)
Summary
An old antibiotic derived from Streptomyces alboniger as a structural analog of tyrosyl tRN 1 [1], is known to induce apoptosis in breast cancer cells by insulin-like growth factor 1 (IGF-I)and exert antitumor activity in MDA-MB-231 cells via the suppression of 45S pre-ribosomal RNA and upstream binding factor (UBF) [2,3], since it terminates the ribosomal protein synthesis process by causing the premature release of a polypeptide from the ribosome in malignant cells compared to normal cells [4]. Many puromycin derivatives have been developed for clinical applications [5] The cellular processes such as development, differentiation, cell proliferation, and apoptosis are controlled indirectly or directly by oncogenes and tumor suppressors including c-Myc, PTEN, and p53 [6,7]. The p53 tumor suppressor protein is a major mediator of cell-cycle arrest and/or apoptosis in the response of mammalian cells to cellular stress, including nucleolar stress or ribosomal stress [8]. Emerging evidence reveals that the disruption of ribosome biogenesis and/or the nucleolar structure activates p53-dependent or independent signaling pathways leading to cell cycle arrest, apoptosis, differentiation, and senescence [12,13]. In the present study, the roles of RPL5 and RPL11 were elucidated in association with p53/MDM2 signaling in the puromycin-induced antitumor effect in p53 sensitive and deficient cancer cells
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