Cannabidiol (CBD), a primary bioactive phytocannabinoid extracted from hemp, is reported to possess potent anti-tumorigenic activity in multiple cancers. However, the effects of CBD on bladder cancer (BC) and the underlying molecular mechanisms are rarely reported. Here, several experiments proved that CBD promoted BC cells (T24, 5637, and UM-UC-3) death. For example, T24 cells were treated with 12 µM CBD for 48 h, flow cytometry analysis demonstrated that early and late apoptotic cells were accounted for by 49.91%, indicating CBD enhanced cell apoptosis ability. To deeper explore molecular mechanisms, the CBD-treated T24 cell transcriptome libraries were established. KEGG analysis implied that the significantly changed genes were enriched in the PI3K/Akt pathway. qRT-PCR and Western blot assays verified that CBD regulated BC cells growth and migration and induced apoptosis by inactivating the PI3K/Akt pathway. Meanwhile, the developed chitosan to wrap CBD-loaded PLGA nanoparticles can significantly enhance the adhesion of the material to the mouse bladder wall, and the binding efficiency of mucin to chitosan-PLGA nanoparticles reached 97.04% ± 1.90%. In summary, this work demonstrates that CBD may become a novel reliable anticancer drug and the developed intravesical adhesion system is expected to turn into a potential means of BC chemotherapy drug delivery.

Background: Precise regulation of partial critical proteins in cancer cells, such as anti‐apoptotic proteins, is one of the crucial strategies for treating cancer and discovering related molecular mechanisms. Still, it is also challenging in actual research and practice. The widely used CRISPR/Cas9‐based gene editing technology and proteolysis‐targeting chimeras (PROTACs) have played an essential role in regulating gene expression and protein function in cells. However, the accuracy and controllability of their targeting remain necessary.Methods: Construction of UMUC‐3‐EGFP stable transgenic cell lines using the Sleeping Beauty system, Flow cytometry, quantitative real‐time PCR, western blot, fluorescence microplate reader and fluorescence inverted microscope analysis of EGFP intensity. Characterization of Survivin inhibition was done by using Annexin V‐FITC/PI apoptosis, calcein/PI/DAPI cell viability/cytotoxicity assay, cloning formation assay and scratch assay. The cell‐derived xenograft (CDX) model was constructed to assess the in vivo effects of reducing Survivin expression.Results: Herein, we established a synergistic control platform that coordinated photoactivatable split‐Cas9 targeted gene editing and light‐induced protein degradation, on which theSurvivingene in the nucleus was controllably edited by blue light irradiation (named paCas9‐Survivin) and simultaneously the Survivin protein in the cytoplasm was degraded precisely by a nanobody‐mediated target (named paProtacL‐Survivin). Meanwhile, in vitro experiments demonstrated that reducing Survivin expression could effectively promote apoptosis and decrease the proliferation and migration of bladder cancerous cells. Furthermore, the CDX model was constructed using UMUC‐3 cell lines, results from animal studies indicated that both the paCas9‐Survivin system and paProtacL‐Survivin significantly inhibited tumour growth, with higher inhibition rates when combined.Conclusions: In short, the coordinated regulatory strategies and combinable technology platforms offer clear advantages in controllability and targeting, as well as an excellent reference value and universal applicability in controlling the fate of cancer cells through multi‐level regulation of key intracellular factors.

Targeted protein degradation is a powerful tool for determining the function of specific proteins nowadays. Survivin is the smallest member of the inhibitor of the apoptosis protein (IAP) family. It exists in the cytoplasm and nucleus of cells, but the exact function of survivin in different subcellular locations retained unclear updates due to the lack of effective and simple technical means. In this study, we created a novel nanoantibody-based molecular toolkit, namely, the ubiquitin–proteasome system (Nb4A-Fc-T2A-TRIM21), that can target to degrade survivin localized in cytoplasmic and cell nuclear by ubiquitinating, and by which to verify the potential roles of survivin subcellular localization. Also, the results showed that the cytoplasmic survivin mainly plays an anti-apoptotic function by directly or indirectly inhibiting the caspase pathway, and the nuclear survivin mainly promotes cell proliferation and participates in the regulation of the cell cycle. In addition, the Nb4A-Fc-T2A-TRIM21 system can degrade the endogenous survivin protein in a large amount by the ubiquitin–proteasome pathway, and the system can provide theoretical support for ubiquitination degradation targeting other endogenous proteins.