RESUMEN
PURPOSE: High-intensity focused ultrasound (HIFU/FUS) has expanded as a noninvasive quantifiable option for hyperthermia (HT). HT in a temperature range of 40-47⯰C (thermal dose CEM43â¯≥ 25) could work as a sensitizer to radiation therapy (RT). Here, we attempted to understand the tumor radiosensitization effect at the cellular level after a combination treatment of FUS+RT. METHODS: An in vitro FUS system was developed to induce HT at frequencies of 1.147 and 1.467â¯MHz. Human head and neck cancer (FaDU), glioblastoma (T98G), and prostate cancer (PC-3) cells were exposed to FUS in ultrasound-penetrable 96-well plates followed by single-dose Xray irradiation (10â¯Gy). Radiosensitizing effects of FUS were investigated by cell metabolic activity (WST1 assay), apoptosis (annexin V assay, sub-G1 assay), cell cycle phases (propidium iodide staining), and DNA double-strand breaks (γH2A.X assay). RESULTS: The FUS intensities of 213 (1.147â¯MHz) and 225â¯W/cm2 (1.467â¯MHz) induced HT for 30â¯min at mean temperatures of 45.20⯱ 2.29⯰C (CEM43â¯= 436⯱ 88) and 45.59⯱ 1.65⯰C (CEM43â¯= 447⯱ 79), respectively. FUS improves the effect of RT significantly by reducing metabolic activity in T98G cells 48â¯h (RT: 96.47⯱ 8.29%; FUS+RT: 79.38⯱ 14.93%; pâ¯= 0.012) and in PC-3 cells 72â¯h (54.20⯱ 10.85%; 41.01⯱ 11.17%; pâ¯= 0.016) after therapy, but not in FaDu cells. Mechanistically, FUS+RT leads to increased apoptosis and enhancement of DNA double-strand breaks compared to RT alone in T98G and PC-3 cells. CONCLUSION: Our in vitro findings demonstrate that FUS has good potential to sensitize glioblastoma and prostate cancer cells to RT by mainly enhancing DNA damage.
Asunto(s)
Glioblastoma/terapia , Neoplasias de Cabeza y Cuello/terapia , Neoplasias de la Próstata/terapia , Línea Celular Tumoral , Terapia Combinada , Daño del ADN/efectos de la radiación , Glioblastoma/genética , Glioblastoma/radioterapia , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/radioterapia , Humanos , Hipertermia Inducida , Masculino , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/radioterapia , Ultrasonografía , Terapia por Rayos XRESUMEN
Transcription factor-driven cell fate engineering in pluripotency induction, transdifferentiation, and forward reprogramming requires efficiency, speed, and maturity for widespread adoption and clinical translation. Here, we used Oct4, Sox2, Klf4, and c-Myc driven pluripotency reprogramming to evaluate methods for enhancing and tailoring cell fate transitions, through directed evolution with iterative screening of pooled mutant libraries and phenotypic selection. We identified an artificially evolved and enhanced POU factor (ePOU) that substantially outperforms wild-type Oct4 in terms of reprogramming speed and efficiency. In contrast to Oct4, not only can ePOU induce pluripotency with Sox2 alone, but it can also do so in the absence of Sox2 in a three-factor ePOU/Klf4/c-Myc cocktail. Biochemical assays combined with genome-wide analyses showed that ePOU possesses a new preference to dimerize on palindromic DNA elements. Yet, the moderate capacity of Oct4 to function as a pioneer factor, its preference to bind octamer DNA and its capability to dimerize with Sox2 and Sox17 proteins remain unchanged in ePOU. Compared with Oct4, ePOU is thermodynamically stabilized and persists longer in reprogramming cells. In consequence, ePOU: 1) differentially activates several genes hitherto not implicated in reprogramming, 2) reveals an unappreciated role of thyrotropin-releasing hormone signaling, and 3) binds a distinct class of retrotransposons. Collectively, these features enable ePOU to accelerate the establishment of the pluripotency network. This demonstrates that the phenotypic selection of novel factor variants from mammalian cells with desired properties is key to advancing cell fate conversions with artificially evolved biomolecules.