Fisetin Exerts Anti-tumorigenic Activity via Inhibition of Trypsin Activity in Colorectal Cancer

doi:10.21203/rs.3.rs-350861/v1

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Fisetin Exerts Anti-tumorigenic Activity via Inhibition of Trypsin Activity in Colorectal Cancer

https://doi.org/10.21203/rs.3.rs-350861/v1

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Background: Colorectal cancer (CRC) is a malignant tumor with high incidence and bad prognosis. Therapies, which are more safety and effective, are urgent needed in clinical. Trypsin is proved to be crucial to cancer proliferation and migration, therefore, it’s possible to control cancers through modulating the activity of trypsin. Fisetin is a flavone with excellent trypsin inhibition that screened from more than 45 compounds derived from traditional Chinese medicine. However, the effects and the mechanisms of fisetin on CRC have not been well investigated.

Methods: In this study, we firstly evaluated the expression of trypsin on CRC cells. The effects of fisetin on proliferation and migration of CRC cells were also investigated in vitro. The inhibitory effects on cell cycle and apoptosis were assayed using flow cytometry. The therapeutic activity and toxicity were evaluated in vivo.

Results: Fisetin remarkably inhibited CRC cell proliferation and migration, as well as induced cell apoptosis and Go/G1 phase arrest in a dose‐dependent manner. Mechanistic studies revealed these effects were mediated partially through signaling pathways involving cell cycle regulators p21, p27, cyclinD1 and NF kappa B (NF-κB) p65. Administration of fisetin also significantly suppressed the tumor growth in tumor-bearing NOG mice inoculated with human HCT116 cells. Fisetin at the given dosage did not induce significant acute or chronic toxicity in rats.

Conclusions: Fisetin can inhibit the growth and migration of CRC by inhibiting the trypsin both in vitro and in vivo. Our results revealed fisetin is a potent therapy for CRC.

Cancer Biology

Internal Medicine

Translational Medicine

Fisetin

colorectal cancer

NF kappa B signaling pathways

anti-tumor

Colorectal cancer (CRC) is the second most common cancer diagnosed in women and third most in men, and the world’s fourth most deadly cancer with almost 900,000 deaths annually [1]. Unfavorable risk factors, such as abnormal dietary habits, lack of physical exercise, smoking and so on, increase the incidence of CRC [2–4]. Although the clinical treatment to CRC, including prevention, early detection, screening, diagnosis and therapeutic have improved, the prognosis is still poor [5]. Therefore, it is essential to find more effective and safety agent for prevention and treatment of CRC.

Trypsin is a digestive enzyme which linked to the development of neoplasia, invasion and metastasis in several cancers [1, 6, 7]. Trypsin-positivity was significantly related to depth of invasion, lymphatic and venous invasion, lymph node and distant metastasis, and recurrence of CRC [8, 9]. Previous studies have shown that patients with trypsin-positive CRC had significantly shorter overall survival (OS) and disease-free survival (DFS) periods than those with trypsin-negative. Meanwhile, cetuximab resistance was found to be related to the level of trypsin (PRSS1) expression secreted by CRC cells [10]. Ttrypsin (PRSS) inhibited the blocking of cetuximab on epidermal growth factor receptor (EGFR) and diminished the therapeutic effects. But the combination of cetuximab with SPINK1 (a trypsin inhibitor, TI) inhibited tumor growth more efficiently than cetuximab in xenograft model. TIs may reduce the drug resistance and improve the prognosis in patients with CRC.

Recently, many flavonoids have been found to exhibit trypsin inhibitory activity [11]. The naturally occurring flavonoids are the most sought after due to their safety, affordability, and ease of availability [12]. Thus, we have established a new immobilized trypsin reactor based on poly(GMA-co-PEGDA) monolithic column and screened trypsin inhibitors from 45 kinds of flavonoid monomers extracted from traditional Chinese medicine [13, 14]. Fisetin (3, 7, 3′, 4′-tetrahydroxyflavone) has been shown to display the best trypsin inhibitory activity (62%) among these Chinese herbs derived flavonoids. Fisetin is one of dietary flavonoid found in Chinese traditional herbs, such as Cotinus coggygria scop. [15, 16]. With polyphenols structure, fisetin is a potent antioxidant agent and free radical scavenger both in vitro and in vivo [17–19]. Fisetin also showed promising therapeutic effects in several cancers, such as breast, leukemia, liver and prostate [20–22].

In the study, we investigated the anti-tumor via trypsin inhibition pathway of fisetin on two different CRC cells (HCT116 and HT-29), and further evaluated the therapeutic effects in vivo. The underline mechanisms of interaction among fisetin, trypsin, and tumor cells were also explored. Finally, we also evaluated the safety of fisetin on normal rats.

Cell culture

Two CRC cell lines (HCT116 and HT-29), which were purchased from American Tissue Type Culture Collection (Manassas, VA, USA), were maintained in Dulbecco's modified Eagle medium (DMEM, Gibco BRL, Rockville, MD, USA) containing 10% fetal bovine serum (Gibco BRL) and 100 U/ml penicillin/streptomycin at 37 ℃ under 5% CO₂.

Transwell cell migration assays induced by trypsin

The transwell assays were performed according to the manufacturer's instructions. The transwells (Costar, USA) with 8 µm pore polycarbonate membranes (Corning, NY, USA) were used. The 1.5×10⁴ cells were seeded in the transwell upper chamber and incubated with trypsin (100 nM) and indicated concentration of fisetin. After incubated for 24 h at 37°C in a humidified incubator with 5% CO₂, remove the cultural medium and stained cells with crystal violet in the lower chamber. Cell migration was evaluated by counting the cells that had migrated into the filters. Each assay was performed in triplicate and repeated three times.

Cell proliferation assay induced by trypsin

For cell proliferation, the cells were seeded into 96-well culture plates at a density of 2 × 10⁴ cells per well. After cultured for 24 h, trypsin (100 nM), fisetin at different concentrations (30, 60 and 120 µM, final concentration) were added into the medium and incubated. At the indicated time points (0, 24, 48 and 72 h after incubation), 20 µl of 5 mg/ml MTT (Sigma, St. Louis, USA) was added, and the plates were incubated at 37°C for an additional 4 h. Then, the supernatant was removed and the residue was dissolved with 100 µl of dimethyl sulfoxide. The optical density (OD) at 490 nm was determined with a microplate reader. Cell viability was represented by the absorbance.

Three independent experiments were performed and the average data was obtained for analysis.

Cell cycle analysis

Cells (2.5 ml/well) were plated at a density of 1 × 10⁶ cells/ml in 6 well plates followed by treatment with different concentration of fisetin for 48 h. After removed the culture mixture, the cells were fixed with 70% ethanol at 4°C overnight, and then incubated with 50 µg/ml PI and 100 µg/ml RNase (Sigma-Aldrich, St. Louis, MA, USA) at room temperature in the dark for 30 min. Cells were analyzed by Flow cytometer (Beckman Coulter, Brea, CA, USA). The experiment was independently repeated three times.

Cell apoptosis analysis

For cell apoptosis, the cells were seeded into 96-well culture plates at a density of 1 × 10⁴ cells per well. Fisetin at different concentrations were added into the medium 24 h later. Following cultured for 48 h, the medium was removed and cells were washed with PBS for three times. Harvested the cells and resuspended in the binding buffer, and incubated with propidium iodide (PI) and Annexin V/fluorescein isothiocyanate (FITC) (Sigma-Aldrich, St. Louis, MA, USA) for 15 min in the dark. Apoptosis was then detected by a flow cytometer (Beckman Coulter, Brea, CA, USA). The experiment was independently repeated for three times.

Western blot

HCT116 and HT-29 cells were seeded in 6 well plants and cultured for 24 h. Trypsin (100 nM), fisetin (60 and 120 µM, final concentration) and NF-κB p65 expression vector (10µM, final concentration) were added into the medium and cultured for 48 h. Protein extraction from the cell lines was performed as standard protocol. Equal amounts of protein were subjected to SDS-PAGE followed by transfer of protein to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). The membranes were first incubated with the primary antibodies against PRSS2, p21, p27, cyclinD1, NF-κB p65 and GAPDH (Abcam, Cambridge, UK), then followed by incubation with the HRP-conjugated secondary antibodies. The results were visualized using an enhanced chemiluminescence detection system (Amersham Biosciences, Piscataway, NJ, USA). Each experiment was repeated thrice and all reactions were carried out in triplicate.

Human CRC xenograft tumor models in NOG mice

The following animal experiments were performed to study the therapeutic effect and safety of fisetin on CRC in vivo. NOG mice and the following Kunming mice, Wistar rats were purchased from the Guangzhou Jennio Biotech Co., Ltd (Guangzhou, China). All animal experiments were approved by the medical ethics committee of the First Affiliated Hospital of Jinan University, and carried out in accordance with the guidelines for Care and Use of Laboratory Animals of the First Affiliated Hospital of Jinan University.

A total of 16 male NOG mice (weight 26–32 g, 4–6 weeks old) were injected subcutaneously into the right axillary with 5 × 10⁶ HCT116 cells. Once the tumor volume growths to 100 mm³, the mice were randomized into four groups (N = 4/group): control group (treated with saline) and experimental groups (treated with 30, 60 and 120 mg/kg of fisetin). NOG mice in experimental groups were injected intraperitoneally with indicated dosage of fisetin, while mice in control group were administrated with isovolumetric saline. The tumor size was monitored every other day. The largest (a) and smallest (b) superficial diameters of tumors were measured with vernier calipers, and the tumor volume was calculated as follow: tumor volume (mm³) = a × b² × 0.5. After the last injection, NOG mice were sacrificed and the tumors were collected.

Acute and chronic toxicity test

Forty Kunming mice (weight 18–22 g, 4–6 weeks old) were randomly divided into two groups (N = 20, male: female = 50:50). Mice in the fisetin group were injected intraperitoneally with 223 mg/kg of fisetin dissolved in 0.1 ml of PEG200: DMSO (7:3; v:v) as described previously, while in the control group injected with 0.1 ml saline[23]. The mice’ general condition (changes in skin, mucous membrane, fur color, eyes, conscious behavior, respiration, circulation, and central nervous system) was observed daily. Body weight was also recorded weekly.

A total of 160 Wistar rats (weight 75–100 g, 4–6 weeks old) were randomly divided into four groups (N = 40, half male and female), which including control group, low dose group (L, fisetin 15 mg/kg), medium dose group (M, fisetin 30 mg/kg) and high dose group (H, fisetin 60 mg/kg). Rats in experimental groups were injected the indicated dosage of fisetin once a day for successive 6 weeks, respectively, followed by a recovery period of 2 weeks. Those in the control group were administered normal saline in a same procedure. Rats were observed daily for abnormal behavior and other adverse signs of toxicity. Body weight and food consumption were measured and recorded once a week. All animals were euthanized by CO₂ inhalation at the end of experiment, blood samples and organs (brain, lung, heart, liver, kidney, spleen, ovaries and testes) were collected for the biochemical assays (hematological assessments, liver and renal function, and serum electrolytes tests).

Statistical analysis

Data was analyzed using Graphpad 8.0 and expressed as the means ± SEM. One-way ANOVA followed by Tukey-Kramer post hoc tests and two-way ANOVA followed by Tukey's multiple comparisons test were used to evaluate statistical differences. The value of statistical significance was set at p < 0.05.

Expression of PRSS2 was increased in CRC cells

The expression of trypsinogen-2 (PRSS2) was firstly examined in 6 different tumor cell lines including non-small-cell lung cancer cell line A549, CRC cell lines HCT116 and HT-29, pancreatic carcinoma cell line Panc-1, gastric carcinoma cell line SGC-7901, and hepatocellular carcinoma cell line Hepg2. As shown in Fig. 1, the PRSS2 protein was high expression in Hepg2, HCT116 and HT-29 compared with A549, Panc-1 as well as SGC-7901. These data suggest that trypsin may be involved in the development of CRC, and trypsin inhibitors may be medicative for CRC.

Fisetin inhibits the migration of CRC cells induced by trypsin

To explore whether fisetin was able to affect the migration capability of CRC cells induced by trypsin, the transwell assays were carried out. Trypsin (100 nm) significantly promotes the migration of both HCT116 and HT-29 compared with control group (Fig. 2). However, fisetin (30 µM, 60 µM and 120 µM) obviously inhibited the migration of both HCT116 and HT-29 cells induced by trypsin.

Fisetin inhibits the proliferation of CRC cells induced by trypsin

The inhibitory effect on proliferation is crucial for cancer treatment. To evaluate the effect of fisetin on the proliferation of CRC cells induced by trypsin, we determined the cell viability of HCT116 and HT-29 cells with three concentrations of fisetin (30, 60 and 120 µM) at different times (0 h, 24 h, 48 h, and 72 h), respectively. Trypsin treatment significant increases the cell proliferation after incubated for 24 h, while fisetin dramatically interrupts the proliferation induced by trypsin in a time-dependent (from 24 h to 72 h after incubation) and dose-dependent manner (Fig. 3). This finding indicates that fisetin can effectively inhibit the proliferation of CRC cells.

Fisetin suppresses the cell cycle progression of CRC cells

Cell cycle arrest is an important mechanism of cancer treatment drugs, so the effects of fisetin on cell cycle were also analyzed. Flow cytometry results revealed that fisetin (60 µM and 120 µM) inhibited cell cycle progression by increasing the proportion of G1/G0 phase and decreasing the number of S phase cells in HCT116 and HT-29 cell lines compared to control group (Fig. 4). These results indicate that fisetin obstructs cell transition from G1 to S phase and inhibits cell cycle progression.

Fisetin induces the apoptosis of CRC cells

Then, the effect of fisetin on CRC cell apoptosis was also investigated. The results showed in Fig. 5. After an incubation of 48 h, fisetin treatment increased the cells in early apoptosis and later apoptosis both in HCT116 and HT-29 cells (Fig. 5C). The statistical results (Fig. 5A and 5B) demonstrated that fisetin significant promoted the CRC cells apoptosis in a concentration-dependent manner. This outcome indicates that fisetin promotes the apoptosis of the CRC cells.

Fisetin and trypsin affect CRC cells proliferation and apoptosis through the modulation of NF-κB pathways

To explore the molecule mechanisms by which fisetin pro-apoptosis CRC cells, the cell proliferation and apoptosis-related proteins were examined. As demonstrated in Fig. 6, the expression of p21 and p27 was markedly elevated, while that of cyclinD1 and NF-κB p65 were significantly decreased in both HCT116 and HT-29 cells treated with fisetin (60 µM and 120 µM), the opposite results were observed in the trypsin-treated group, although there were no statistical difference compared with the control group (Fig. 6A and 6B). We then explored the role of NF-κB in CRC cell proliferation and apoptosis mediated by fisetin and trypsin. After loaded NF-κB expression vector into HCT116 cells, the increased expression of p21, p27, cyclinD1 and NF-κB p65 induced by fisetin were significantly repressed (Fig. 7). These findings suggested that trypsin may promote the growth of CRC cell through the NF-κB signaling pathway, and the inhibitory effect of fisetin on cell growth is also through this signaling pathway.

Fisetin suppresses the growth of cancer in tumor-bearing NOG mice

To assess the in vivo antitumor activity of fisetin, subcutaneous tumor-bearing NOG mice model was performed using HCT116 cells. Fisetin at different dosage (30, 60 and 120 mg/kg) and saline were inject intraperitoneally for 6 weeks, respectively. As showed in Fig. 8, administration of fisetin significantly decreased the tumor growth compared with the control group, especially at high dosage. The morphology of tumor showed in Fig. 8 revealed that the tumor in mice treated with fisetin was obviously smaller compared with the saline treated mice. These findings indicating that fisetin induces a forceful antineoplastic activity in CRC in vivo.

Fisetin causes no acute or chronic toxicity in animals

To investigate the safety of the clinical use of fisetin, acute and chronic toxicity tests were conducted in Kunming mice and Wistar rats, respectively. At the dose of 223 mg/kg, fisetin showed no significant toxicity in mice without mortality or other abnormal changes, including in skin, mucous membrane, changes in fur color, eyes, circulation, central nervous system, respiration, and conscious behavior. In addition, the growth of mice in experimental group has no significant difference to that in control group (Table 1). After a long-term administration of 6 successive weeks, fisetin also did not cause significant differences in body weight (Table 2) and food consumption (Table 3), neither the noteworthy abnormalities in the hematology (Table 4) and biochemistry (Table 5), or obvious changes in the major organs of the rats. The results indicate that fisetin at the studied dosage levels does not cause acute and chronic toxicity in animal models.

Table 1

Body weight changes of mice in the acute toxicity test.
Group	Dose (mg/kg)	n	Body weight (g)
Group	Dose (mg/kg)	n	Day 0	Day 7	Day 14
Fisetin	223	20	20.81 ± 0.53	23.16 ± 0.93	24.88 ± 1.51
Control	/	20	20.90 ± 0.54	22.89 ± 0.88	25.27 ± 1.93
Values are expressed as mean ± SEM. Fisetin-treated group showed non-significant changes as compared with control group.

Table 2

Body weight changes of rats in chronic toxicity test.
Group	Dose	Gender	Body weight (g)
Group	Dose	Gender	0 w	1 w	2 w	3 w	4 w	5 w	6 w	7 w	8 w
H	60 mg/kg	M	89.30 ± 6.75	116.78 ± 8.60	149.75 ± 7.67	147.89 ± 9.37	172.22 ± 7.86	205.55 ± 8.44	221.30 ± 6.54	261.38 ± 7.02	294.26 ± 9.36
H	60 mg/kg	F	85.62 ± 6.20	95.83 ± 4.35	116.47 ± 4.59	134.52 ± 5.71	154.85 ± 6.67	176.45 ± 6.61	185.07 ± 5.87	216.23 ± 4.23	234.64 ± 5.86
M	30 mg/kg	M	86.99 ± 6.02	115.07 ± 8.46	146.29 ± 8.84	143.51 ± 9.82	176.92 ± 9.46	207.51 ± 11.19	234.96 ± 8.63	267.63 ± 7.62	296.77 ± 8.33
M	30 mg/kg	F	86.24 ± 7.41	93.95 ± 5.64	114.90 ± 5.59	135.61 ± 6.49	155.85 ± 5.86	175.94 ± 6.14	193.23 ± 4.48	213.38 ± 5.59	235.81 ± 6.08
L	15 mg/kg	M	87.71 ± 7.34	112.21 ± 7.92	145.53 ± 7.96	141.56 ± 8.24	177.64 ± 9.97	203.94 ± 8.44	235.73 ± 8.16	267.70 ± 8.08	300.52 ± 6.74
L	15 mg/kg	F	87.74 ± 7.53	95.77 ± 5.08	116.14 ± 5.76	135.07 ± 6.12	155.41 ± 5.38	173.97 ± 6.55	196.99 ± 5.97	216.64 ± 5.85	235.81 ± 5.98
Ctrl	/	M	88.88 ± 8.05	113.80 ± 9.24	144.09 ± 9.10	144.06 ± 8.89	170.77 ± 7.96	204.68 ± 9.46	232.46 ± 7.54	265.41 ± 9.03	296.61 ± 6.87
Ctrl	/	F	85.25 ± 5.67	95.05 ± 5.87	114.14 ± 5.41	136.06 ± 5.51	155.64 ± 6.57	173.33 ± 5.23	197.67 ± 4.92	215.90 ± 5.70	236.72 ± 6.37
Note: Ctrl: control group; w: week; H, M, L represents high-dose, medium dose, low dose group, respectively. The values are expressed as mean ± SEM.

Table 3

Food consumption of rats in chronic toxicity test.
Group	Dose	Gender	Food consumption (g)
Group	Dose	Gender	1 w	2 w	3 w	4 w	5 w	6 w	7 w	8 w
H	60 mg/kg	M	16.52 ± 0.23	17.36 ± 0.28	18.63 ± 0.27	19.81 ± 0.59	20.26 ± 0.71	20.82 ± 0.39	22.43 ± 0.73	23.31 ± 0.71
H	60 mg/kg	F	15.94 ± 0.55	16.41 ± 0.30	16.92 ± 0.29	17.83 ± 0.19	18.75 ± 0.24	18.72 ± 0.37	20.27 ± 0.35	20.61 ± 0.46
M	30 mg/kg	M	16.47 ± 0.24	17.54 ± 0.32	18.52 ± 0.29	19.98 ± 0.59	20.47 ± 0.88	21.67 ± 0.82	22.32 ± 0.99	23.19 ± 0.83
M	30 mg/kg	F	15.86 ± 0.58	16.56 ± 0.35	17.05 ± 0.27	17.84 ± 0.23	18.63 ± 0.21	19.42 ± 0.35	20.25 ± 0.43	20.66 ± 0.41
L	15 mg/kg	M	16.56 ± 0.25	17.47 ± 0.32	18.52 ± 0.29	19.96 ± 0.53	20.39 ± 0.77	21.41 ± 0.79	22.16 ± 0.46	23.52 ± 0.95
L	15 mg/kg	F	15.88 ± 0.60	16.58 ± 0.32	16.97 ± 0.29	17.87 ± 0.21	18.79 ± 0.21	19.45 ± 0.31	20.20 ± 0.38	20.67 ± 0.48
Ctrl	/	M	16.47 ± 0.33	17.50 ± 0.29	18.44 ± 0.32	19.97 ± 0.57	20.59 ± 0.75	21.56 ± 0.88	22.48 ± 0.82	23.62 ± 0.86
Ctrl	/	F	16.14 ± 0.53	16.44 ± 0.31	16.94 ± 0.30	17.88 ± 0.19	18.68 ± 0.24	19.29 ± 0.28	20.39 ± 0.34	20.70 ± 0.42
Note: Ctrl: control group; w: week; H, M, L represents high dose, medium dose, low dose group, respectively. The values are expressed as mean ± SEM.

Table 4

Hematological parameters of rats in chronic toxicity test.
Group	Dose	Gender	Hematological parameters
Group	Dose	Gender	WBC (× 10⁹/L)	RBC (× 10¹²/L)	HGB (g/L)	PLT (× 10⁹/L)	NEUT (× 10⁹/L)	LYMPH (× 10⁹/L)	MONO (× 10⁹/L)
H	60 mg/kg	M	14.03 ± 0.74	6.89 ± 0.56	126.17 ± 4.61	470.78 ± 51.38	1.50 ± 0.24	9.64 ± 0.88	2.35 ± 0.16
H	60 mg/kg	F	11.85 ± 0.55	6.67 ± 0.32	125.37 ± 3.81	441.21 ± 33.62	1.21 ± 0.14	8.24 ± 0.53	2.18 ± 0.09
M	30 mg/kg	M	13.52 ± 0.94	6.55 ± 0.36	125.89 ± 2.91	505.57 ± 35.54	1.38 ± 0.21	9.21 ± 0.76	2.35 ± 0.21
M	30 mg/kg	F	12.39 ± 0.89	6.87 ± 0.33	123.40 ± 3.29	428.57 ± 52.66	1.26 ± 0.16	8.57 ± 0.85	2.20 ± 0.10
L	15 mg/kg	M	13.95 ± 0.64	6.71 ± 0.44	128.80 ± 3.76	496.68 ± 39.85	1.50 ± 0.25	9.53 ± 0.79	2.32 ± 0.20
L	15 mg/kg	F	11.81 ± 0.83	6.63 ± 0.37	124.47 ± 3.69	423.08 ± 36.32	1.25 ± 0.12	8.37 ± 0.60	2.21 ± 0.11
Ctrl	/	M	13.38 ± 0.92	6.76 ± 0.33	128.40 ± 4.96	464.72 ± 51.00	1.61 ± 0.26	9.28 ± 0.70	2.23 ± 0.14
Ctrl	/	F	12.03 ± 0.81	6.61 ± 0.28	125.34 ± 2.60	447.04 ± 54.24	1.24 ± 0.15	8.32 ± 0.61	2.14 ± 0.09
Note: Ctrl: control group; H, M, L represents high-dose, medium dose, low-dose group, respectively. WBC: total white blood cell count; RBC: red blood cell; HGB: hemoglobin; PLT: blood platelet; Neut: neutrophil; Lymph: lymphocyte; Mono: mononucleosis. The values are expressed as mean ± SEM.

Table 5

Biochemical parameters of rats in chronic toxicity test.
Group	Dose	Gender	Biochemical parameters
Group	Dose	Gender	ALT (IU/L)	AST (IU/L)	ALB (g/L)	BUN (mmol/L)	Cre (µmol/L)	Glu (mmol/L)	TC (mmol/L)
H	60 mg/kg	M	40.36 ± 1.38	190.77 ± 8.21	32.04 ± 1.39	6.62 ± 0.29	71.44 ± 1.38	6.71 ± 0.31	1.85 ± 0.03
H	60 mg/kg	F	39.30 ± 0.81	181.68 ± 4.92	32.65 ± 0.72	7.11 ± 0.36	69.43 ± 1.86	6.52 ± 0.45	1.78 ± 0.06
M	30 mg/kg	M	39.16 ± 1.82	189.76 ± 5.84	31.95 ± 1.06	6.73 ± 0.44	72.91 ± 1.71	7.10 ± 0.31	1.85 ± 0.03
M	30 mg/kg	F	39.19 ± 1.19	185.40 ± 4.52	32.77 ± 0.94	6.71 ± 0.47	68.90 ± 1.42	6.56 ± 0.37	1.78 ± 0.07
L	15 mg/kg	M	39.20 ± 1.60	188.39 ± 6.41	31.87 ± 1.19	6.66 ± 0.28	72.82 ± 1.46	6.72 ± 0.31	1.84 ± 0.03
L	15 mg/kg	F	39.26 ± 1.15	184.17 ± 4.78	32.89 ± 0.81	6.75 ± 0.46	69.30 ± 1.71	6.50 ± 0.44	1.79 ± 0.07
Ctrl	/	M	39.91 ± 1.90	190.45 ± 5.52	30.81 ± 0.68	6.76 ± 0.41	72.82 ± 2.03	6.82 ± 0.44	1.85 ± 0.03
Ctrl	/	F	38.37 ± 0.88	183.57 ± 4.35	32.69 ± 0.88	6.78 ± 0.43	69.43 ± 1.74	6.41 ± 0.44	1.83 ± 0.07
Note: Ctrl: control group; H, M, L represents high dose, medium dose, low dose group, respectively. ALT: alanine aminotransferase; AST: aminotransferase; ALB: albumin; BUN: blood urea nitrogen; CRE: creatinine; Glu: glucose; TC: total cholesterol. The values are expressed as mean ± SEM.

CRC is notorious for its high incidence and poor prognosis, which characterized by rapid growth, migration, invasion and recurrence [24]. About 20% of CRC patients staged in TNM stage II will develop cancer recurrence [25]. Due to the unsatisfactory efficacy, and the side effects of chemotherapy, targeted therapy and immunotherapy in clinical CRC treatment, there is increasing interest in screening more efficient and tolerable anti-cancer drugs from natural products. The main efficiency limitation of CRC treatment is primary and secondary drug resistance, the underlying mechanism of which requires extensive investigation urgently. Tan et al. [10] have demonstrated PRSS can cleave cetuximab, leading to drug resistance. Cetuximab or bevacizumab combined with SPINK1 inhibited cell growth more efficiently than cetuximab or bevacizumab alone in xenograft models. Fisetin exhibits various activities such as anti-invasive, anti-inflammatory, anti-angiogenic, anti-oxidant, anti-diabetic, cardioprotective and neuroprotective activities [26]. Despite the ever-rising support for anti-tumorigenic activities, its clinical application is still limited partially due to the lack of comprehensive evidence. Our study systematically investigated the anti-tumorigenic activity and safety of fisetin in vitro and in vivo, and preliminarily explored its possible mechanisms.

In the current study, we first showed that trypsinogen PRSS2 expression was significantly up-regulated in CRC cell lines HCT116 and HT-29. Williams et al. [27] also showed high levels of trypsinogen in the CRC cell line SW620. Koivunen et al. [28] has reported that the CRC cell line COLO-205 produces tumor-associated trypsinogen. Tan et al. [10] demonstrated the possibility of using PRSS (a trypsin) as a predictive marker of the mCRC response to cetuximab treatment. Those findings suggest the possibility that trypsin may play an important role in the development of CRC, and trypsin inhibitor fisetin may affect CRC tumor behavior.

Next, we performed in vitro and in vivo experiments to further explore the effects of fisetin on the development and progression of CRC. In vitro function study displayed that fisetin treatment led to decreased migration capability and proliferation induced by trypsin, G1/G0 phase arrest, apoptosis induction of HCT116 and HT-29 cells in a dose-dependent manner. The animal experiment showed that fisetin treatment led to significant tumor growth inhibition in an established HCT116 xenograft model. To our knowledge, this is the first study that demonstrates the therapeutic value of fisetin in vivo for CRC treatment. Taken together, these in vitro and in vivo results suggest that fisetin may have an important influence on the development, progression, and metastasis of CRC, and that fisetin may possess the therapeutic value for CRC. Similar to our findings, previously studies exploring the effect of fisetin on CRC indicated that fisetin significantly suppressed proliferation and cell cycle progression of HT-29 cells, and induced HCT116 cells apoptosis [29–31]; Liao et al. [32] revealed that fisetin remarkably inhibited adhesion, migration, and invasion of lung cancer A549 cells; Khan et al. [33] showed that treatment with fisetin resulted in significant inhibition of tumor growth in athymic nude mice implanted with CWR22Rν1 prostate cancer cells. However, we first report that fisetin plays an anti-tumor role by inhibiting trypsin activity and cytotoxic dual-acting targets, and has the potential to address the problem of targeted drug resistance. Then, we investigated the molecular mechanism involved in inhibition of CRC cell growth and migration induced by fisetin or/and trypsin treatment. The expression of the cell proliferation or apoptosis-related proteins p21 and p27 (pro-apoptotic protein) as well as cyclinD1 and NF-κB p65 (anti-apoptotic protein) was examined between both HCT116 and HT-29 cells treated with fisetin or not. The results showed that treatment with fisetin could increase p21 and p27 protein levels and decrease cyclinD1 and NF-κB p65 protein levels, treatment with trypsin could increase cyclinD1 and NF-κB p65 protein levels, suggesting that suggesting that activation of the NF-κB p65 and cyclinD1 plays an important role in trypsin-induced CRC cell growth, while fisetin plays an anti-tumor role by inhibiting this effect. Moreover, we also found that supplement of NF-κB remarkably remediedhe changes in anti-apoptotic, pro-apoptotic protein levels induced by fisetin treatment, implying that both fisetin and trypsin regulate CRC cell growth partially through the NF-κB signaling pathway. Similarly, Suh et al. [34] also reported that fisetin induced cell apoptosis in CRC by inhibiting the activation and transcriptional activity of NF-κB. However, the role of NF-κB in cancer remains contradictory [35, 36]. For example, several studies have demonstrated that activation of NF-κB signaling pathway promotes inflammation-associated cancer, and inhibition of it in hepatocytes attenuates the onset of HCC related to inflammation [37, 38]. On the contrary, other studies have shown that NF-κB inactivation can stimulate hepatocarcinogenesis and induce spontaneous development of HCC, implying a tumor suppressor role for it in hepatocytes [39]. Further investigation by cell and animal model is therefore necessary to elucidate the causative mechanism on how fisetin exerts its anti-carcinogenic effects in addition to the NF-κB pathway.

Finally, the safety of fisetin was evaluated in Kunming mice and Wistar rats. In the acute and chronic toxicity test, no statistically significant alterations were found in behavior, organ weight, biochemistry, or hematological parameters. These findings demonstrated that fisetin at the studied dosage levels did not induce obvious harmful effects in animals. Thus, it is reasonable to conclude that fisetin is safe at the given doses. Our data have shown that fisetin not only displays non-toxic properties, but that it can also significantly exert the in vitro and in vivo anti-tumors effect of CRC, indicating that the non-toxic dietary flavonoid fisetin might advantageously be applied to the clinical treatment of patients with CRC.

In conclusion, fisetin can significantly suppress CRC growth and migration partially through modulating NF-κB pathways and inhibiting trypsin activity, and fisetin is a promising drug for CRC therapy, trypsin can be used as an important target to study drug resistance in cancer therapy. Our findings provide useful information for understanding the therapeutic effects of fisetin on CRC, and the possible mechanism involved in inhibition of CRC growth and migration induced by fisetin needs further study. Furthermore, the result of this research also reminded that the nature derived TI maybe one of the treatments of CRC.

CRC: colorectal cancer; NOG mouse: NOD.Cg-PrkdcscidIl2rgtm1Sug/JicCrl; PD: Parkinson’s disease; AD: Alzheimer’s disease; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; OD: optical density; MTT: 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide;

Ethics approval and consent to participate

All animal experimental protocols were performed in accordance with the National Institutes of Health guidelines (NIH, USA), and approved by the Ethics Committee of the Institute of Laboratory Animal Science at Jinan University, Guangzhou, China.

Consent for publication

Not applicable.

Availability of data and materials

The datasets used during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare no conflicts of interest.

Funding

This work was financially supported by the National Natural Science Foundation of China (Grant: 82073806 and 81872832), the Guangdong Basic and Applied Basic Research Foundation (Grant: 2019A1515010806), the Key Field Projects (Intelligent Manufacturing) of General Universities in Guangdong Province (Grant: 2020ZDZX2057), the International Science & Technology Cooperation Program of Guangzhou, China (Grant: 201807010022) and the Scientific Research Projects (Characteristic Innovation) of General Universities in Guangdong Province (Grant: 2019KTSCX195).

Authors’ contributions

GJ and JZ conceived and designed the study; LZ wrote the main manuscript text; LL and WM performed cell experiments; LL and HX performed animal experiments; LY analyzed and collected data; LZ prepared figures and tables. All authors reviewed the manuscript. All authors read and approved the final manuscript.

Acknowledgements

We gratefully appreciate the financial support from the National Natural Science Foundation of China (Grant: 82073806 and 81872832), the Guangdong Basic and Applied Basic Research Foundation (Grant: 2019A1515010806), the Key Field Projects (Intelligent Manufacturing) of General Universities in Guangdong Province (Grant: 2020ZDZX2057), the International Science & Technology Cooperation Program of Guangzhou, China (Grant: 201807010022) and the Scientific Research Projects (Characteristic Innovation) of General Universities in Guangdong Province (Grant: 2019KTSCX195).

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Fisetin Exerts Anti-tumorigenic Activity via Inhibition of Trypsin Activity in Colorectal Cancer

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Abstract

Figures

Background

Materials And Methods

Cell culture

Transwell cell migration assays induced by trypsin

Cell proliferation assay induced by trypsin

Cell cycle analysis

Cell apoptosis analysis

Western blot

Human CRC xenograft tumor models in NOG mice

Acute and chronic toxicity test

Statistical analysis

Results

Expression of PRSS2 was increased in CRC cells

Fisetin inhibits the migration of CRC cells induced by trypsin

Fisetin inhibits the proliferation of CRC cells induced by trypsin

Fisetin suppresses the cell cycle progression of CRC cells

Fisetin induces the apoptosis of CRC cells

Fisetin and trypsin affect CRC cells proliferation and apoptosis through the modulation of NF-κB pathways

Fisetin suppresses the growth of cancer in tumor-bearing NOG mice

Fisetin causes no acute or chronic toxicity in animals

Discussion

Conclusion

Abbreviations

Declarations

Ethics approval and consent to participate

Consent for publication

Availability of data and materials

Competing interests

Funding

Authors’ contributions

Acknowledgements

References

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