Ultrasound-triggered microbubble destruction enhances the radiosensitivity of glioblastoma by inhibiting PGRMC1-mediated autophagy in vitro and in vivo

Abstract

Background Ultrasound-triggered microbubble destruction (UTMD) is a widely used noninvasive technology in both military and civilian medicine, which could enhance radiosensitivity of various tumors. However, little information is available regarding the effects of UTMD on radiotherapy for glioblastoma or the underlying mechanism. This study aimed to delineate the effect of UTMD on the radiosensitivity of glioblastoma and the potential involvement of autophagy. Methods GL261, U251 cells and orthotopic glioblastoma-bearing mice were treated with ionizing radiation (IR) or IR plus UTMD. Autophagy was observed by confocal microscopy and transmission electron microscopy. Western blotting and immunofluorescence analysis were used to detect progesterone receptor membrane component 1 (PGRMC1), light chain 3 beta 2 (LC3B2) and sequestosome 1 (SQSTM1/p62) levels. Lentiviral vectors or siRNAs transfection, and fluorescent probes staining were used to explore the underlying mechanism. Results UTMD enhanced the radiosensitivity of glioblastoma in vitro and in vivo (P < 0.01). UTMD inhibited autophagic flux by disrupting autophagosome-lysosome fusion without impairing lysosomal function or autophagosome synthesis in IR-treated glioblastoma cells. Suppression of autophagy by 3-methyladenine, bafilomycin A1 or ATG5 siRNA had no significant effect on UTMD-induced radiosensitization in glioblastoma cells (P < 0.05). Similar results were found when autophagy was induced by rapamycin or ATG5 overexpression (P > 0.05). Furthermore, UTMD inhibited PGRMC1 expression and binding with LC3B2 in IR-exposed glioblastoma cells (P < 0.01). PGRMC1 inhibitor AG-205 or PGRMC1 siRNA pretreatment enhanced UTMD-induced LC3B2 and p62 accumulation in IR-exposed glioblastoma cells, thereby promoting UTMD-mediated radiosensitization (P < 0.05). Moreover, PGRMC1 overexpression abolished UTMD-caused blockade of autophagic degradation, subsequently inhibiting UTMD-induced radiosensitization of glioblastoma cells. Finally, compared with IR plus UTMD group, PGRMC1 overexpression significantly increased tumor size [(3.8 ± 1.1) mm2 vs. (8.0 ± 1.9) mm2, P < 0.05] and decreased survival time [(67.2 ± 2.6) d vs. (40.0 ± 1.2) d, P = 0.0026] in glioblastoma-bearing mice. Conclusion UTMD enhanced the radiosensitivity of glioblastoma partially by disrupting PGRMC1-mediated autophagy.

Vitamin D3 attenuates SARS‐CoV‐2 nucleocapsid protein‐caused hyperinflammation by inactivating the NLRP3 inflammasome through the VDR‐BRCC3 signaling pathway in vitro and in vivo

“…Cells and tissues including lung, kidney, spleen, and liver were collected, lysed, and homogenized for protein extraction and subjected to western blotting, as described previously 42 . Briefly, 10%–15% SDS‐PAGE was used to resolve the protein (40–120 μg) and electroblotted onto polyvinylidene difluoride membranes.…”

“…At 21 days after AAV‐N injection, the lung tissues were collected, embedded in optimal cutting temperature compound at −20°C, and sectioned (6–8 μm). Then, the expression of IL1β and IL6 was detected by immunofluorescence analysis accordingly to our previously published methods 42 . Briefly, antibodies of IL1β and IL6 were diluted at 1:300 and 1:200 and were incubated with the sections at 4°C for 12 h, respectively.…”

“…Then, the expression of IL1β and IL6 was detected by immunofluorescence analysis accordingly to our previously published methods. 42 Briefly, antibodies of IL1β and IL6 were diluted at 1:300 and 1:200 and were incubated with the sections at 4 • C for 12 h, respectively. After three times washing with phosphate-buffered saline for 5 min, the sections were incubated with Alexa Fluor 555 antibody or Alexa Fluor 647 antibody for 2 h at 20-25 • C. The nuclei were stained by 4′,6-diamidino-2-phenylindole (DAPI) for 10 min at room temperature.…”

PPARγ controls ESCRT-dependent fibroblast-like synoviocyte exosome biogenesis and Alleviates Chondrocyte Osteoarthritis Mediated by Exosomal ANXA1

“…Furthermore, mRFP-GFP-LC3 adenovirus double labeling (Figure 3C and 4E) and transmission electron microscopy (TEM) (Figure 5C) revealed that autophagosomes (APs), characterized by double-layer or multilayer membranes encapsulating portions of the cytoplasm and organelles slated for degradation [38], were increased by OE-PPARγ exosomes, but decreased by KD-PPARγ exosomes. Flow cytometry results showed that apoptosis decreased following intervention with OE-PPARγ exosomes, but increased with KD-PPARγ exosomes (Figure 3E and 4F).…”

Targeted microbubbles combined with low-power focused ultrasound promote the thrombolysis of acute deep vein thrombosis

“…The combination of a microbubble contrast agent and ultrasound targeted microbubble destruction (UTMD) technology can deliver bioactive substances to various targets non-invasively and specifically ( Liu et al, 2022b ). At present, it is mainly used in the treatment of heart disease, tumors, and atherosclerotic plaque ( Zhang et al, 2021 ; He et al, 2022 ). UTMD technology breaks up targeted microbubbles and releases large amounts of therapeutic substances in the target area, which optimizes the local treatment effect while reducing systemic side effects ( Yang et al, 2019 ; Xu et al, 2022 ).…”