RPMI1640 (Jinuo, Jiangsu, China); Fetal bovine serum (BiologicalIndustrie, Israel); ER Stress Antibody Sampler Kit (CST, United States); ATF-4; Caspase-3 (CST, United States); Cleaved caspase-3(CST, United States); LC3B (Zenbio, Chengdu, China); Anti-mouse (CST, United States); Anti-rabbit (CST, United States); JC-1 (Beyotime, Shanghai, China); LDH Cytotoxicity Assay Kit (Beyotime, Shanghai, China); Autophagy Antibody Sampler Kit (CST,United States); FITC Annexin Ⅴ Apoptosis Detection Kit (BD, US); Sodium 4-phenylbutyrate (4-PBA) (Bidepharm, Shanghai, China); Spautin-1; 4% Paraformaldehyde (4% PFA) (Solarbio, Beijing, China); 0.5% Crystal violet (Solarbio, Beijing, China); spautin-1; Immunohistochemistry (IHC) detection system kit (Rabbit) (Bioss, Beijing, China); Cryptotanshinone (Herbest, Shanxi, China); 0.003% tricaine (Sigma, United States); DiI (Invitrogen, Carlsbad, CA, United States); Nanoliter Injector (Drummond Scientific Company, Broomall, PA); Cisplatin Injection (Xinnuo, Jiangsu, China); dimethyl sulfoxide (DMSO) (Solarbio, Beijing, China); RIPA buffer (Solarbio, Beijing, China); Acridine orange detection kit (Leagene, Beijing, China).

CRC cells (HCT116, SW480, and Sw620) were cultured in RPMI1640 supplemented with 10% FBS and 1% penicillin/streptomycin at 37°C in a humidified atmosphere with 5% CO₂.

The procedures in the current study has been formally approved by the ethics committee of Qingdao University (Qingdao, China) and written consent forms were approved by all patients. In this study, tumor tissues were collected from 56 patients with colorectal cancer who underwent surgery between June 2019 and February 2020. Diagnosis and staging were carried out by 2 independent senior oncologists blinded to the data. Colorectal cancer tissues and adjacent non-cancer tissues were collected and stored at −80°C until further uses.

Cells at a density of 5 × 10³ per well were seeded in 96-well plates, treated for 24 or 48 h, and then the viability of treated cells was determined with the MTT assay according to the supplier’s instructions.

The cells were seeded in a 6-well plate at a density of 600 cells/well for 24 h, and then stimulated with drugs for 8 h. After the treatment, incubation was continued for 14 days in a complete culture medium at 37°C and 5% humidified CO₂. At the end of incubation, the cells were washed twice with PBS, fixed with methanol for 15 min, and stained with 0.5% crystal violet for 15 min at room temperature. A colony is defined as accumulation of at least 50 cells. The visible colonies were counted, and the colony formation rate was calculated with the following equation: Colony   formation   rate = ( number   of   colonies / number   of   inoculated   cells ) × 100 % .

LDH assay was used to detect cytotoxicity following different treatments, using a LDH assay kit. Studies were performed following the instructions provided by the manufacturer.

After drug treatment, the cells were washed with PBS, and digested with trypsin. After the cells were collected, they were washed with PBS for 2 times and centrifuged. The cells were resuspended with Annexin Ⅴ-FITC binding buffer; Add Annexin Ⅴ-FITC to the cell suspension and incubate for 15 min in the dark The PI was added, and the cells were filtered and detected by flow cytometry. Data were analyzed with the CytExpert software (Beckman Coulte, United States).

Wash cells with precooled PBS. RIPA cell lysate is used to lyse cells. After the protein was heated and denaturated, 15–30 μg of protein was taken from each sample for protein electrophoresis. After membrane transfer, the PVDF membrane was sealed with 5% skimmed milk at room temperature for 60 min. Then, they were incubated overnight in a buffer containing the antibodies at 4°C. Wash 3 times with PBST (PBS with 0.1% Tween-20), and the corresponding HRP-linked Antibody was selected and incubated at room temperature for 1 h. After washing again with PBS for 3 times, drop ECL chemiluminescence solution. Photographs were taken in a chemiluminescence imager. Densitometry analysis was performed with Image J software (NIH, United States).

Cells at 5 × 10³ per well were seeded in 96-well plates, treated with different concentrations of CPT for 48 h. The cells were washed twice with warm PBS and incubated with JC-1 (2 µM final concentration) for 30 min in the dark. After two more washes with PBS, images were captured with an fluorescence microscope. Densitometry analysis was performed with Image J software (NIH, United States).

Cells were cultured in a 24-well plate at a density of 5–10 × 104 per well overnight; treatments were then performed as described in triplicate for 48 h. Then the supernatant was aspired, cells were rinsed with PBS for three times, and then 0.5 ml of 0.1 mg/ml acridine orange working solution was added to each well. Cells were incubated in a 5% CO₂, 37°C constant temperature incubator for 15 min (avoid light), and then the supernatant was aspired, pictures were taken with an inverted fluorescence microscope after three washes with PBS.

Continuous sections were acquired from formalin fixed, paraffin-embedded tumor tissues, dewaxed in xylene, rehydrated in gradient ethanol, immersed in deionized water, and heated in 0.01 M sodium citrate antigen-repair buffer in microwave oven for 15 min. The Sections were then treated with 3% hydrogen peroxide in methanol (endogenous peroxidase blocker) for 30 min, and then blocked with 5% bovine serum albumin (BSA) to block non-specific binding for 15 min. Place in PBS buffer for 10 min and repeat 3 times. Then the sections were incubated with primary antibody (BIP [1:200], LC3B [1:100], cleaved caspase-3 [1:250]) overnight at 4°C. After incubation with a secondary antibody, 3, 3’-diaminobenzidine tetrachloride was used to visualize the stainings. After counterstaining with hematoxylin, sections were dehydrated in ethanol, cleared in xylene, and sealed with resin. The sections were photographed under a microscope and analyzed with Image J software (NIH, United States).

At the night before injection, female and male zebrafishes were matched at a ratio of 1:2 and placed in the mating aquarium; 2 h after fertilization, the zebrafishes were treated with PTU (1-phenyl-2-thiourea, 0.2 mM) for lucidification. HCT116 and SW620 cells were labeled with DiI. 48 2 hpf zebrafish were anesthetized with 0.003% tricaine. Then microinjections of the cell suspensions into the yolk sac was performed on 48 2 hpf zebrafish embryos with Nanoject II automatic booster syringe. Carbon dioxide injection was used to ensure the 5 nL injection volume. After receiving corresponding treatments, the animals were kept for 3 days. At 0 and 3 days after injection, animals were anesthetized with 70% ethanol, and then subjected to fluorescence microscopy. All experiments were approved by the Animal Research Ethics Committee of the University of Qingdao.

Data were expressed as the means ± SD from three independent experiments. Statistics were performed with Prism 5.0 statistical analysis software. After normality tests, the mean differences of groups were assessed with one way analysis of variance (one-way ANOVA), followed by post hoc Student Newman-Keuls test. All statistical tests were two-sided, and p < 0.05 was considered to be significant. Calculated p-values of p < 0.05, p < 0.01, and p < 0.001 were as indicated.

The incidence of colorectal cancer is among the top three worldwide (Siegel et al., 2020a; Siegel et al., 2020). CPT exhibited potent cytotoxicity on a series of CRC cells while underlying mechanisms are unclear. In the current study, three main types of CRC cells (HCT116, SW620, and SW480) were treated with CPT. As shown in Figures 1A–C, CPT treatment significantly inhibited SW620 and HCT116 cells proliferation and growth in a dose and time- dependent manner (optimum stimulation time:48 h; stimulation dose:10 μM), while no remarkable cytotoxicity were observed on SW480 cells. Additionally, the results of LDH assay and colony-forming assay in both HCT116 and SW620 cells further indicated that CPT treatment selectively induced cytotoxicity in HCT116 and SW620 cells (Figures 1D–F).

To further verify the data from in vitro experiments, xenograft zebrafish model was established to simulate the internal environment to evaluate the anti-tumor effects of CPT. As shown in Figure 2, the growth of the tumor formed with either HCT116 (Figure 2A) or SW620 (Figure 2B) was significantly inhibited after 3 days of CPT treatment at a concentration of 50, 100 nM, which was consistent with the data from in vitro experiments.

Exact mechanisms of CPT-induced CRC cell death remain unknown, and were further explored in the current study. To examine whether apoptosis is associated with the anti-cancer effect of CPT, we used JC-1 staining to detect changes in mitochondria, Western blotting analysis to detect apoptosis-related proteins, and flow cytometry to evaluate the apoptotic ratio using flow cytometry. As shown in Figures 3A–G, CPT treatment significantly induced apoptosis in CRC cells. Increased cleaved caspase-3 expression levels were observed in CPT-treated CRC cells and maximized at 48 h in HCT116 cells and at 12 h in SW620 cells (Figures 3A,B). Besides, based on JC-1 assay, much more green fluorescence was observed in CPT-treated cells and green/red ratio were remarkably increased in cells treated with CPT, indicating severe mitochondrial destruction (Figures 3C,D). IHC was used to detect clinical samples, and the results showed that the expression of cleaved caspase3 in colorectal cancer tissues was significantly lower than that in non-cancerous tissues (Figure 3F). Collectively, CPT inhibited CRC growth by inducing apoptosis accompanied with mitochondrial dysfunction.

Growing evidence has indicated that autophagy is closely linked with apoptosis induction (Jhou et al., 2020). Meanwhile CPT is considered as an important inducer of autophagy (Xu et al., 2017). In the current study, expression levels of autophagy-related proteins were increased with CPT treatment in a time- and concentration-dependent manner in HCT116 cells but not in SW620 cells, suggesting that CPT induced autophagy to regulate HCT116 cells viability (Figures 4A–C). To further explore the role of autophagy in the biological process of HCT116 cells, impacts of autophagy inhibitor treatment on CPT-induced cytotoxicity was tested on HCT116 cells (Figures 5A,B). As shown in Figure 5C–E, autophagy inhibitor Spautin-1 effectively ameliorated CPT-induced cytotoxicity in HCT116 cells. In addition, our study also confirmed that spautin-1 can effectively inhibit CPT induced apoptosis (Figures 5F,G). Therefore, the induction of HCT116 cell death by CPT is likely mediated via autophagy mediated apoptosis. In the clinical samples, the results from IHC staining showed that the expression levels of LC3B were remarkably higher while those of cleaved caspase 3 were significantly lower in CRC tissues relative to matched non-cancerous tissues (Figures 3F, 4D). Above data showed that autophagy was the potential target for CPT in CRC treatment.

It is generally accepted that the generation of endoplasmic reticulum stress plays an important role in apoptosis by changing the internal environment of cells (Alper et al., 2020) (Wu et al., 2020). Recent studies reported that CPT is a crucial ER stress promoter in some colorectal cancers (Wang L. et al., 2020). Therefore, the roles of endoplasmic reticulum stress in growth of HCT116 and SW620 were explored in the current study. First of all, overexpression of ER stress protein was observed in both HCT116 and SW620 cells exposed to CPT (Figures 6A,B), which was further confirmed by the IHC results from human colon cancer tissues (Figure 6I). Next, based on results of MTT assay, LDH assay and plate cloning assay, ER stress inhibitor 4-PBA significantly alleviated CPT-induced damages on HCT116 cells, while no remarkable effects were observed on SW620 cells. Such results further revealed the specific selectivity of CPT on CRC cells and this regulation is likely mediated via ER stress (Figures 6C–G). Furthermore, apoptosis induced by CPT could be abolished by 4-PBA treatment, which indicated that CPT induced ER stress could induce apoptosis on HCT116 cells (Figure 6H).

In the current study, both ER stress and autophagy were shown to be involved in the apoptotic process of HCT116 cells in response to CPT treatment while the association between them still needs to be explored. As shown in Figure 7A,B,D, ER stress inhibitor 4-PBA effectively inhibited the expression of autophagy-related proteins such as Beclin-1 and LC3B in CPT-treated HCT116 cells. However, autophagy inhibitor spautin-1 had little effects on the expression of BIP. We speculated that in HCT116 cells, ER stress is located upstream of autophagy pathway in damaged HCT116 cells (Figure 7C). Our results showed that autophagy induced by CPT could be inhibited by 4-PBA, which was subsequently verified by Acridine orange staining.