Chaetoglobosin A induces apoptosis in T-24 human bladder cancer cells through oxidative stress and MAPK/PI3K-AKT-mTOR pathway

Reagents and antibodies

Trypsin, MTT, and dimethyl sulfoxide (DMSO) were purchased from Sangon Biotech (Shanghai, China). Annexin-V-fluorescein isothiocyanate (FITC), RNase, propidium iodide (PI), 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA), 2′-(4-ethoxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5′-bi-1H-benzimidazole, trihydrochloride (Hoechst 33342), 3,3′-dihexyloxacarbocyanine iodide (DioC6) and NAC were purchased from Sigma Aldrich (St. Louis, MO, USA). Chloroquine (CQ), SB203580, 3-methyladenine (3-MA) and rapamycin (Rap) purchased from Aladdin. Portions of this text were previously published as part of a preprint (https://doi.org/10.21203/rs.3.rs-3258565/v1).

Cell culture

Human bladder cancer cell line T-24 was provided by the cell bank of the Chinese Academy of Sciences in Shanghai. Cells were cultured in DMEM medium (Gibco, Waltham, MA, USA) enriched with 10% FBS (Gibco, Waltham, MA, USA) at 37 °C with 5% CO₂ and passaged at 80–90% confluence. T-24 cell in the logarithmic growth phase was used for the following experiments.

Measurement of cytotoxicity and colony formation assay

Cells (5,000/well) were inoculated into 96-well plates in 100 μL of culture medium. Cytotoxicity was examined using the MTT assay. The cells were treated with ChA at different concentrations (0, 12.5, 25, 50, 75, 100 μM) for 24 h. After the desired treatment, cells were incubated with 10 μL of MTT solution for an additional 4 h. Add 150 μL DMSO to dissolve formazan and incubate at 37 °C overnight. Cell absorbance at 570 nm was measured using the Universal Microplate Spectrophotometer (Bio-Rad, Berkeley, CA, USA).

Cells (600/well) were inoculated into 6-well plates and cultured in fresh medium containing ChA or DMSO, with media changes every 3 days. After 14 days, cells were fixed for 30 min in PFA 4% and crystal violet staining was performed at room temperature (RT). Repeatedly rinsed and photographed under the microscope.

Wound healing assay

For easy observation, we drew horizontal lines at 1 cm intervals on the back of the six-well plate. Cell plate count method was used to adjust the cell concentration to about 5 × 10⁵/mL and the cells were inoculated into a six-well plate. When the cell density reached approximately 80%, the monolayer was wounded by scratching with a 10 µl sterile pipette tip lengthwise along the plate surface, and the media was removed. The cells were then washed three times with PBS and cultured in serum-free media. The cells were treated with ChA at different concentrations (0, 1.56, 3.125, 6.25, 12.5 μM) for 24 h. Imaging was done at 0 and 24 h under light microscopy.

Hoechst 33342 analysis

After ChA treatment in different concentrations for 6 h, cells were collected and washed with PBS, fixed with 4% paraformaldehyde for 30 min at 4 C, and stained with 1 mM Hoechst 33258 for 5 min. Nuclei and apoptotic bodies were observed under a fluorescence microscope (Nikon, Tokyo, Japan) with ×20 objective lens and ×10 eyepiece and photographed.

Apoptosis assay

T-24 cells were firstly stimulated with an increasing concentration of ChA solution (0, 25, 50, 75 µM). After 12 h treatment, the cells were collected by centrifugation after trypsinization, and washed with cold PBS. Cultured cells were resuspended and stained with Annexin V-FITC and PI in binding buffer according to the kit’s protocol and the cells were examined by flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA) within one hour.

Measurement of ROS

T-24 cells were firstly stimulated with an increasing concentration of ChA solution (0, 25, 50, 75 µM) for 2 h. After stimulation, T-24 cells were digested by trypsin and collected by centrifugation. Then the cells were stained with 5 μM DCFH-DA for 30 min. After two washes with PBS to remove the excess DCFH-DA, the population of stained cells was examined by flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA).

Measurement of MMP

T-24 cells were firstly stimulated with an increasing concentration of ChA solution (0, 25, 50, 75 µM) for 4 h. After stimulation, T-24 cells were digested by trypsin and collected by centrifugation. Then the cells were washed in PBS and treated with 40 nM DioC6(3) for 30 min. Subsequently, the percentage of cells with MMP loss was examined by flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA).

Cell cycle analysis

T-24 cells were firstly stimulated with an increasing concentration of ChA solution (0, 25, 50, 75 µM). After 6 h treatment, the cells were collected by centrifugation after trypsinization, and washed with cold PBS. Cells were resuspended in 1 mL PBS containing 20 μg/ml RNase A, incubated at 4 C for 30 min, and resuspended in propidium iodide (PI) solution. Cells at different stages of the cell cycle and sub-G1 phase were analyzed by flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA).

Western blot analysis

T-24 cells were firstly stimulated with an increasing concentration of ChA solution (0, 25, 50, 75 µM). After 6 h treatment, cells were washed with PBS three times, and the total protein was extracted using RIPA lysis buffer or RIPA lysis buffer containing protease and phosphatase inhibitors. After brief sonication, the lysates were centrifuged at 13,000 × g for 10 min at 4 °C, and the protein contents in the supernatant were measured by a BCA kit (Solarbio, Beijing, China). Proteins were separated on dodecyl sulfate, sodium salt-polyacrylamide gel electrophoresis (SDS-PAGE), transferred on poly-vinylidene fluoride (PVDF) membranes, and blocked with 5% skimmed milk for 1 h. The membranes were then probed with primary antibodies overnight at 4 °C, followed by secondary antibody for 2 h at room temperature. For the detection of proteins, the chemiluminescence agents ECL kit (Beyotime, Shanghai, China) was used, and the protein expression was analyzed by Image J software. Western blot analysis was performed using the following specific monoclonal or polyclonal antibodies: anti-Bcl-2 (Cat# sc-73822), anti-Bax (Cat# sc-20067), anti-Bid (Cat# sc-56025), anti-CDK1 (Cat# sc-53219), anti-CCNB1 (Cat# sc-53238), anti-ERK (Cat# sc-81459), anti-JNK (Cat# sc-7345) and anti-GAPDH (Cat# sc-137179) from Santa Cruz, Dallas, Texas, USA; caspase-8 (Cat# AF6442), anti-phospho-ERK1/2 (Cat# AF3240), anti-phospho-JNK (Cat# AF3319), anti-PI3K (Cat# AF6241), anti-p-PI3K p85 alpha (Tyr607) (Cat# AF3241), anti-AKT (Cat# AF0836), secondary anti-mouse (Cat# SA00001-1) and anti-rabbit antibodies (Cat# SA00001-2) from Affinity, Cincinnati, OH, USA; anti-p-AKT (Ser473) (Cat#66444-1-1g), anti-mTOR (Cat#66888-1-1g), anti-p-mTOR (Ser2448) (Cat#67778-1-1g), anti-Calnexin (Cat#10427-2-AP) from Proteintech Group, Chicago USA; anti-p38 (Cat#9212S) and anti-phospho-p38 (Cat#9211S) from Cell Signaling Technology (Danvers, MA, USA).

Statistical analysis

All experiments were repeated at least three times independently. The data were expressed as the mean ± standard deviation (SD). The treated groups were compared by one-way variance (ANOVA) with SPSS 13.0 (SPSS, Chigaco, IL, USA). The statistically significant p values were labeled as follows: *p < 0.05, **p < 0.01, ***p < 0.001.

Inhibitory effect of ChA on T-24 cell proliferation

To investigate the proliferation inhibitory effect of ChA on tumor cells, we treated T-24 cells with different concentrations of ChA (0, 12.5, 25, 50, 75, 100 μM) for 24 h. Figure 1A is the structural formula of ChA. The results showed that ChA could reduce cell viability in a dose-dependent manner as measured by MTT (Fig. 1B). We analyzed the effect of ChA (0, 1.56, 3.12, 6.25, 12.5, 25 μΜ) on the cloning ability of T-24 cells at lower concentration ranges. The results showed that the clonogenic “spots” were reduced at lower concentrations compared to the unspoked group, and almost no clonogenic “spots” were formed after the drug concentration reached 12.5 μM (Fig. 1C). These results indicate that ChA can significantly inhibit the proliferation and division of T-24 cells and has a sustained inhibitory effect.

ChA inhibits T-24 cell migration

The migration ability of the cells could be examined by the number of cells entering the blank area at the edge of the scratch. As shown in the Figs. 2A and 2B, the changes of cell migration at 24 h after different concentrations of ChA treatment. The distance of cell scratch was the same in all groups at 0 h, and after 24 h of treatment, the cells at the edge of the scratch gradually entered the blank area. Compared with the control group, as the concentration of the drug increased, the cell migration ability decreased. The above results indicated that ChA could inhibit the migration of T-24 cells.

ChA induces apoptosis in T-24 cells

It was found that ChA had a high cytotoxic effect on human bladder cancer T-24 cells by pre-MTT experiments. In order to explore whether ChA killed tumor cells through the apoptotic pathway, Hoechst 33342 fluorescence staining was performed on T-24 cells treated with different concentrations of ChA, and the results are shown in Fig. 3A. Compared with the unspiked group, ChA-treated T-24 cells with increasing drug concentration the fluorescence intensity increased and the apoptotic morphological features of cell crumpling and chromatin condensation appeared, indicating that ChA induced apoptosis in T-24 cells (Nitobe et al., 2003). The quantified and the flow through results are shown in Figs. 3B and 3C to determine the specific apoptotic rate. The total apoptotic rate of T-24 cells treated with different concentrations of ChA increased in a concentration-dependent manner compared to the untreated group and increased significantly at drug concentrations up to 75 μM (p < 0.001), which is consistent with the morphological observations.

ChA induces ROS accumulation and MMP reduction in T-24 cells

The accumulation of intracellular reactive oxygen species (ROS) is associated with the development of several diseases and is also closely linked to apoptosis (Wang et al., 2023; Dröge, 2002). In this experiment, the detection of intracellular ROS was performed by fluorescent probe (DCFH-DA). Flow cytometry results showed that intracellular ROS levels were elevated after 2 h of dosing treatment and significantly higher at 75 μM (p < 0.001) compared to the non-dosed group. In addition, the addition of NAC pretreatment restored ROS levels after ChA treatment (Figs. 4A and 4B). The decrease in the MMP is an event that occurs early in apoptosis (Cui et al., 2014). We used DiOC6(3) dye for mitochondrial membrane potential detection. A decrease in green fluorescence intensity indicating MMP loss was detected by flow cytometry (Figs. 4C and 4D). The degree of loss of intracellular mitochondrial membrane potential was significantly increased after the drug concentration reached 50 μM compared to the unspiked group (p < 0.001). This indicates that excessive accumulation of ROS in T-24 cells leads to oxidative stress and causes mitochondrial dysfunction, inducing apoptosis in T-24 cells.

ChA induces T-24 cell cycle blockade

To explore the mechanism of ChA inhibition of T-24 cell growth, the distribution of T-24 cells cycle and the relative changes of each cycle after 6 h of drug treatment were examined and analyzed separately in this study. The flow results (Figs. 5A and 5B) showed that the Sub G1 (subdiploid DNA) cell population increased after ChA treatment, with a slight increase in G1 phase, a less pronounced change in S phase, a slight decrease at 75 μM, and a gradual G2/M phase cell decreased. This indicates that ChA treatment increased DNA fragmentation, caused arrest of G1 phase cells and inhibited cell mitosis.

Changes in the expression of CCNB1 and CDK1, G2/M phase cell cycle regulatory proteins that play an important role in cell cycle progression, were analyzed by western blot. As shown in the Figs. 5C and 5D, the expression of CDK1, CCNB1 was decreased in a dose-dependent manner after ChA treatment of T-24 cells for 6h, which prevented the formation of CCNB1-CDK1 complex and further hindered the cell mitosis.

Effect of ChA on apoptosis-associated proteins in T-24 cells

The effect of ChA on MMP was detected by flow cytometry, and it was found that the dysregulation of Bcl-2 family proteins was found to directly affect MMP (Hassan et al., 2014; Nasser et al., 2017). As shown in Figs. 6A and 6B, increased Bax expression and decreased Bcl-2 expression were observed in ChA-treated T-24 cells, consistent with decreased MMP detected by flow, suggesting that the Bcl-2 family is involved in the mitochondrial apoptotic pathway. Whereas caspase family proteins are closely linked to Bcl-2 family proteins in apoptosis, we examined the initiating caspase (caspase-8) and effector caspase (caspase-3) as well as Bid proteins. The results were as follows: caspase-3 and caspase-8 protein expression showed a decreasing trend, and activated caspase-8 broke Bid in the cytoplasm, leading to Bid protein cleavage further affecting MMP. The results indicate that ChA can induce apoptosis in T-24 cells by way of mitochondrial pathway and death receptor pathway.

ChA induces activation of MAPK pathway in T-24 cells

The mitogen-activated protein kinase MAPK signaling pathway plays an important role in a series of cellular physiological activities such as tumor cell growth, differentiation and apoptosis. Among them, JNK and p38 MAPK functions are associated with inflammatory factors and oxidative stress, and ERK1/2 is mainly involved in cell growth, differentiation and other activities (Wagner & Nebreda, 2009). To verify whether ChA induces apoptosis through MAPK pathway regulation, this experiment detected JNK, p38 as well as ERK1/2 and their phosphorylated protein expression levels by western blot and the results are shown in Figs. 7A and 7B. Compared with the unspiked group, the levels of JNK and p38 phosphorylation were significantly increased after 6 h of drug treatment, and ERK1/2 phosphorylation levels were significantly increased when the drug concentration reached 75 μM. This indicates that ChA induces apoptosis in T-24 cells by regulating the MAPK pathway.

Effect of ChA on PI3K-Akt-mTOR pathway

The PI3K-Akt-mTOR signaling pathway is thought to play a key role in regulating proliferation, migration, and autophagy. Dysregulation of PI3K-Akt-mTOR signaling was found in 40% of bladder cancers (Khan et al., 2020). To detect the effect of PI3K-AKT-mTOR pathway changes on bladder cancer T-24 cell line after compound ChA treatment, the experiment used western blot to detect PI3K, AKT, and mTOR proteins, and selected phosphorylation sites to detect p-PI3K-p58, p-AKT-Ser473, and p-mTOR-Ser2448 expression changes. The experimental results are shown in the Figs. 7C and 7D. Compared with the unspiked group, the compound ChA spiked treatment could increase the levels of p-PI3K-p58 and p-AKT-Ser473, and inhibit the phosphorylation expression of mTOR protein. The above experimental results suggest that the PI3K-AKT-mTOR pathway is involved in regulating compound ChA-induced apoptosis in T-24 cells.

Effect of inhibitors on protein expression in T-24 cells after ChA treatment

To further investigate the key roles played by ROS, p38, mTOR, and autophagy in ChA-induced apoptosis in T-24 cells, we analyzed the effects of drug combination inhibitors on intracellular protein expression of related pathways by western blot. We used co-incubation with the corresponding inhibitors before ChA treatment of cells. The inhibitors and the concentrations used were reactive oxygen species inhibitor NAC (5 mM), mTOR inhibitor Rapamycin (5 μM), p38 MAPK inhibitor SB203580 (20 μM) and PI3K inhibitor 3-MA (5 mM) were incubated for 1 h before ChA treatment, and then cells were treated with 75 μM of ChA for 6 h.

Western blot results (Fig. 8) showed that NAC pretreatment upregulated the expression level of Bcl-2 protein and decreased p38 phosphorylation and increased ERK phosphorylation. ROS was also found to be involved in the mTOR signaling pathway, and the levels of PI3K and p-mTOR were significantly restored after NAC pretreatment with ChA. ROS was found to negatively regulate Bcl-2, MAPK, and PI3K-Akt-mTOR signaling pathways by adding the ROS inhibitor NAC.

Pretreatment with PI3K inhibitor 3-MA and mTOR inhibitor Rapamycin had no effect on the expression levels of Bcl-2 protein in the mitochondrial pathway and ERK in the MAPK pathway, but could up regulate the expression of PI3K. P38MAPK inhibitor SB203580 pretreatment decreased the level of p38 phosphorylation and increased the phosphorylation of ERK. Figs. 8B–F shows the statistical diagram of western blot results. Through the analysis of the above experimental results, we deduced that ChA treatment of T-24 cells resulted in the accumulation of intracellular ROS as an important factor leading to apoptosis. When inhibiting the production of ROS, it could simultaneously affect the MAPK pathway, mTOR pathway and mitochondria-regulated endogenous pathways jointly involved in the regulation of ChA-induced apoptosis in T-24 cells. Among where the interaction of the MAPK pathway and the mTOR pathway was not confirmed, but the joint participation of multiple pathways amplifies the apoptotic cascade response in T-24 cells.