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. 2025 Aug 18;10(34):38675–38685. doi: 10.1021/acsomega.5c03715

Phosphorus(V) Tris(ethoxycarbonyl)corrole As a Safe and Effective Photosensitizer for Hepatocellular Carcinoma Treatment in Zebrafish Xenograft Models

Yan Liu †,‡,*, Liangliang Wang †,‡, Hao Lu §, Rongrong Chen †, Kangdi Zheng ∥, Zhao Zhang †,∥, Haiying Zhan §,*, Haitao Zhang †,*
PMCID: PMC12409558  PMID: 40918364

Abstract

Corrole-based photosensitizers show great potential for tumor photodynamic therapy (PDT). While their photodynamic activity has been extensively studied at the cellular level, evaluation in mouse xenograft models remains challenging due to prolonged experimental timelines, complex drug administration, and high costs. To address these limitations, we developed a novel in vivo hepatocellular carcinoma model using wild-type AB zebrafish embryos as a xenograft platform. This model was employed to assess the antitumor efficacy, acute toxicity, and biodistribution of phosphorus­(V) tris­(ethoxycarbonyl)­corrole (1-P), an electron-deficient photosensitizer. Under red-light irradiation, 1-P exhibited strong phototoxicity in zebrafish, inducing apoptosis in xenografted tumor cells. Biodistribution studies revealed 1-P accumulation in the liver and digestive tract, demonstrating favorable tumor-targeting properties. Mechanistic investigations via qPCR indicated that 1-P-mediated PDT activated the c-JUN N-terminal kinase pathway, upregulated SIRT1 expression, and suppressed tumor cell proliferation. This work not only supports the therapeutic potential of corroles in hepatocellular carcinoma PDT but also establishes zebrafish as an efficient model for photosensitizer screening and mechanistic analysis, offering significant translational and research value.

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1. Introduction

Hepatocellular carcinoma (HCC) is one of the major cancers with a high mortality rate in globally, which is prone to develop resistance to anticancer drugs, resulting in poor efficacy of systemic chemotherapy. , Thus, there remains a need for optimization of novel treatment strategies. Photodynamic therapy (PDT) is a noninvasive treatment for tumors wherein cytotoxic reactive oxygen species (ROS) are generated via photosensitizer (PS) irradiation, showing the advantages of trauma reduction, low toxicity to surrounding tissues, and high selectivity. − With regard to the PSs used in PDT, corrole, a porphyrin analogue, is able to form more stable complexes with high-valence metals, exhibiting wider prospective applications.

Liu’s research group has been investigating corrole PSs for clinical applications in tumor treatment. In 2003, we first reported that monohydroxycorrole is a potential PS in PDT for nasopharyngeal carcinoma (NPC) and its ability to induce cell apoptosis in NPC was found to be associated with the activation of the mitochondrial apoptosis pathway under light conditions. , In 2016, the photocytotoxicity of cationic metal corrole complexes was initially reported. Following this, three types of representative corroles and their metal complexes, including water-soluble, amphiphilic, and lipid-soluble corroles, were designed, synthesized, and characterized. Additionally, many studies have been undertaken to assess the photodynamic anticancer activities of different types of corroles on various tumor cells, and the photodynamic anticancer activities of the same type of corrole complexes with different metal coordination. − Although many studies have been conducted regarding the detection of the photodynamic activity of corrole photosensitizers, most of these results were verified at the cellular level. A pioneering work was undertaken in our research in 2017 by employing a mouse xenograft tumor model to assess the photodynamic activity of corrole PSs during the investigation of the molecular mechanism behind the photodynamic anticancer activity of corroles.

We systematically assessed the in vivo toxicity of corrole using a mouse xenograft tumor model. Phosphorus-coordinated corrole compounds may endow the complexes with suitable energy level structures, facilitating the absorption of specific wavelengths of light and enabling efficient electron transitions. This can lead to the highly effective generation of cytotoxic ROS such as singlet oxygen, thereby exerting photodynamic antitumor effects. Additionally, phosphorus coordination can enhance the stability of corrole compounds, making them less prone to degradation in biological systems. This improved stability allows them to maintain their photodynamic activity and achieve better antitumor efficacy. However, we used the intratumoral route for drug administration, considering the experimental conditions and limitations at that time. In addition to the intratumoral administration of drugs in the clinical treatment of liver cancer, the experimental period of using a mouse xenograft model is long when evaluating the photodynamic activity of corrole. Both of these factors contribute to an unfavorable sensitivity analysis of patient-specific corrole PSs in clinical practice. Therefore, zebrafish was introduced as a novel animal model to establish a xenograft model to assess the photodynamic activity and other parameters of phosphorus­(V) tris­(ethoxycarbonyl)­corrole (1-P). Compared with the rodent model, many advantages have been shown in zebrafish xenograft models including simple drug administration, a short tumor transplantation cycle, transparent visualization of embryonic tumors, and no need for immunosuppressive therapy.

Herein, the zebrafish model was employed to assess the safety of 1-P under different light conditions and drug distribution in vivo. The in vivo photodynamic activity evaluation of 1-P was completed in a zebrafish xenograft tumor model in just 1 week, showing remarkable efficiency of the zebrafish model in the evaluation of the photodynamic activity of corrole PSs in vivo. The new model provides a novel approach for sensitivity analysis and screening of photosensitizer drugs. To the best of our knowledge, this study is the first attempt to investigate the safety and antitumor activity of corrole PSs and verify the enrichment of corrole PSs in zebrafish. The outcome of this study is expected to provide a novel method to evaluate the safety and photodynamic activity of corrole PSs and a new vision for the development of corrole PSs for clinical application in tumor treatment.

2. Materials and Methods

2.1. Reagents and Materials

Tricaine, methylene blue, 1-phenyl-2-thiourea (PTU), DMEM medium, PBS, penicillin-streptomycin, fetal bovine serum (FBS), and 0.25% Trypsin-EDTA were purchased from Sigma (St. Louis, USA). Dio and DCFH-DA were purchased from Beyotime (Shanghai, China). All reagents and solvents were commercially available and used directly without further treatment, unless otherwise specified. Phosphorus­(V) tris­(ethoxycarbonyl)­corrole (1-P) was synthesized following the procedure described previously as shown in Figure S1.

2.2. Cell Lines and Culture

Human hepatoma cell lines (HepG2) were provided by ATTC. Cells were maintained at 37 °C in 5% CO2 and cultured in T75 flasks filled with 10 mL of DMEM medium. The medium was supplemented with 10% heat-activated FBS and 1% penicillin-streptomycin. Following this, cells were harvested 0.25% Trypsin-EDTA, resuspended in complete medium and then counted through a Leica DM6000 B optical microscope (Leica Microsystems, Wetzlar, Germany) using a standard Beckman Coulter Z2 hemocytometer (Beckman Coulter, Brea, CA, USA) before plating. Cells used in all experiments were below five passages.

2.3. Zebrafish Line and Maintenance

Zebrafish of the wild-type AB and Tg (−1.7apoa2:GFP) transgenic zebrafish lines were obtained from the China Zebrafish Resource Center and maintained according to European (2010/63/EU and 86/609/EEC) standard procedures. Fish were kept under a 14/10 h (day/night) photoperiod and fed live brine shrimp twice daily. Zebrafish embryos derived from natural mating of adult zebrafish were maintained in E3 water (NaCl 11.7 g, KCl 0.506 g, CaCl2 1.456 g, MgSO4 1.584 g, total dissolved solids (TDS) 287.17 mg/L, pH 7.46). To inhibit melanin formation, 0.003% PTU was added to the bathing solution medium after 10–12 h postfertilization (hpf). The embryos were examined under an SZ680 continuous magnification stereomicroscope (OPTEC, Chongqing, China) at 4 hpf. The embryos that had developed normally to the blastula stage were selected for subsequent experiments. All zebrafish experiments were approved and met the ethical standards of the Institutional Review Board of the Laboratory Animal Ethics Committee, Center of Human Microecology Engineering and Technology of Guangdong Province (approval number: IACUC MC 0130-01-0204).

2.4. 1-P Acute Toxicity and Safety Experiments

The phototoxicity of 1-P on zebrafish embryos was evaluated using a method inspired by the OECD guideline 236 and previously published literature. , Postspawning, healthy zebrafish embryos were selected under a microscope to perform the following experiments. 1-P was prepared into 10 mM mother liquor with DMSO, maintained in dark conditions, and diluted with E3 water (NaCl 11.7 g, KCl 0.506 g, CaCl2 1.456 g, MgSO4 1.584 g, TDS 287.17 mg/L, pH 7.46) at the concentration required for the experiment when used. The embryos were most sensitive to teratogenic agents at the gastrula stage (0–8 hpf after fertilization); therefore, the drug exposure experiment was performed at 3 hpf after collecting fertilized eggs. Zebrafish embryos (wild type; AB line) were selected and randomly divided into 64, 32, 16, 8, 4, 2, and 1 μM 1-P medium in dark intervention groups and 0.1% DMSO as the blank control group; each concentration group consisted of three composite wells with five embryos in each well (N = 15). After intervention, the hatching rate and mortality rate of each group were collected and counted every 24 h for five consecutive days; subsequently, statistical charts for both rates were drawn.

In the safety evaluation experiment of 1-P under red light, wild-type AB zebrafish embryos at 3 hpf were selected and evenly distributed into 6-well plates, with five embryos in each well. Each concentration group was set up with three compound wells (N = 15). A control group was established using 0.1% DMSO. There were eight different concentrations of 1-P solution used as the drug groups, viz. 800, 400, 200, 100, 50, 25, and 12.5 nM, and zebrafish underwent a 96 h intervention in the darkness. At 24, 48, 72, and 96 h, the zebrafish were transferred to E3 feedwater for cleaning, placed in new feedwater, and continuously exposed to red light (625 nm, 5 W, 15.5 cm) for 1 h. The status and number of dead zebrafish were observed and recorded every 15 min during the irradiation period to determine the effect of compound 1-P on zebrafish mortality under light conditions. Moreover, the 50% lethal concentrations (LC50) of 1-P at 96 hpf were calculated under dark and light conditions, respectively.

2.5. 1-P Distribution Experiments in Zebrafish

1-P was prepared into 10 mM mother liquor with DMSO, preserved in dark conditions, and diluted with E3 water at the concentration required for the experiment until used. Tg (−1.7apoa2:GFP) liver green fluorescent transgenic zebrafish treated at 72 hpf were randomly divided into intervention groups of 50, 100, and 200 nM 1-P medium, along with a control group using 0.1% DMSO (N = 3/group). The zebrafish were placed in a 48-well plate, and each concentration was set up in five multiple wells, with one zebrafish larva in each well. The zebrafish models were reared in dark conditions, and the photos of zebrafish models were taken under fluorescence and light field under a fluorescence microscope at 48 hpa. The distribution of 1-P in zebrafish was observed by synthesizing pictures from both light field and fluorescence field images.

2.6. Experiments to Evaluate the Antitumor Efficacy of 1-P

Three concentrations below the LC50 were selected to detect the antitumor effect of 1-P. HepG2 tumor cells were washed with PBS, stained with DiO away from light, incubated in a dark room for 10 min, and washed again with PBS. The cells were digested with 0.25% trypsin, the cell suspension was prepared with PBS, and green fluorescence was observed under a fluorescence microscope. Zebrafish embryos (wild type; AB line) at 2 days postfertilization (dpf) were demembranized with 0.25% trypsin, then treated with PTU until 48 hpf, anesthetized with 0.02% tricaine, placed on AGAR plate, and injected with tumor cells into yolk sac under stereoscopic microscope. Next, 500–1000 cells per embryo were injected and transferred to E3 water. The WPI-PV850 microinjector was used for injection. The cell distribution was observed under fluorescence microscope 1 h after injection. Zebrafish tumor models with uniform distribution and similar number of injected cells were randomly assigned to the 50, 100, or 200 nM 1-P medium intervention groups or the 0.1% DMSO control group and placed on 48-well plates with 10 wells in each group and one zebrafish in each well. 1-P drug intervention and red-light irradiation were performed as described in the schedule in Section . The histomorphological features of the zebrafish xenograft tumors were observed under a microscope (Leica DMi8), and the images were captured and analyzed. Images were analyzed by using FIJI, an image processing package based on ImageJ2. According to the DiI-positive signals (red fluorescence) in zebrafish, the tumor area was selected using the freehand tool in the software, and both the area and intensity were measured for analysis. Finally, the zebrafish in the blank control group and the drug intervention group were fixed in 4% formaldehyde fixative overnight at 4 °C to maintain cell morphology and structure. After washing, dehydration, and embedding in paraffin, pathological sections were prepared. After the sections were stained with hematoxylin and eosin (HE), the histomorphology of xenografted zebrafish tumors in the model group was observed under a microscope and the images were collected for analysis.

2.7. ROS Detection

According to the schedule in Section , 3 dpf zebrafish embryos (wild type, AB line) were exposed to 1-P (200, 100, and 50 nM) and red light irradiation, respectively. Following this, 1-P groups and the control group were incubated with DCFH-DA (5 μM) in the dark for 30 min (N = 15/group). After staining and incubation, the zebrafish were washed with E3 water 3–5 times to remove excess DCFH-DA. The corresponding fluorescence pictures were taken under a fluorescence microscope, and the average fluorescence intensity of each group was analyzed and counted by ImageJ software. The fluorescence intensity was measured in arbitrary units (au).

2.8. Quantitative Real-Time PCR (qPCR)

The experiment set up a control group injected with PBS and a model group injected with HepG2 cells, and the difference analysis between the two groups proved whether the tumor model was successfully constructed. HepG2 cells (500–1000 cells) were injected into the yolk sac of 2 dpf wild-type zebrafish embryos (AB line). According to the schedule in Section , after 1-P drug intervention and red light irradiation, zebrafish larvae were collected for total RNA extraction (N = 20/group). Twenty zebrafish were homogenized in an RNA Easy Fast Tissue Kit (TIANGEN, Beijing, China) using a tissue homogenizer. Total RNA was extracted using RNA Easy Fast Tissue Kit (TIANGEN, Beijing, China) following manufacturer’s protocol. Reverse transcription and qPCR were performed according to the manufacturer’s protocols. cDNA was generated using a S1000 Thermal Cycler (Bio-Rad, California, USA). qPCR was performed using a MA6000 (Molarray, Suzhou, China) and a Bio-Rad CFX96 Real-Time System. Runs were carried out in triplicate using the housekeeping gene β-actin to normalize the mRNA level of the target genes. Three biologically independent experiments were performed by real-time fluorescence quantitative PCR. 2–ΔΔCt was used to analyze the relative expression levels of target genes. The primer sequences are shown in Table S1.

2.9. Statistical Analysis

The statistical analysis of all experiments was performed using SPSS 25.0 software, and the data were expressed as mean ± standard error (mean ± SEM). The significance of survival and hatching rates was determined by the chi-square (χ2) test. One-way analysis of variance (ANOVA) was used to analyze the differences of ROS fluorescence intensity and gene expression levels. p < 0.05 was considered statistically significant.

3. Results

3.1. Acute Toxicity and Safety Evaluation of 1-P under Dark Conditions

The photosensitizer 1-P plays a critical role in PDT by inducing ROS formation under illumination. Therefore, we treated zebrafish embryos with 1-P under different dark and light conditions to investigate the acute toxicity and safety of 1-P (N = 15/group). Under dark conditions, the acute toxicity and safety of 1-P in zebrafish embryos was evaluated for 4 days. As shown in Figure A, zebrafish embryos treated with 1-P (1–16 μM) exhibited no morphological abnormalities in dark conditions. In addition, zebrafish embryos treated with low concentrations of 1-P (1–8 μM) showed survival rates comparable to those of the control group under dark conditions (Figure B). However, exposure of zebrafish embryos to high concentrations of 1-P (16–64 μM) significantly reduced survival rates in the absence of light (p < 0.01). At 48–96 hpf without light, the cumulative hatching rate of the zebrafish embryos treated with 1-P at concentrations of 1–16 μM was not significantly different from that of the control group (Figure C). However, at 48–96 hpf without light, hatching was significantly delayed in the zebrafish embryos exposed to 64 μM 1-P compared with that of the controls (p < 0.01). Moreover, the LC50 obtained was 18.41 μM for 1-P at 96 hpf under dark conditions. These findings suggest that 1–8 μM 1-P induces low or almost no toxic effects on the developmental stages of zebrafish embryos under dark conditions.

1.

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Acute toxicity and safety evaluation of 1-P on zebrafish embryos in the dark. (A) Morphological changes in zebrafish embryos treated with 1-P (0, 1, 2, 4, 8, and 16 μM). (B) The survival rate of zebrafish embryos treated with 1-P (0, 1, 2, 4, 8, 16, 32, and 64 μM) (N = 15). (C) The cumulative hatching rate of zebrafish embryos treated with 1-P (0, 1, 2, 4, 8, 16, and 32 μM) (N = 15). The experiment was repeated in three independent experiments. Data are expressed as mean ± SEM. Statistical significance was assessed using chi-square (χ2). Values versus the control group: **p < 0.01, ***p < 0.001.

3.2. Acute Toxicity and Safety Evaluation of 1-P under Red-Light Irradiation

In this experiment, the zebrafish embryos were treated with 1-P under red-light irradiation for 1 h/day for 4 days to evaluate the acute toxicity and safety of 1-P under light conditions (N = 15/group). At 200 nM 1-P, zebrafish embryos exhibited normal development, indicating that no obvious toxic effect was observed in the presence of 200 nM corrole on the zebrafish embryos (Figure A). The survival rate of zebrafish embryos treated with 1-P at concentrations of 400 and 800 nM was significantly reduced compared to the control group under red-light irradiation (Figure B) (p < 0.001). Moreover, the LC50 of 1-P was determined to be 285.71 nM at 96 hpf under red-light irradiation. Therefore, 200 nM was selected as the experimental therapeutic concentration to evaluate the antitumor activity of the drugs.

2.

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Safety evaluation of 1-P on zebrafish embryos under red-light irradiation. (A) Morphological changes of zebrafish embryos treated with 1-P (0, 12.5, 25, 50, 100, and 200 nM). Mortality of zebrafish embryos treated with 1-P (0, 12.5, 25, 50, 100, 200, 400, and 800 nM) (N = 15) for 24 h (B), 48 h (C), 72 h (D), and 96 h (E) after exposure to red light for 1 h each day. Statistical significance was assessed using chi-square (χ2). Values versus the control group: **p < 0.01, ***p < 0.001.

3.3. Distribution of 1-P in Zebrafish

A drug distribution experiment was performed to determine the distribution of 1-P at therapeutic concentrations in zebrafish (N = 3/group). As depicted in Figure A, the red area indicated by an arrow shows the distribution of 1-P in zebrafish. The green area indicated by an arrow in Figure B is the location of the zebrafish liver labeled with green fluorescence. Moreover, as shown in Figure C, which is a composite of Figure A and Figure B, the yellow area indicated by an arrow is the site where the distribution of 1-P and zebrafish liver overlap. Hence, 1-P was enriched in the zebrafish liver and served as a tumor tissue-targeting anticancer PS in PDT.

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Distribution of 1-P in zebrafish. (A) The red area is the distribution of 1-P in zebrafish. (B) The green area is the liver location of the Tg (−1.7apoa2:GFP) liver green fluorescein-labeled transgenic zebrafish. (C) The composites of (A) and (B) (N = 3/group).

3.4. Antitumor Activity of 1-P in Zebrafish Ectopic Tumor Models

To examine the antitumor activity of 1-P in zebrafish, we established the zebrafish xenograft liver cancer model and treated it with 1-P under red-light irradiation as described in the schedule of zebrafish embryonic tumor injection and 1-P administration shown in Figure A (N = 15/group). As illustrated in Figure B, the tumor fluorescence intensity was significantly reduced in zebrafish in the 1-P PDT group compared to that in the control group, and this decrease positively correlated with the concentration of 1-P. As shown in Figure C, the average fluorescence intensity of tumors in the control group was 2043.86 ± 274.25 au after 72 h, whereas that of tumors in the PDT group was 1088.00 ± 229.38 and 844.18 ± 147.80 au in the presence of 100 and 200 nM 1-P, respectively. These values were significantly different from those in the control group (p < 0.01). Furthermore, the fluorescence area in the control group considerably increased with time, and after 1-P PDT, significant reductions of the tumor area were observed in the groups treated with 100 and 200 nM 1-P (Figure D, p < 0.001). These results have shown the excellent antitumor ability of 1-P in inhibiting the growth of zebrafish liver tumors, suggesting good potential for clinical application owing to its low therapeutic dose and high phototoxicity compared with those of other antitumor drugs.

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Growth inhibition of HepG2 cells in zebrafish liver cancer xenograft models inhibited by 1-P. (A) 1-P intervention, red-light irradiation treatment, and data collection schedule. (B) The proliferation of HepG2 cells (the cell membrane was stained with green fluorescence) in zebrafish after treatment with 1-P (0, 50, 100, and 200 nM) for 72 h (N = 15/group). (C) Tumor fluorescence intensity and (D) tumor area of zebrafish liver cancer xenograft models with different concentrations of 1-P. Data were plotted as means ± SEM. Statistical significance was assessed using one-way ANOVA. Values versus the control group: *p < 0.05, **p < 0.01, ***p < 0.001, ns: not significant.

3.5. Photodynamic Therapy Triggers ROS Elevation and Tumor Cell Cytomorphological Remodeling in Zebrafish Xenograft Models

We evaluated the level of ROS produced by 1-P in zebrafish embryos under light conditions in both the control and 1-P PDT groups (N = 15/group). Figure A shows a typical fluorescence micrograph of the zebrafish. The control group generated a clear image, whereas 1-P groups (50, 100, and 200 nM) generated a fluorescence image, suggesting that 1-P induced ROS production in zebrafish under red light irradiation. Moreover, the fluorescence intensity in the zebrafish treated with 0.1% DMSO (control) was 663.16 ± 30.48 au, whereas that for the zebrafish treated with 50, 100, and 200 nM of 1-P was 1552.48 ± 205.73, 5395.51 ± 426.19, and 8653.63 ± 368.46 au (Figure B), respectively. The ROS levels in zebrafish embryos exposed to 1-P PDT were significantly higher than those in the control group, and the distribution was coincided with the distribution of 1-P in zebrafish (p < 0.01). In summary, 1-P produced ROS in zebrafish embryos under light conditions, inhibiting tumor cell growth in zebrafish. In addition, the ROS levels in vivo were positively correlated with the 1-P concentration.

5.

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Photodynamic therapy with 1-P significantly increased reactive oxygen species (ROS) levels and induced distinct cellular morphological changes in zebrafish models. (A) Fluorescence imaging of ROS in zebrafish after treatment with 200, 100, and 50 nM 1-P PDT. Fluorescence imaging of ROS in zebrafish of the control group (N = 15). (B) Changes of ROS fluorescence intensity in zebrafish in each group. Statistical significance was assessed using one-way ANOVA. Values versus the control group: **p < 0.01, ***p < 0.001. (C) HE staining of zebrafish tumor model in the 200 nM 1-P PDT group. (D) HE staining of zebrafish tumor model in the control group. The red squares in the figure indicates the morphology of the tumor cells in the zebrafish, and the black squares indicates the morphology of the zebrafish caudal fin tissue.

After 1-P PDT, apoptosis of zebrafish tumor cells was observed by light microscopy with HE staining. As shown in Figures C and D, the zebrafish in the 1-P PDT group showed tumor apoptosis (nuclear shrinkage and dark color), while normal morphology and proliferation of tumor cells were observed in zebrafish in the control group. The effect of 1-P PDT on other tissues was also observed, revealing that 1-P PDT had no effect on the other tissues of zebrafish.

3.6. Potential Antitumor Mechanism of 1-P

To investigate the potential antitumor mechanism of 1-P in zebrafish, total RNA was extracted and the relative expression of 10 previously reported antitumor-mechanism-related genes following 1-P PDT was detected via real-time fluorescence quantitative PCR analysis (N = 15/group). As shown in Figure , compared with the control group, decreased expression of four genes (SIRT1, JNK, ERK, and p21) in zebrafish was observed after tumor injection (p < 0.05). After 1-P PDT, the expression of these four genes was increased in the treatment group, among which the expression levels of SIRT1, JNK, and p21 were significantly increased in a drug-dose-dependent manner (p < 0.05). Interestingly, a nonsignificant increase in the expression of two genes (p53 and Nrf2) was observed following PDT. No significant differences could be detected in the expression levels of another four genes (caspase-2, caspase-8, caspase-9, and FoxO3).

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Expression of related genes in zebrafish after 1-P photodynamic therapy. The control group was not treated, and the 1-P treatment groups were treated with 0, 50, 100, or 200 nM 1-P for 72 h (N = 15/group). The gene expressions of JNK16 (A), ERK (B), SIRT1 (C), p21 (D), p53 (E), and Nrf2 (F) in different groups of zebrafish were detected. Data were plotted as means ± SEM. Statistical significance was assessed using one-way ANOVA. Values versus the model group: *p < 0.05, **p < 0.01, ***p < 0.001.

4. Discussion

Our previous research has focused on the synthesis of corrole PSs and their metal complexes as well as the verification of the photodynamic activity of corrole PSs at a cellular level. − However, there remain some problems in verifying the photodynamic activity of corrole PSs in animals, such as long experimental periods, difficulty in drug administration, and susceptibility to infection during immunosuppression due to xenograft rejection. Importantly, in our previous works, we have demonstrated the toxicity and antitumor efficacy of 1-P through mouse xenograft models. However, the method of intratumor 1-P injection was limited by the experimental conditions at that time, thereby resulting in difficulties in achieving this drug delivery mode in clinical applications. Here, we investigated the safety and antitumor activity of 1-P using the zebrafish model and feeding 1-P in water to address this deficiency, which is expected to contribute to the transformation and application of corrole PSs in human studies.

In the safety experiment of 1-P in zebrafish, we adjusted the time schedule of corrole PS administration and light irradiation to evaluate the safety of 1-P. The zebrafish model was transferred to the culture water without the drug for red-light irradiation after the drug intervention period, allowing the corrole PS in the zebrafish body to exhibit photodynamic activity and eliminate the influence of PS in the culture water on the drug screening experiment. We found that the safe concentration of 1-P was 8 μM under dark conditions and 200 nM under the red-light irradiation conditions. Following this, the safe concentration of 1-P under red-light irradiation (200 nM) was selected as the therapeutic concentration to explore its antitumor activity. A good antitumor effect was observed with 1-P when exposed to 200 nM, and the tumor fluorescence intensity and fluorescence area in zebrafish were significantly reduced compared to those in the control group. According to relevant studies, the Ruthenium-based photosensitizer TLD1433, which has completed Phase I trials of PDT for bladder cancer, had low cytotoxicity in the dark and extremely high phototoxicity when tested with photoactivation on a wide range of human cancer cells. , The maximum tolerated dose of TLD1433 in zebrafish embryos was only 9.2 nM after light activation. However, zebrafish embryos tolerated light-activated 1-P without any effect on mortality or malformation at a maximum tolerated dose of 200 nM, which was considerably higher than the safe concentration of TLD1433 after light activation. Therefore, these results indicate that 1-P could be further developed as a potential antitumor drug candidate for future research.

In addition to measuring the fluorescence intensity and area of the tumor in the zebrafish tumor model, we designed a HE staining experiment using paraffin-embedded sections of the zebrafish liver cancer model to visually observe the histological characteristics of the transplanted tumors in both the model and treatment groups. Previous tumor treatment methods, such as radiotherapy and chemotherapy, are often accompanied by serious side effects that impact patients’ quality of life. However, the results of HE staining revealed that 1-P exerted a killing effect on tumor cells in zebrafish without affecting the surrounding cells, such as heart and caudal fin cells, demonstrating the potential of 1-P to be developed into a targeted therapeutic agent. Moreover, we investigated the distribution of 1-P in zebrafish. The fluorescence of 1-P in zebrafish and the green-fluorescence-labeled liver of Tg (−1.7apoa2:GFP) zebrafish was observed by concurrent photographing and overlapping. It was found that 1-P was enriched in the liver and digestive tract of zebrafish following drug intervention. After clarifying the distribution of 1-P in zebrafish, we explored the ROS enrichment in zebrafish and found that the level of ROS produced in zebrafish via red-light irradiation following 1-P intervention was positively correlated with the concentration of 1-P. The outcome reaffirms the mechanism of action of PDT, which is in line with previous findings and involves reactions mediated by a photosensitizer, light, and oxygen, ultimately leading to the generation of cytotoxic ROS and the induction of apoptosis. ,

In our previous study regarding the photodynamic mechanism of 1-P in mice, we found that the PDT activity of 1-P can degrade the important deacetylase SIRT1. Herein, considering the small amount of protein in zebrafish larvae, we extracted the total RNA from zebrafish following some modifications based on previous reports. Subsequently, we detected the relative expression of genes related to the previously reported antitumor mechanism following 1-P PDT via quantitative real-time PCR analysis. , Among those genes, the expression levels of SIRT1, JNK, ERK, and p21 decreased following tumor cell transplantation but increased after PDT. Similar results have been reported in relevant studies, showing that the SIRT1 and JNK signaling pathways are involved in inhibiting resveratrol-mediated activation of hepatic stellate cells by regulating autophagy and apoptosis; SIRT1 may be involved in the induction of autophagy, while JNK affects autophagy and apoptosis. In human patients with cerebral ischemia (CI), SIRT1 activation upregulates the phosphorylation of the JNK/ERK/MAPK/AKT signaling pathway and the expression of caspase-3. However, this result differs from our previous findings on the photodynamic mechanism of 1-P in a mouse xenograft model. Garten reported that SIRT1 overexpression may be a potential mechanism for the resistance of liver cancer cells to drugs belonging to the class of multikinase inhibitors. Hao et al. also confirmed that in both liver cancer cells and animal models of liver cancer, increasing the expression level of SIRT1 can induce epithelial–mesenchymal transition (EMT) and promote the metastasis of liver cancer. SIRT1 can also promote the proliferation and metastasis of HCC cells by regulating the initiation of telomerase reverse transcriptase gene expression, promoting YAP/TEAD4 synergy, and stabilizing c-Myc protein. −

Herein, we speculated that 1-P could degrade SIRT1 protein in tumor cells through photodynamic activity, causing changes in cellular gene expression patterns and affecting cell proliferation, differentiation, and death. In zebrafish, the photodynamic activity of 1-P can activate SIRT1 expression, thereby upregulating apoptosis-related gene expression and promoting tumor cell apoptosis in vivo. Therefore, it would be worth comparing the relationship between SIRT1 expression in 1-P-treated tumor cells in zebrafish and the relationship between SIRT1 and the JNK/ERK signaling pathway activation. In addition, SIRT1 is a conserved nicotinamide adenine dinucleotide (NAD+)-dependent histone deacetylase that plays a crucial role in various cellular processes, including gene regulation, cell proliferation, and apoptosis. P53, one of the most extensively studied tumor suppressors, was the first identified nonhistone target of SIRT1. SIRT1 deacetylates p53 at its C-terminal residues in a NAD+-dependent manner, ultimately suppressing p53-mediated transcription-dependent apoptosis. The SIRT1 inhibitor tenovin-1 has been found to enhance cancer cell apoptosis by increasing p53 acetylation levels. The expression level of SIRT1 increased after 1-P PDT, indicating that the effect of 1-P PDT on the growth of HepG2 cells may involve various signaling pathways associated with both pro-cancer and anticancer mechanisms, which warrants further investigation in the future. Interestingly, ROS production in zebrafish led to tumor cell apoptosis under 1-P treatment and light conditions. A large number of studies have shown that ROS can induce apoptosis through caspase-dependent and caspase-independent mitochondrial pathways. ,

Our research results showed that 1-P did not significantly alter the expression of caspase-2, caspase-8, and caspase-9 genes in zebrafish, indicating that 1-P induces apoptosis through a caspase-independent signaling pathway. Many studies have shown that high levels of ROS can alter the mitochondrial permeability transition pore, leading to the translocation of apoptosis-inducing factor (AIF) from mitochondria to the nucleus. , It has been reported that AIF plays a central role in regulating caspase-independent pathways in cells. After being released from mitochondria, AIF is transported to the nucleus and is involved in chromatin condensation and DNA degradation. Hou et al. reported that MeHg had a toxic effect on SH-SY5Y cells, which activated downstream apoptotic signaling pathways through oxidative stress and mitochondrial damage, including the PARP/AIF pathway and the caspase-3 apoptosis complex pathway. However, blocking the caspase-3 pathway failed to inhibit the expression of PARP/AIF protein, indicating that the function of the PARP/AIF pathway was independent of caspase. Therefore, we speculated that 1-P exerted PDT activity by inducing AIF-mediated caspase-independent apoptosis. The mechanisms of AIF-mediated apoptosis induced by 1-P need to be further studied in the future.

5. Conclusion

In summary, the findings from our study revealed that 1-P showed low toxicity under dark conditions and high photodynamic activity under red-light irradiation. Moreover, 1-P effectively suppressed tumor growth in the HepG2 xenograft zebrafish model under red-light irradiation. Furthermore, the antitumor mechanisms might be attributed to ROS induction by 1-P PDT, which triggered the ROS-mediated c-JUN N-terminal kinase pathway and significantly increased SIRT1 expression.

Supplementary Material

ao5c03715_si_001.pdf (105.7KB, pdf)

Acknowledgments

We acknowledge Ziyi Li from Jihua Laboratory for language polishing and technical support provided by the Longseek high-throughput zebrafish screening platform for drug and probiotic evaluation.

All data supporting the findings of this study are available within the article and its Supporting Information. Additional raw data sets generated during the current study are available from the corresponding author upon reasonable request.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c03715.

  • Synthesis route of phosphorus­(V) tris­(ethoxycarbonyl)­corrole (1-P) and primer sequences used in this study (PDF)

∇.

Y.L. and L.-L.W. contributed equally to this work.

This work was supported by the National Natural Science Foundation of China (82003786), the Guangdong Province Basic and Applied Basic Research Fund (2019A1515110338), and the Postdoctoral Initial Foundation of Guangdong Medical University (4SG24185G and 4SG24187G).

Institutional Review Board Statement: The animal study protocol was approved by the Institutional Review Board of the Laboratory Animal Ethics Committee, Center of Human Microecology Engineering and Technology of Guangdong Province (approval number: IACUC MC 0130-01-0204).

The authors declare no competing financial interest.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

ao5c03715_si_001.pdf (105.7KB, pdf)

Data Availability Statement

All data supporting the findings of this study are available within the article and its Supporting Information. Additional raw data sets generated during the current study are available from the corresponding author upon reasonable request.

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