Injectable pegylated niclosamide (polyethylene glycol-modified niclosamide) for cancer therapy

Rui Ma 1, Zhen-Gang Ma 1,Jin-Lai Gao 2, Yu Tai 2, Lan-Jun Li 2, Hai-Bin Zhu 2, Li Li 1, De-Li Dong 2, Zhi-Jie Sun 1,*
1. Institute of Materials Processing and Intelligent Manufacturing, Center for Biomedical Materials and Engineering, Harbin Engineering University, 145 Nantong Street, Nangang District, Harbin 150001, P.R.China.
2. Department of Pharmacology (the State-Province Key Laboratories of Biomedicine-Pharmaceutics of China, Key Laboratory of Cardiovascular Research, Ministry of Education), College of Pharmacy, Harbin Medical University; Translational Medicine Research and Cooperation Center of Northern China, Heilongjiang Academy of Medical Sciences, Harbin Medical University, Harbin 150086, P.R.China.

*Corresponding author
Professor. Zhi-Jie Sun,
Institute of Materials Processing and Intelligent Manufacturing, Center for Biomedical Materials and Engineering,
Harbin Engineering University,
145 Nantong Street, Nangang District,Harbin 150001,

People’s Republic of China
E-mail: [email protected]

ABSTRACT

Niclosamide is an antihelminthic drug. Recent studies show that niclosamide exerts antitumor activity through inhibiting multiple signals including Wnt/β-catenin, mTORC1, STAT3, NF-κB, Notch signals; however, the insolubility and poor bioavailability limits its potential clinic use, the aim of the present work is to synthesize an injectable pegylated niclosamide (polyethylene glycol-modified niclosamide) and investigate its antitumor activity in vitro and in vivo.
Methods: The pegylated niclosamide (mPEG5000-Nic) was synthesized and the chemical structure was identified by FTIR and 1H NMR spectra. The anti-tumor activity was evaluated in CT26 and HCT116 colon cancer cells in vitro and nude mouse xenograft model of CT26 cells in vivo.
Results: The water solubility of niclosamide in mPEG5000-Nic was significantly increased. Niclosamide could be released from mPEG5000-Nic nanoparticles in PBS solution. mPEG5000-Nic inhibited the cell viability of CT26 and HCT116 cells in vitro. No animal death was observed in mice with intraperitoneal injection of mPEG5000-Nic (equivalent to 1000 mg/kg niclosamide) within 24 h, indicating that mPEG5000-Nic was less toxic. In nude mouse xenograft model of CT26 colon

carcinoma, intraperitoneal injection of mPEG5000-Nic (equivalent to niclosamide 50 mg/kg) inhibited tumor growth but had no effect on animal body weight and heart, liver, kidney, and lung weight in vivo. Meanwhile, in the same model, intraperitoneal injection of the positive clinic drug 5-fluorouracil (5-FU) not only inhibited the tumor growth, but also reduced the animal body weight.
Conclusion: Our study demonstrates that pegylated niclosamide is novel niclosamide delivery system with clinical perspective for cancer therapy .

Key words: Niclosamide; pegylation; drug delivery; cancer therapy

INTRODUCTION

Cancer is the leading cause of diseases related with death in the world. The treatments for cancer include chemotherapy, radiotherapy, surgery, and immunetherapy. Among these therapies, chemotherapy plays a major role clinically. However, drug-resistance is a big challenge for classical chemotherapy and newer targeted agents. Therefore, development of novel antitumor drugs or therapeutic ways is extremely important.
Niclosamide is an FDA-approved antihelminthic drug. Niclosamide is insoluble and has poor oral bioavailability, these properties permit it keep higher concentration

in intestinal tract, which is advantageous for antihelminthic treatment. Recently, numerous studies show that niclosamide exerts antitumor activity through inhibiting Wnt/β-catenin, mTORC1, STAT3, NF-κB, Notch signals and inducing mitochondrial uncoupling1,2. Furthermore, niclosamide is reported to overcome multidrug-resistance and radio-resistance in types of cancers3-7.
Theoretically, niclosamide should be an ideal antitumor drug for its multiple-targets and broad-spectrum effects, however, the insolubility and poor absorption limit its clinical use for cancer treatment. Therefore, several studies attempt to improve the pharmacokinetics of niclosamide through establishing novel niclosamide delivery system, for instance, Russo et al proposed targeted Pluronic(®) P123/F127 mixed micelles (PMM) delivering niclosamide to treat multidrug resistant non-small lung cancer cell lines8, Lin et al prepared a nanosuspension of niclosamide against ovarian cancer9. Our previous study synthesized the poly (methacrylic acid-niclosamide) polymer (PMAN) to increase the solubility of niclosamide10, nevertheless, the disadvantage is that polymethacrylic acid as control showed a little toxic in vivo.
Polyethylene glycol (PEG) is a type of soluble polymer widely used in multiple formulations. Pegylation technique is to modify the target drug with mPEG, thus, improves the drug hydrophilicity, enhances the drug bioavailability, stability, and

circulation half-life in vivo, and reduces the drug potential toxicity. Numerous PEG-based prodrug conjugates have been developed, some have received market approval and others are under clinical trials. For example, PEG-asparaginase, PEG-adenosine deaminase, PEG-Interferon alpha 2b, PEG-rhGCSF, PEG-anti-TNF Fab et al have entered the clinic with FDA approval; pegylated compounds including PEG-camptothecin, PEG-irinotecan, PEG-SN38, PEG-naloxone are under clinical trials 11.
Based on the advantages of pegylation technique, we put forward the prodrug strategy of modified niclosamide with mPEG to keep niclosamide active, improve niclosamide hydrophilicity and solubility. Here, we synthesized pegylated niclosamide with mPEG-5000 (mPEG5000-Nic) by RAFT (Reversible Addition Fragmentation Chain Transfer Polymerization) method, characterized its microstructure and studied its anti-tumor effect in vitro and in vivo. Results show that pegylated niclosamide is novel niclosamide delivery system with clinical perspective. MATERIALS AND METHODS
Materials

Methoxypolyethylene glycols (mPEG5000), Carbon disulfide, phenylmagnesium bromide and alpha-bromo-p-toluic acid was purchased from aladdin@ industrial corporation (Shanghai, China). Niclosamide was purchased from

Jianglai Reagent Company (Shanghai, China). Acryloyl chloride(AC) , anhydrous tetrahydrofuran (THF), 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and 4-dimethylaminopyridine (DMAP) was purchased from energy chemical (Shanghai,China). All other chemicals were purchased from commercial suppliers.
Synthesis of 5-Chloro-N-(2-chloro-4-nitrophenyl)-2-(2-acryloyloxy)-benzamide (Nic-AC)
5-Chloro-N-(2-chloro-4-nitrophenyl)-2-(2-acryloyloxy)-benzamide (Nic-AC) ynthesized according to our previous study with modified10. In brief, 0.01 mol niclosamide was dissolved in 200 ml anhydrous THF containing 0.015 mol triethylamine (TEA), and then 0.018 mol AC was dropwise added into the above solution. The mixture was reacted at room temperature for 5 h in dark. At the end of the reaction, the solution was poured into deionized water. The product was filtered and purified by recrystallization in dichloromethane/petroleum ether (1/1 v/v).
Synthesis of the RAFT agents

4-toluic acid dithiobenzoate (TAD) was synthesized according to previous work12. In brief, 0.045 M carbon disulfide reacted and 0.03 M phenylmagnesium bromide in dry THF at 10 °C for 6 h, and the unreacted reagents were removed by distillation. The reaction products and alpha-bromo-p-toluic acid were added to

methanol at a molar ratio of 1.5:1 and reacted at 60 ℃ for 24 h. Again, unreacted reagent and methanol were removed by distillation. The red crude product was obtained.
The RAFT agents was synthesized by the esterification reaction. 1 mmol mPEG5000 and 3 mmol TAD were dissolved in 500 ml N,N-dimethylformamide (DMF), 15 mmol 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 15 mmol 4-dimethylaminopyridine (DMAP) were used as catalysts, the reaction processed at 35 ℃ for 48 h. The reactant solution was precipitated with cold diethyl ether before filtration. The products were dissolved in ethanol and the excess catalyst and reactant were removed by dialysis method. Finally, the pink pure product (mPEG5000-TDA) was obtained by precipitating with cold diethyl ether and used as the RAFT agent.
Synthesis of pegylated niclosamide (mPEG5000-Nic).

mPEG5000-Nic was synthesized by RAFT method. The mPEG-TAD and Nic-AC was dissolved in DMF, azodiisobutyronitrile (AIBN) was used as catalyst, the amount of AIBN (0.03 g) was fixed at 1 wt% relative to Nic-AC, the molar ratio of mPEG5000-TAD RAFT agent to Nic-AC was 1:5. The polymerization was carried out under nitrogen condition at 70 ℃ for 24 h. The product was precipitated with cold diethyl ether before filtration and the purification process was similar to

mPEG5000-TAD.

Determination of niclosamide content and drug release

Niclosamide content of mPEG5000-Nic was determined using UV-Vis spectrophotometry (UV-2550, Shimadzu corporation, Japan). mPEG5000-Nic were hydrolyzed for 2.5 h at 50 ℃ in 6 M HCl and methanol (1:8 v/v) and the wavelength
analyzed was 331 nm, the equations of standard curve was Y=0.01802×X-0.00244
(R2=0.9901).

The drug release was analyzed by UV-Vis spectrophotometry (UV-2550, Shimadzu corporation, Japan). mPEG5000-Nic polymer (100 mg) was dissolved in 10 mL PBS (Phosphate Buffer Saline, pH 7.4, 0.01 M) containing 0.5 % (w/w) tween80, and then the solution was transferred into a dialysis tube (MWCO: molecular weight cutoff =1 kDa) and immersed in 90 mL PBS (pH 7.4) containing 0.5 % (w/w) tween80 at 37 ℃. At pre-designed time intervals, the outside solution (4 mL) was taken out for UV-Vis spectrometer detection and replaced with 4 mL fresh medium, the absorbency was analyzed at around 341 nm, the equations of standard curve of niclosamide was Y (mg/ml) = 0.05373× X-3.052e-4 (R2 = 0.99706).
Fourier transform infrared spectra and 1H nuclear magnetic resonance spectra

Fourier transform infrared spectra (FT-IR) were detected with FT-IR spectrometer (spectrum 100, Perkin Elmer, USA). 1H Nuclear Magnetic Resonance

(1H NMR) spectra were measured by using Bruker AM 300 apparatus.

Cell culture

Colon cancer cell lines CT26, HCT116 were maintained in RPMI 1640 (Roswell Park Memorial Institute) medium supplemented with 10 % fetal bovine serum, 1 % penicillin/streptomycin. CT26 and HCT116 were passaged every 2 days. All cell lines were incubated in a humidified incubator at 37 °C supplied with 5 % carbon dioxide. Cell Viability
Cell viability was measured by colorimetric MTT assay. The optical density (OD) values were read at 490 nm using an Infinite M200 microplate reader (Tecan, Salzburg, Austria). The method in detail was described in our previous work13.
Western blot

Total protein extraction and separation methods were described in our previous studies14, 15. The membranes were incubated with anti-p-STAT3 (Y705, #9131; S727, #9134, 1:1000), anti-STAT3 (#4904, 1:1000) (Cell Signaling Technology, Danvers, MA, USA) and fluorescence-labeled secondary antibodies (LI-COR Biosciences, Lincoln, NE, USA). β-actin (ZSBIO, #TA-09, 1:1000) was used as loading control. Band densities were quantified with Odyssey v3.0 software.
In vivo tumorigenesis assay

Male BALB/c nude mice (Six- to eight-week-old) were purchased from Beijing

Vital River Laboratory Animal Technology Co., Ltd. Tumor cells were harvested from flasks and resuspended with phosphate buffer saline. 200 μL tumor cell suspension containing 3×106 cells were injected subcutaneously into the right flank region of BALB/c nude mice. When tumor size reached to ~50 mm3, mice were randomly grouped to vehicle group and drug treatment group. Body weights were measured
every day and tumor volume was assessed by caliper measurements every two days.

Statistical analysis

Data were presented as mean ± standard error (SE). Significance was determined by using Student’s t-test or one-way ANOVA followed by Holm-Sidak. P < 0.05 was considered significant.
RESULTS

Synthesis and identification of pegylated niclosamide (mPEG5000-Nic)

mPEG5000-Nic was synthesized by RAFT method. Niclosamide was conjugated on mPEG5000 by ester bond. The synthetic route of mPEG5000-Nic was shown in Figure.1. The chemical structure of Nic-AC, mPEG5000-Nic was identified by FT-IR and 1H NMR. The FT-IR spectra of niclosamide and Nic-AC were shown in Figure.2A, as shown, the extension vibration of C=O on ester bond was observed at 1749cm-1. The chemical structure of Nic-AC was confirmed from the proton 1H NMR
spectrum as shown in Figure.2B. In the 1H NMR spectrum, the signal peaks of each proton in the Nic-AC structure were attributed, and there were almost no stray peaks,

indicating that the impurity content of the product was low. The 1H NMR spectra of mPEG5000-TAD and mPEG5000-Nic was shown in Figure.3 and Figure.4, respectively. To deduce the structural composition, the alphabetical letters assigned for the corresponding protons were marked. The typical peaks of mPEG was distributed at around 3.6-3.9ppm (“a” ,“b” and “c”). In Figure.3, the alphabetical letters “d”, “e”, “g” and “h” were assigned for TAD unit in mPEG5000-TAD, which consisted with the previous study about chemical structure of TDA12. In Figure.4, the multiplet around 2.8 ppm-3.0 ppm belonged to the resonance of the methyne protons and methylene protons (“g” and “h”), 1H NMR spectra from 7 ppm to 11 ppm (“k-q”) showed the characteristic peaks of niclosamide. Overall, niclosamide was grafted on mPEG5000 through ester bond.
Niclosamide content in mPEG5000-Nic was measured by using UV-Vis spectrophotometry. The UV spectrum of mPEG5000-Nic hydrolyzed in 6 M HCl and methanol (1:8 v/v) solution from 0 h-2.5 h were shown in support information, as it was shown, the maximum UV absorbance of the polymer was 298 nm at 0.5 h, but around 331 nm at 2.5 h, this is due to the hydrolysis of niclosamide from the polymer, indicating that niclosamide was grafted in the polymer successfully. The niclosamide
content was 6.73±0.5%, that is to say, the number of Nic-AC unit (“m” in Figure.1) conjugated in mPEG5000 was 1-2.

Morphology of mPEG5000-Nic aqueous solution

It was found that the solubility of niclosamide in water could be significantly improved by grafting niclosamide into water soluble macromolecules, the solubility of the modified niclosamide was over 1.8 mg/ml, which was more than 8000 times higher than that of pure niclosamide. In the 1H NMR spectrum of mPEG-Nic D2O solution (support information), only the signal peak of protons on mPEG5000 was observed, but the signal peak of niclosamide was not observed, which indicated that niclosamide grafted on mPEG5000-Nic formed hydrophobic kernel in water. As illustrated in Figure.5A and Figure.5B, mPEG5000-Nic could self-assemble in water to improve the water solubility of niclosamide, the hydrophilic chain segment mPEG mutual entanglement wrapped the hydrophobic group niclosamide inside, and niclosamide was relatively evenly distributed in the water without precipitation, the solution of mPEG5000-Nic was obviously transparent, while the solution of niclosamide was visibly insoluble, and the image of transmission electron microscope explain the mechanism.
Niclosamide release from mPEG5000-Nic in vitro

Since niclosamide was grafted in mPEG5000 through easter bond, the niclosamide release from mPEG5000-Nic, thus playing a role in the biological activity of drugs as shown in Figure. 5C. The in vitro release experiment was carried

out in PBS (pH 7.4, 0.01 M) containing 0.5 % (w/w) tween 80. The drug release profile was shown in Figure. 6. Niclosamide grafted in the copolymer was released slowly and continuously until 11 days, and the cumulative drug release reached more than 80 %.
Effect of mPEG5000-Nic on cell viability of CT26 and HCT116 colon cancer cells

in vitro.

Niclosamide has anti-cancer activities through the mechanism of inhibiting Wnt/β-catenin, mTORC1, STAT3, NF-κB, Notch signals and inducing mitochondrial uncoupling. Since mPEG5000-Nic could release niclosamide in PBS solution, we speculated that it could release niclosamide in the cell culture medium because serum in the cell culture medium contained enzyme which would hydrolyze mPEG5000-Nic to release niclosamide more easily. We compared the effect of niclosamide and mPEG5000-Nic on cell viability of CT26 and HCT116 colon cancer cells. As shown in Figure 7, both niclosamide and mPEG5000-Nic inhibited the cell viability of CT26 and HCT116 cells, however, the potency of mPEG5000-Nic was significantly less than that of niclosamide. Because that there was a gradual release process for niclosamide from mPEG5000-Nic, the results were reasonable and suggested that niclosamide could be released from mPEG5000-Nic to show its antitumor activity indeed.

Evaluation of mPEG5000-Nic activity in vivo.

Because the solubility of niclosamide in mPEG5000-Nic was significantly improved, we further measured the acute toxicity of mPEG5000-Nic administered by intraperitoneal injection in mice. No death was observed in the animals with accumulative amount of mPEG5000-Nic equivalent to 1000 mg/kg niclosamide within 24 h, indicating that mPEG5000-Nic was less toxic in vivo.
Niclosamide is a STAT3 (Signal Transducer and Activator of Transcription 3) inhibitor. Therefore, we used STAT3 activity as the marker to evaluate the activity of niclosamide and mPEG5000-Nic in vivo. We compared the effect of oral niclosamide and intraperitoneal injection of mPEG5000-Nic on STAT3 activity in different organs in mice. Niclosamide was orally administered at 300 mg/kg and mPEG5000-Nic equivalent to 50 mg/kg was intraperitoneally injected for 18 days in mice, and the STAT3 activity in heart, lung and liver was measured. As shown in Figure 8, oral niclosamide (300 mg/kg) showed no significant effect on STAT3 activity in heart, lung and liver; however, intraperitoneal injection of mPEG5000-Nic equivalent to 50 mg/kg niclosamide significantly inhibited STAT3 activity in liver, though STAT3 activity in heart and lung was not affected. These results indicated that intraperitoneal injection of mPEG5000-Nic showed in vivo activity.
Anti-tumor activity of mPEG5000-Nic in vivo

Since mPEG5000-Nic was less toxic and had biological activity in vivo, we established nude mouse xenograft models. CT26 colorectal cancer cells (3 × 106) were subcutaneously inoculated into the right flank region of BALB/c nude mice. When tumor size reached to ~50 mm3, mice were randomly grouped to vehicle group and mPEG5000-Nic (equivalent to niclosamide 50 mg/kg) for 16 days. As shown in Figure. 9A -9C, mPEG5000-Nic treatment significantly reduced the size, volume and weight of tumor, compared with the control group, suggesting that mPEG5000-Nic (i.p) treatment reduced the growth of tumor in vivo. Meanwhile, intraperitoneal injection of mPEG5000-Nic for 16 days had no effect on animal body weight and heart, liver, kidney, and lung weight in vivo (Figure. 9D and 9E).
We further comparatively studied the antitumor activity of 5-fluorouracil (5-FU), the clinically chemotherapeutic agent, in the same nude mouse xenograft model. Results showed that intraperitoneal injection of 5-FU significantly inhibited tumor growth in vivo (Figure 10A and 10B), however, at the same time, 5-FU significantly reduced the animal body weight (Figure 10C), indicating the potential toxicity of 5-FU in vivo. Collectively, the comparison of anti-tumor activity of mPEG5000-Nic and 5-FU in vivo suggests that PEG5000-Nic has the equivalent anti-tumor activity and less toxicity.
DISCUSSION

Recent studies have indicated that niclosamide may have broad clinical applications for the treatment of diseases including cancer, bacterial and viral infection, metabolic diseases, artery constriction, and systemic sclerosis16. However, because of its insolubility and poor bioavailability, the potential clinic use of niclosamide for the diseases mentioned above was limited, and niclosamide is still used as an oral antihelminthic drug for treating parasitic infections. In order to overcome the shortage of pharmacokinetics of niclosamide, several niclosamide delivery systems were developed, for instance, the niclosamide-conjugated polypeptide nanoparticles, poly(methacrylic acid-niclosamide) polymer, and Niclosamide loaded biodegradable chitosan nanocargoes et al. 10, 17, 19
Pegylation technique modifies the target drug with PEG, thus, improves the drug hydrophilicity and enhances the drug bioavailability 18. Small chemical drugs can also be modified with PEG to improve their pharmacokinetics, for instance, the PEGylated-paclitaxel20. Therefore, we designed the pegylated niclosamide to improve the solubility of niclosamide. As shown in Figure. 5, mPEG5000-Nic is a prodrug of niclosamide which could greatly improve the solubility and stability of niclosamide in water. Niclosamide is STAT3 inhibitor, our results showed that the STAT3 activity of liver was very sensitive to niclosamide and the liver STAT3 activity could be as a marker to monitor the activity of niclosamide in vivo (Figure 8).

CONCLUSION

The antihelminthic niclosamide not only exerts direct antitumor activity, but also overcomes drug-resistance and radio-resistance in multiple types of cancers. However, the insolubility and poor absorption of niclosamide limit its clinical use for cancer treatment. Based on the advantages of pegylation technique, we designed an injectable pegylated niclosamide and proved its antitumor activity in vitro and in vivo. In conclusion, pegylated niclosamide is novel niclosamide delivery system with clinical perspective for cancer therapy.

Competing Interests
The authors have declared that no competing interest exists.

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Figure-1. The synthetic route of mPEG5000-Nic.

Figure 2. (A) The FTIR spectra of niclosamide (Nic) and 5-chloro-N-(2-chloro-4-nitrophenyl)-2-(2- acryloyloxy)-benzamide (Nic-AC). (B) The 1H NMR spectrum of mPEG5000-Nic.

Figure-3. The 1H NMR spectrum of mPEG5000-TDA.

Figure 4. The 1H NMR spectrum of mPEG5000-Nic.

Figure-5. Schematic illustration of mPEG5000-Nic aqueous solution. (A) The illustration the presence of mPEG-Nic in water. (B) The water solubility and micromorphology of mPEG5000-Nic. (C) Niclosamide inhibits STAT3 transcriptional activity by blocking itsvphosphorylation and nuclear translocation

Figure-6. Niclosamide release from mPEG5000-nic in vitro.

Figure-7. Effects of niclosamide and mPEG5000-Nic on cell viability of CT26 and HCT116 colon cancer cells in vitro. Cells were treated for 24 hours and cell viability was measured by using MTT method. The dose of mPEG5000-Nic was presented with the equivalent niclosamide amount. n=6 in each group. mPEG5000-Nic, pegylated niclosamide with mPEG-5000.

Figure-8. Effects of oral niclosamide and intraperitoneal injection of mPEG5000-Nic on STAT3 activity of heart, lung and liver tissues of mice.
(A) Intraperitoneal injection of mPEG5000-Nic inhibited STAT3 activity of liver tissues. Intraperitoneal injection (i.p.)of mPEG5000-Nic was equivalent to 50mg/kg/d niclosamide for 18 days.(B) Oral administration of niclosamide showed no effect on STAT3 activity of heart, lung, liver tissues. Oral administration(i.g.) of niclosamide was 300mg/kg/d for 18days. n=5 in each group. mPEG5000-Nic, pegylated niclosamide with mPEG-5000. *P<0.05 vs control.

Figure-9. Intraperitoneal injection of mPEG5000-Nic inhibited tumor growth in nude mice. CT26 cells were subcutaneously injected into nude mice. When tumor size reached to ~50 mm3, mice were randomly grouped into control and mPEG5000-Nic groups. Intraperitoneal injection of mPEG5000-Nic was equivalent to 50 mg/kg niclosamide. Equal volume of saline was administered in model group. All animals were treated for 16 days. (A) Dissected colorectal xenograft tumors after 16 day treatment. (B) Analysis of nude mice xenograft tumor volumes. *P<0.05, **P<0.01 vs control. (C) Tumor weight decreased in nude mice treated with mPEG5000-Nic for 16 days. **P<0.01 vs model. (D, E) Intraperitoneal injection of mPEG5000-Nic for 16 days had no effect on animal body weight and heart, liver, kidney, and lung weight. mPEG5000-Nic, pegylated niclosamide with mPEG-5000.

Figure-10. Intraperitoneal injection of 5-Fluorouracil (5-FU) inhibited tumor growth in nude mice. CT26 cells were subcutaneously injected into nude mice. When tumor size reached to ~50 mm3, mice were randomly grouped into control and 5-FU groups. (A) 5-FU treatment reduced body weight of mice. **P<0.01 vs control.(B) Analysis of nude mice xenograft tumor volumes. **P<0.01 vs control. (C) Tumor weight decreased in nude mice treated with 5-FU for 20 days. *P<0.05, **P<0.01 vs control.BAY2353