SC66 inhibits the proliferation and induces apoptosis of human bladder cancer cells by targeting the AKT/β‐catenin pathway

Abstract Bladder cancer (BC) is a major disease of the genitourinary tract, and chemotherapy is one of the main treatments commonly used at present. SC66 is a new type of allosteric AKT inhibitor that is reported to play an effective inhibitory role in the progression of many other types of tumours, but there is no reported research on its role in BC. In this study, we found that SC66 significantly inhibited the proliferation and EMT‐mediated migration and invasion of T24 and 5637 cells. In addition, experiments confirmed that SC66 achieved its antitumour effect by inducing cell apoptosis and affecting the cell cycle. Luciferase assays confirmed that SC66 exerted an antitumour effect through the AKT/β‐catenin signalling pathway, and this inhibitory effect was reversed after the addition of the β‐catenin signalling pathway activator, CHIR‐99021. In addition, animal studies have shown that, compared with the control group, the experimental group with SC66 intraperitoneal injection showed significantly reduced the tumour weight and volume in nude mice with T24 tumours and that SC66 combined with cisplatin achieved better inhibition on tumours. Western blot analysis and immunohistochemistry staining confirmed that SC66 inhibited the EMT process in vivo and induced apoptosis through the AKT/β‐catenin signalling pathway. In conclusion, our study demonstrated that SC66 exerts a significant antitumour effect through the AKT/β‐catenin signalling pathway, thereby providing a new potential treatment for BC.

As a highly effective anti-cancer drug, cisplatin has been proved to have great potential in the treatment of various cancers, including urological cancers, head and neck cancers and thoracic cancers.
After internalization in cells, due to the aquation of one of the two chloride leaving groups, cisplatin is stimulated and can be covalently bound to DNA, which subsequently activates many molecular mechanisms involving DNA modification, cell cycle arrest and apoptosis to exert its anti-cancer effect. [4][5][6][7] In addition, damage to important organelles such as the endoplasmic reticulum and mitochondria is another important mechanism of cisplatin's anti-cancer effect.
However, the abnormal activation of autophagy, unfolded protein response and other protective processes promotes the chemical resistance of cisplatin and limits its functions. 8,9 Therefore, it is extremely urgent to explore the molecular mechanisms in the progression of BC and identify new and effective treatments.
Many studies have confirmed that the phosphatidylinositol-3 kinase (PI3K)/AKT signalling pathway plays a vital role in regulating cell proliferation, differentiation and survival. [10][11][12] Abnormal activation of the PI3K/AKT signalling pathway plays an important role in the progression of many human cancers. [13][14][15] AKT is a serine/threonine kinase that is activated by phosphorylation. Activated AKT regulates cell proliferation, differentiation and apoptosis through multiple downstream targets, such as mTORC1, GSK-3β and CASP9.
Therefore, AKT is also regarded as a key regulator of the PI3K/AKT signalling pathway. [16][17][18] In recent years, many studies have confirmed that AKT-targeted therapy significantly inhibits the progression of many cancers, including BC. 19,20 Therefore, drug research targeting AKT has also become an important focus in the treatment of BC.
SC66 is a new type of allosteric AKT inhibitor. Jo et al. 21 showed that SC66 inhibits AKT activation by interfering with the binding of the pleckstrin homology domain to phosphatidylinositol-3,4,5-triphosphate and directly promotes AKT ubiquitination to enhance PI3K inhibition-mediated antitumour effects. In recent years, an increasing number of studies have demonstrated that SC66 plays an inhibitory role in the development of many cancers. Yeying Liu et al. reported that SC66 mediates the apoptosis of colon cancer cells by targeting AKT. 22 Xu et al. 23 confirmed that SC66 inhibits the proliferation, migration and invasion of renal cell carcinoma cells by targeting AKT. Another report has shown that SC66 inhibits the proliferation of human glioblastoma and induces apoptosis in tumour cells by inhibiting AKT. 24 However, there is still no research on the role of SC66 in BC.
Thus, the present study investigated the specific role of SC66 in BC and its molecular mechanism.

| Transwell migration and invasion assay
To perform migration assays, T24 or 5637 cells were cultured with different concentrations of SC66 (0, 5 and 10 µmol/L) for 24 h, and cells were then trypsinized and resuspended in serum-free medium.
After adjusting the cell density, 200 μl of serum-free medium con- software was used to analyse and process the results. Each experiment was repeated three times.

| Apoptosis assay
An Annexin V-PE/7-ADD kit (Becton Dickinson) was used to detect Finally, flow cytometry (BD FACSCalibur) was used to detect the apoptotic rate of the cells. Each experiment was repeated three times.

| TUNEL detection
The TUNEL assay was performed using the In Situ Apoptosis Detection kit (Roche Applied Science) according to the manufacturer's protocol. In brief, cell and tissue samples were prepared, and apoptotic cells were accurately labelled by terminal transferasemedicated dUTP nick-end labelling. Finally, an upright fluorescence microscope (Olympus BX51) was used to acquire images. In tissue samples, apoptotic cell nuclei were stained brown, and negative cell nuclei were stained blue.

| Western Blot analysis
Cell and tissue samples were collected and lysed on ice in RIPA buffer (Beyotime) containing 0.1 mM PMSF and a protease inhibitor (Roche) for approximately 30 min. After centrifugation of the lysate, the supernatant was collected, and the protein concentration was determined by the BCA method (Beyotime). The samples were subjected to 12% or 15% SDS-PAGE, and the protein was transferred to a PVDF membrane, which was then blocked with 5% skimmed milk at room temperature for 1 h. After washing three times with TBS-T, the PVDF membrane was incubated with the corresponding antibody overnight at 4°C. After washing three times with TBS-T, the membrane was then incubated with goat anti-mouse Proteintech) at room temperature for 1 h. Finally, a ChemiDoc™ Touch Imaging System (BIO-RAD) was used to scan protein bands and acquire images, and ImageJ software was used to analyse the results. The experiment was repeated three times for each group. Finally, cells were counterstained with DAPI (ab104139, Abcam), and the coverslip was fixed on a glass slide. An upright fluorescence microscope (Olympus BX51) was used to obtain the images. were acquired using a microscope (Olympus BX51).

| Luciferase assays
T24 and 5637 cells were seeded on a six-well plate at a density of 5 × 10 4 cells per well. After treating the cells with different con-

| Statistical analysis
All statistical graphs in this manuscript were constructed using GraphPad Prism 5.0 software. SPSS 19.0 software (SPSS Inc.) was used to perform the one-way analysis of variance (ANOVA) to evaluate whether the differences between the experimental data of each group were statistically significant. p < 0.05 was considered significant. All data are expressed as the mean ± SD based on three independent experiments.

| SC66 inhibits the proliferation of BC cells in vitro
SC66 is a new type of allosteric AKT inhibitor, and the chemical formula of SC66 is shown in Figure 2A. control group was significantly higher than that in the SC66 group ( Figure 2G,H). In summary, these results indicated that the proliferation of T24 and 5637 cells is significantly inhibited after SC66 treatment and that the degree of inhibition is positively correlated with the drug concentration of SC66.

| SC66 inhibits the EMT of BC cells in vitro
Studies have shown that epithelial-mesenchymal transition (EMT) plays an important role in promoting tumour invasion and metastasis in many cancers. Based on the inhibitory effect of SC66 on the proliferation of BC cells, we investigated whether SC66 also inhibits the EMT of BC cells. A wound-healing assay was performed to quantify the effect of SC66 on the motility of BC cells, and the results showed that SC66 significantly inhibited wound healing (within the red line area) in a concentration-dependent manner ( Figure 3A-C).
The percentage of wound healing in the control group was significantly higher than that of BC cells treated with SC66. In addition, a Transwell assay was used to evaluate cell invasion and migration.
The results showed that SC66 significantly inhibited the migration ability of T24 and 5637 cells. Consistent with this result, the invasive ability of BC cells treated with SC66 was significantly lower than that of control cells ( Figure 3D-H). Western blot analysis veri-

| SC66 induces BC cells apoptosis in vitro
In addition, we further explored whether SC66 achieves its antitu-

| SC66 inhibits AKT/β-catenin signalling pathways in BC cells in vitro
To explore whether the antitumour effect of SC66 is achieved by inhibiting the AKT signalling pathway, we used Western blot-  Figure 6E). The above results indicated that SC66 exerts its antitumour effect by inhibiting AKT/β-catenin signalling pathways in T24 and 5637 cells.

| Enhancing β-catenin activity rescues the anti-BC effect of SC66
To further prove that SC66 exerts antitumour effects through

| CON CLUS ION
The data from GLOBOCAN show that an estimated 570,000 people were diagnosed with BC in 2020, accounting for nearly 3% of all newly diagnosed cancers. 1 BC originates from the urothelium and is divided into non-muscle-invasive bladder cancer and muscle-invasive bladder cancer. Surgical treatment is currently the main treatment for local BC. 25,26 Recent studies have shown that compared with cystectomy alone, platinum-based neoadjuvant chemotherapy combined with cystectomy has a 33% reduction in the risk of death, 27 and the 10-year survival rate is increased by 6%. 28 However, the drug resistance and adverse reactions of chemotherapeutics often limit the chemotherapy for BC. 29,30 Immortal cell proliferation is the main feature of tumour cells and is related to cell cycle disorders. 31,32 Our study confirmed that SC66  After confirming that SC66 inhibits EMT-mediated metastasis, promotes apoptosis and affects the cell cycle, we further explored the molecular mechanism of the antitumour effect of SC66. β-catenin is a multifunctional protein that plays an important role in maintaining normal physiological functions. 43 Under normal circumstances, In animal experiments, we also found that the combined use of SC66 and cisplatin exerted a better antitumour effect than SC66 or cisplatin alone. This phenomenon may be related to SC66 increasing the sensitivity of BC cells to cisplatin, but further experiments are needed to prove this conjecture.
In conclusion, the present study confirmed that SC66 exerts its anti-BC effect in vivo and in vitro through the AKT/β-catenin signalling pathway, thereby providing a new potential drug for the treatment of BC. group as well as representative images of IHC staining of P-AKT, P-GSK-3β, vimentin, Bax and β-catenin. All data are expressed as the mean ± SD based on three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 versus the control group; Δ p < 0.05 and ΔΔ p < 0.01 versus the cisplatin group; # p < 0.05 and ## p < 0.01 versus the SC66 group [Colour figure can be viewed at wileyonlinelibrary.com]

ACK N OWLED G EM ENTS
This work was strongly supported by Professor Fan Cheng and Professor Hongfei Song.

CO N FLI C T O F I NTE R E S T
We declare that we have no commercial or associative interests that conflict with the work submitted. Funding acquisition (equal). Fan Cheng: Funding acquisition (equal).

CO NTR I B UTI O N TO TH E FI E LD S TATE M E NT
The data from GLOBOCAN show that an estimated 570,000 people were diagnosed with BC in 2020, accounting for 3% of all newly diagnosed cancers. Surgery combined with immunotherapy is currently the main treatment for local BC. Studies have shown that compared with cystectomy alone, platinum-based neoadjuvant chemotherapy combined with cystectomy has a 33% reduction in the risk of death, and the 10-year survival rate is increased by 6%. However, the drug resistance and adverse reactions of chemotherapeutics often limit the treatment of BC. In recent years, many studies have confirmed that AKT-targeted therapy significantly inhibits the progression of many cancers, including BC, and SC66 is a new type of AKT inhibitor.
This study confirmed that SC66 exerts its anti-BC effect in vivo and in vitro through the AKT/β-catenin signalling pathway, specifically including inhibiting EMT-mediated metastasis, inducing cell cycle redistribution and promoting tumour cell apoptosis. This study provides a new potential drug for the treatment of BC.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data supporting the conclusions of this study can be obtained from the corresponding author.