To validate the effects of Cu(sal)(phen) on CRC, two human primary adenocarcinoma colon cancer cell lines, SW480 and HCT116, whose tumor biology has been extensively studied in the literature (29), were selected for use in the present study. Since these cells are derived from two different patients with CRC, the two investigated cell lines exhibit some differences at the genetic level (30), which made the results more reliable to a certain extent. The HCT116 (CCL-247EMT) and SW480 (CCL-228) human CRC cell lines were obtained from The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences and cultured in DMEM containing 10% FBS and 1% penicillin/streptomycin (all purchased from Hyclone; Cytiva) at 37°C in an atmosphere of 5% CO₂. Cu(sal)(phen) and Oxa were provided by Hubei Dinglong Chemical Co., Ltd. and Selleck Chemicals, respectively.

Cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay with CellTiter 96^® AQueous One Solution Cell Proliferation Assay (Promega Corporation). Accordingly, the cells were grown in 96-well plates overnight and then treated with Cu(sal)(phen) and Oxa at various concentrations (1, 2.5, 5, 10, 25 µM) with dimethyl sulfoxide (DMSO) used as the control. Following 48 or 72 h of treatment, MTS was added to the plates and incubated for 3 h at 37°C. Subsequently, the optical density was recorded at 492 nm using a Synergy 2 plate reader (BioTek Instruments, Inc.). The half-maximal inhibitory concentration (IC₅₀) was calculated based on the percentage decrease in the optical density at 492 nm relative to the control group using GraphPad prism 7.0 (Dotmatics).

A colony formation assay was conducted in order to evaluate cell proliferation. Briefly, the cells were seeded in six-well plates at a density of 500 cell/well for 48 h, followed by treatment with 1 and 2.5 µM Cu(sal)(phen) and Oxa for 14 days. The medium was changed every 3 days, and the colonies were fixed with 4% paraformaldehyde for 50 min at room temperature. Finally, 0.5% crystal violet dye (Sinopharm Chemical Reagent Co., Ltd.) for 50 min was used to stain cell colonies, and the colonies were counted using the Fiji (ImageJ 2.1.0) software (National Institutes of Health).

An Annexin V-FITC/PI Apoptosis Assay kit (Beijing Zoman Biotechnology Co., Ltd.) was used to examine cell apoptosis after HCT116 and SW480 cells were treated with Cu(sal)(phen) (25 µM) or Oxa (25 µM) with or without 20 µM carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-FMK; GlpBio) solution in DMSO at 37°C. In brief, the cells were collected (200 × g, 5 min, 4°C) following treatment and staining with Annexin V-FITC and propidium iodide (PI) for 15 min in the dark. The samples were then measured using a BD Accuri™ C6 Flow Cytometer (BD Biosciences), and the data were analyzed using FlowJo 10.6.2 software (BD Biosciences).

A Reactive Oxygen Species Assay kit (Beyotime Institute of Biotechnology) was used to detect ROS accumulation. According to the manufacturer's instructions, the cells were pre-treated with or without N-acetylcysteine (NAC; 5 mM, Selleck Chemicals) or glutathione (GSH; 1 mM, GlpBio) for 1 h at 37°C prior to the addition of Cu(sal)(phen) and harvested (200 × g, 5 min, 4°C), followed by staining with 2′,7′-dichlorofluorescein diacetate (DCFH-DA) for 20 min at 37°C. Samples were detected, and the data were analyzed using a BD Accuri™ C6 Flow Cytometer (BD Biosciences) and FlowJo 10.6.2 software (BD Biosciences), respectively.

The depolarization of Δψm was measured using a Mitochondrial Membrane Potential Assay kit with JC-1 (Bestbio). According to the manufacturer's instructions, the treated and collected cells were stained with JC-1 for 30 min at 37°C and observed using a flow cytometer (BD Biosciences). Finally, the data were gathered and analyzed using FlowJo 10.6.2 software (BD Biosciences).

Cells treated with Cu(sal)(phen) and Oxa were lysed with RIPA lysis buffer (Beyotime Institute of Biotechnology) supplemented with complete protease inhibitor cocktail (Roche Applied Science) and phenylmethane sulfonyl fluoride. The protein concentration was determined using the BCA method. Following heating for 10 min; at 95°C in loading buffer, 25 µg proteins were separated by 10% SDS-PAGE and transferred onto a PVDF membrane blocked with a blocking buffer [Odyssey Blocking Buffer (PBS); LI-COR Biosciences] for 1 h at room temperature. Western blot analysis was performed using primary and secondary antibody incubation. Subsequently, blots were acquired using Odyssey SA (LI-COR Biosciences) and images analyzed using Image Studio ver 5.2 software (LI-COR Biosciences). Primary antibody incubation was carried out overnight at 4°C. The following primary antibodies were used: Anti-Bcl-2 (1:1,000, cat. no. ab32124; Abcam), anti-survivin (1:500, cat. no. 2808S; Cell Signaling Technology, Inc.), anti-cleaved poly (ADP-ribose) polymerase (PARP; 1:300, cat. no. 9541S; Cell Signaling Technology, Inc.), anti-γ-H2A histone family member X (γ-H2AX; 1:5,000, cat. no. ab81299; Abcam), anti-caspase-3 (1:1,000, cat. no. 9662s; Cell Signaling Technology, Inc.), anti-cleaved casepase-3 (1:1,000, cat. no. 9664; Cell Signaling Technology, Inc.), anti-p-JAK2 (1:1,000, cat. no. 3771s; Cell Signaling Technology, Inc.), anti-p-STAT3 (1:500, cat. no. ab76315; Abcam), anti-p-STAT5 (1:1,000, cat. no. 68000-1-Ig; ProteinTech Group, Inc.) and anti-actin (1:2,000, cat. no. EM21002; Huabio). The following secondary antibodies were used (incubation for 1 h at room temperature): Odyssey Blocking Buffer (PBS), fluorescently-labeled goat anti-rabbit (cat. no. 926-32211) or goat anti-mouse antibody (cat. no. 926-32210) (both 1:10,000; LI-COR Biosciences).

All animal experiments were carried out in accordance with the protocols of the Institutional Animal Care and Use Committee of Jianghan University. The animal experiments followed the rules of the Animal Ethics Committee of Jianghan University (Wuhan, China) and were performed in accordance with relevant guidelines and regulations, including the ARRIVE guidelines (ethics permission no. JHDXLL:2019-001). BALB/c-nu female mice [5 weeks old; specific pathogen-free (SPF); Beijing, n=25) Biotechnology Co., Ltd.] were domesticated for 1 week and then used to construct a xenograft tumor model. All mice were maintained in a specific pathogen-free state, with a regulated 12-h light/dark cycle, and constant temperature (22±2°C) and relative humidity (50±10%). HCT116 cells (5×10⁶) were suspended in 100 µl medium containing 50% Matrigel and injected subcutaneously into the right flanks of the mice. After the tumors grew to ~100 mm³, the mice selected according to the criteria (n=21, tumor volume ~100 mm³; 4 mice were excluded as their tumor size exceeded the standard deviation of mouse tumor volume by 3-fold) were randomly divided into three groups (n=7) and intraperitoneally administered either 70% DMSO solution in saline (control solution) as the vehicle control, Cu(sal)(phen) (5 mg/kg in control solution) or Oxa (5 mg, in saline) every other day. The mouse tumor volumes and body weights were monitored every other day for 18 days. Tumor volumes were calculated based on the following formula: V=0.5 × l × w², where l is the length (mm), w is the width (mm), and V is the volume of tumor. The mice were sacrificed with isoflurane. Briefly, the mice were placed in a plexiglass chamber with 5% isoflurane until respiration ceased. The death of the mice was confirmed by a lack of active paw reflex and no heartbeat. The tumor weights were recorded and the tumor tissues were fixed in Bouin's (100%) fixative solution (Phygene) for 48 h at room temperature. The tumor tissues were then embedded into paraffin blocks. After sectioning the tissue blocks into 4-µm-thick slices, the sections were stained with a hematoxylin and eosin (H&E) staining solution (Wuhan POWERFUL Biotechnology Co., Ltd.; http://boerfu.net/) for 4 min 15 sec at room temperature. An upright microscope (ECLIPSE Ni-U, Nikon Corporation) was used to observed the sections and the images were captured and analyzed at ×200 magnification using NIS-Elements D software (5.3.00, Nikon Corporation).

For immunohistochemistry (IHC), the aforementioned tissue sections (4-µm-thick) were deparaffinized with xylene, rehydrated with a decreasing ethanol series, and then washed three times with distilled water for 5 min at room temperature. Subsequently, these sections were blocked using 3% BSA (cat. no. A8010, Beijing Solarbio Science & Technology Co., Ltd.) for 30 min at room temperature. The slides were then incubated with anti-Ki67 (1:1,000, cat. no. ab32124; Abcam), anti-Bcl-2 (1:100, cat. no. ab32124; Abcam) and anti-survivin (1:400, cat. no. 2808S; Cell Signaling Technology, Inc.) antibodies overnight at 4°C. The sections were then stained with DAB (cat. no. K3468, Dako; Agilent Technologies, Inc.) at room temperature and the color development time was controlled using a microscope by the positive brownish yellow color and the section was washed with tap water to terminate the color development. Subsequently, the nuclei were counterstained with hematoxylin (cat. no. BH0001, Wuhan POWERFUL Biotechnology Co., Ltd.; http://boerfu.net/) at room temperature for 3 min. Finally, the sections were dehydrated with increasing concentrations of ethanol and xylene. A PANNORAMIC Digital Slide Scanner (3D Histech) were used to scan the sections. The images were analyzed using the Fiji (ImageJ 2.1.0) (National Institutes of Health) and CaseViewer (2.2, 3D Histech) software.

Furthermore, humane endpoints were set when the tumor diameter >20 mm, tumor necrosis, ulceration, or tumor infection. Mice that reached the humane endpoint were euthanized. No mice were sacrificed due to reaching the humane endpoints.

Results from three independent experiments were presented as the mean ± SEM. One- or two-way ANOVA (Tukey's multiple comparisons test) were used to compare the differences among groups. Statistical differences were evaluated using GraphPad prism 7.0 (GraphPad Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.

To evaluate the potential effect of Cu(sal)(phen) on CRC cells, an MTS assay was conducted with various concentrations of Cu(sal)(phen). Oxa was used as a positive control. Cu(sal)(phen) displayed a potent ability to inhibit cell viability in a concentration- and time-dependent manner in the HCT116 and SW480 cell lines (Fig. 1A). Oxa exhibited a moderate inhibitory effect on the viability of HCT116 cells, even though it is one of the most commonly used chemotherapeutic drugs for the clinical treatment of CRC (3,7). Furthermore, Oxa had a minimal inhibitory effect on the SW480 cell line, confirming that it was indeed an Oxa-resistant cell line, as previously reported (31). However, the SW480 cells were sensitive to Cu(sal)(phen) treatment (Fig. 1A). The IC₅₀ values of Cu(sal)(phen) at 48 and 72 h were 4.28 and 3.48 µM, respectively, while the IC₅₀ values of Oxa at 48 and 72 h were >100 and 66.55 µM, respectively. These findings suggested that Cu(sal)(phen) was more effective than Oxa in inhibiting CRC cell viability.

To further validate the inhibitory effects of Cu(sal)(phen) on CRC cell viability, a colony formation assay was performed. As shown in Fig. 1B, the proliferation was markedly lower than that of the control groups in the Cu(sal)(phen)-treated HCT116 and SW480 cells. Oxa treatment exerted similar effects as Cu(sal)(phen) treatment. The aforementioned results demonstrated that Cu(sal)(phen) effectively inhibited CRC cell growth in vitro.

Since Cu(sal)(phen) was more effective than Oxa in decreasing cell viability, the role of Cu(sal)(phen) in inducing cell apoptosis was then investigated. The HCT116 and SW480 cells were treated with various concentrations of Cu(sal)(phen) or Oxa for 24 h, and cell apoptosis was then measured using an apoptosis assay. As shown in Fig. S1, Cu(sal)(phen) effectively induced the apoptosis of the two cell lines in a concentration-dependent manner. At a concentration of 25 µM, the apoptotic rate was as high as 26.4% (HCT116 cells) and 25.8% (SW480 cells). However, the apoptotic rate of the two cell lines only remained at ~10% following treatment with 25 µM Oxa (Fig. 2A). Using an apoptosis inhibitor, Z-VAD-FMK, it was found that pre-treatment of the CRC cells with 20 µM Z-VAD-FMK for 1 h prior to the addition of Cu(sal)(phen) significantly attenuated the apoptosis of both the HCT116 and SW480 cells (Fig. 2B and C). These results indicated that Cu(sal)(phen) was more effective than Oxa in promoting the apoptosis of the two selected CRC cell lines at a concentration of 25 µM.

A number of reports on copper (II) complexes have revealed the crucial role of ROS in promoting cell apoptosis (13,20,27,32). In order to elucidate the mechanisms through which Cu(sal)(phen) induces higher levels of apoptosis than Oxa, a ROS generation assay was conducted. The results shown in Fig. 3A revealed a significantly higher generation of ROS in both CRC cell lines treated with Cu(sal)(phen), as compared with the control group. The changes in ROS generation were 5.5- and 2.7-fold in the HCT116 and SW480 cells, respectively. Pre-treatment with the ROS scavenger, NAC (33), significantly reduced ROS generation in both the HCT116 and SW480 cell lines (Fig. 3A). By comparison, ROS production did not differ significantly compared to the control following treatment of the two CRC cell lines with Oxa (Fig. S2A). To determine the association between ROS production and cell apoptosis, NAC was added to the CRC cells to reassess Cu(sal)(phen)-mediated apoptosis. As was expected, the addition of NAC to the Cu(sal)(phen)-treated HCT116 cells led to a decreased level of apoptosis, as compared with the Cu(sal)(phen) group (Fig. 3B). Unexpectedly, the combination of NAC and Cu(sal)(phen) markedly increased SW480 cell apoptosis (Fig. S2B). Another antioxidant, GSH, was used to determine Cu(sal)(phen)-induced ROS production and apoptosis. As shown in Fig. 3A and C, GSH attenuated Cu(sal)(phen)-induced ROS generation and SW480 cell apoptosis. It was thus found that Cu(sal)(phen) initiated apoptosis through ROS production.

Mitochondria are considered to be a major source of ROS (17,18,34), which is closely associated with cell apoptosis. In addition, cell apoptosis is often accompanied by Δψm depolarization. For that reason, in the present study, a Δψm assay was performed, and there was a notable decrease in Δψm in the HCT116 and SW480 cells following treatment of the two cell lines with 25 µM Cu(sal)(phen) (Fig. 3D). Pretreatment with NAC partially reversed Δψm reduction in Cu(sal)(phen)-treated HCT116 cells. In addition, GSH treatment also reversed the reduction in Δψm in the Cu(sal)(phen)-treated SW480 cells (Fig. 3D). In line with the results of ROS assay, the Δψm of the two cell lines treated with Oxa was not markedly altered compared with the controls (Fig. S3). Thus, as demonstrated by the results, the Cu(sal)(phen)-induced apoptosis was closely associated with the release of ROS by the mitochondria.

In order to further elucidate the mechanisms of Cu(sal)(phen)-induced cell death, a variety of proteins involved in apoptosis were detected using western blot analysis. Bcl-2 is a member of the Bcl-2 family which confers a survival advantage to cancer cells by maintaining mitochondrial integrity (22,35). Bcl-2 expression in the HCT116 and SW480 cells was downregulated following treatment with Cu(sal)(phen) at 25 µM compared with the control group, as shown in Fig. 4A. However, Bcl-2 expression in the HCT116 cells was not decreased in the Oxa group. In the SW480 cells treated with Oxa, Bcl-2 expression only decreased to a level comparable to that of Cu(sal)(phen). Unlike Bcl-2, survivin belongs to the IAP family; it is overexpressed in cancer cells, but rarely in normal adult tissues and plays a crucial anti-apoptotic role in cancer cells (24,36). Survivin expression was downregulated by Cu(sal)(phen) at a concentration of 25 µM, but not by Oxa in the two CRC cell lines, as compared with the control (Fig. 4A and B).

PARP is a substrate of caspases, and its product, cleaved PARP, is generally used to detect cancer cell apoptosis (37). The levels of cleaved PARP were increased in both CRC cell lines in the Cu(sal)(phen) group (Fig. 4A and B). Consistent with this observation, the levels of cleaved caspase-3 were increased in both cell lines following treatment with Cu(sal)(phen). In order to elucidate the association between Cu(sal)(phen) and apoptosis, the level of γ-H2AX, a biomarker of DNA double-strand breaks (DSBs) often caused by ROS (38), was analyzed. γ-H2AX expression was significantly increased in both the HCT116 and SW480 cells following treatment with Cu(sal)(phen) compared with the control. As a drug which directly acts on DNA, Oxa was found to upregulate γ-H2AX expression in the two CRC cell lines; however, its effect was less prominent compared with that of Cu(sal)(phen).

The JAK2/STATs signaling pathway (33) is involved in the regulation of apoptosis through Bcl-2 in cancer cells. Therefore, the present study examined whether Cu(sal)(phen) can affect the JAK2/STAT5 pathway in CRC cells. As shown in Fig. 4C and D, the expression of p-STAT5 and p-JAK2 was significantly inhibited following treatment of the HCT116 and SW480 cells with 25 µM Cu(sal)(phen), while the expression of p-STAT5 and p-JAK2 remained unaltered following treatment with 25 µM Oxa. The level of p-STAT3 remained unaltered following treatment of the two CRC cell lines with Cu(sal)(phen) or Oxa. These results suggested that Cu(sal)(phen) induces CRC cell apoptosis by downregulating the JAK2/STAT5 pathway.

Due to the results obtained from the in vitro experiments, the antitumor effect of Cu(sal)(phen) was investigated in vivo using a HCT116 cell xenograft model. The tumor volumes and body weights of the mice were recorded following the first administration of the drugs and until the mice were euthanized. The tumor growth curves illustrated in Fig. 5A suggested that Cu(sal)(phen) and Oxa markedly attenuated tumor growth in the mice following 18 days of treatment, as compared with the control group. Of note, the antitumor effects of Cu(sal)(phen) were almost comparable to those of Oxa. The results were further supported by the images and weights of the tumors (Fig. 5C and D). There was no statistically significant difference in body weight among the three groups of mice (Fig. 5B). This suggested that Cu(sal)(phen) can effectively suppress HCT116 cell xenograft tumor growth without any evident toxicity in vivo.

To investigate the possible mechanisms of the antitumor effects of Cu(sal)(phen) in vivo, H&E staining and IHC of the tumors were performed. H&E staining revealed evident necrotic areas in the tumors from the drug-treated group that were not observed in the control tumors (Fig. 5E). Consistent with the results obtained in vitro, the levels of the two apoptotic proteins, Bcl-2 and survivin, were decreased, and the level of the proliferation marker, Ki-67, was decreased (Fig. 5F and G). Collectively, Cu(sal)(phen) inhibited tumor growth in the HCT116 cell xenograft model, and the mechanism of the antitumor activity appeared to involve the downregulation of Bcl-2 and survivin.