Colorectal cancer (CRC) is the third most commonly diagnosed cancer worldwide. It was estimated that there were over 1.9 million new CRC cases and 935,000 deaths in 2020 according to Global Cancer Statistics (Sung
FoxM1 is an oncogenic transcription factor that plays a significant role in the initiation, progression, metastasis, and drug resistance of a variety of human tumors, including CRC (Chu
Thus, the objective of this study was to investigate the anti-cancer activity of urushiol V and explore its underlying mechanisms using SW480 colon cancer cells. In addition, the efficacy of urushiol V on restoring the antitumor activity of 5-FU in a 5-FU resistant SW480 colon cancer (SW480/5-FUR) cells was determined.
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Human colon cancer SW480 cells were purchased from Korean Cell Line Bank (KCLB; Seoul, Korea). 5-FU-resistant SW480/5-FUR cells were established by repeatedly culturing SW480 cells with constant treatment by 1 μM 5-FU for three months. SW480 and SW480/5-FUR cells were cultured with RPMI1640 (Corning Inc., Corning, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; TCB, Tulare, CA, USA), penicillin (100 U/mL), and streptomycin (10 µg/mL). All cells were maintained in a humidified atmosphere with 5% CO2 at 37°C.
SW480 and SW480/5-FUR cells were seeded into 96-well plates at density of 2×103 cells/well and allowed to adhere overnight. They were incubated with various concentrations of urushiol V or 5-FU. After treatment, MTT solution (0.5 mg/mL) was added and incubated for 2 h at 37°C. Then, 100 µL dimethyl sulfoxide (DMSO) was added to each well and the plate was shaken for 5 min. Absorbance was measured at 540 nm with a microplate reader (Molecular Devices, Sunnyvale, CA, USA).
Cells were seeded into 6-well plates at 200 cells/well and treated with indicated concentrations of urushiol V. After 72 h of treatment, cells were maintained with drug free medium for 12-14 days with media changes every 3 days. Colonies were fixed with 4% formaldehyde and stained with crystal violet (0.05%). The plate was then photographed and the number of colonies was counted using Image J software (NIH, Bethesda, MD, USA).
Treated cells were dispersed and washed with cold phosphate buffered saline (PBS) before adding pre-cooled 70% ethanol. Cells were then washed with PBS and incubated with 500 µL propidium iodide (PI)/RNase solution for 30 min in the dark. Prepared cellular samples were immediately analyzed using a FACS Calibur flow cytometer (BD Biosciences, San Jose, CA, USA).
Total RNA was isolated from the cell pellet using TRIzolTM Reagent (Invitrogen, Carlsbad, CA, USA). First-stand cDNA was synthesized using LabopassTM cDNA synthesis kit (Cosmogenetech, Seoul, Korea) and amplified using a PCR thermal cycler (GeneAmp PCR System 2700, Applied Biosystems, Foster City, CA, USA). The primer sequences used for RT-PCR were as follows: FoxM1 forward, 5′-ATGGCAAAT TTTTCGCTCC-3′; FoxM1 reverse, 5′-ATGTCACCAGAAATTCCCAGTT-3′; β-actin forward, 5’-AAGGGACTTCCTGTAACAACG-3’; β-actin reverse, 5’-AGGATGCAGAAGGAGATCACT-3’. Amplified DNA was separated on 2% agarose gels and stained with ethidium bromide.
Cell lysis, sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE), and Western blotting were performed as described previously (Jeong and Ryu, 2020). Antibodies used were FoxM1 (A301–533A, Bethyl Laboratories, Montgomery, TX, USA), cyclin E1 (ab71535, Abcam, cambridge, UK), cyclin D1, cyclin B1, survivin, phosphorylated AMPK (p-AMPK), total AMPK, phosphorylated mTOR (p-mTOR), total mTOR, c-Myc (#2922, #4138, #2808, #2535, #5831, #5536, #2972 #9402, Cell Signaling Technology, Danvers, MA, USA), p21, TS (sc-10736, sc-33679, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and β-actin (A2066, Sigma Aldrich, St. Louis, MO, USA).
Results are expressed as mean ± standard deviation (SD). The significance of differences among groups was evaluated using Student’s t-test.
To assess the effect of urushiol V on cell proliferation, SW480 cells were exposed to different concentrations (0-150 µM) of urushiol V. Cell proliferation was then measured by MTT assay. Urushiol V inhibited the rate of cell proliferation of SW480 with IC50 values of 33.1, 14.7, and 10.2 µM after treatment for 24, 48, and 72 h, respectively (Fig. 1B). The suppression effect of urushiol V on cell proliferation was confirmed by colony formation assay. Compared with the control group, groups treated with urushiol V at 5, 10, and 20 µM showed decreased numbers of colony formation by 43.1 ± 1.4, 53.9 ± 0.9, and 98.6 ± 0.1%, respectively (Fig. 1C). Taken together, these results indicate that urushiol V can significantly inhibit SW480 colon cancer cell proliferation.
To further elucidate the anti-proliferative mechanisms of urushiol V in SW480 cells, flow cytometry was performed to analyze the distribution of cell cycle phases. As shown in Fig. 2A, urushiol V reduced the number of cells in the G0/G1 phase with a corresponding accumulation in the S phase. The S phase cell cycle population was 22.1%, 28.6%, and 35.8% at 0, 10, and 30 µM urushiol V, respectively.
Moreover, treatment with urushiol V decreased the levels of cyclin E1 and thymidylate synthase (TS) in a concentration-dependent manner. On the other hand, the expression level of p21, known as a tumor suppressor (el-Deiry
FoxM1 is known to be closely related to cell proliferation and cell cycle in cancer cells (Zhang
We next examined effects of urushiol V on mRNA expression of FoxM1 using RT-PCR and found that urushiol V did not affect the mRNA level of FoxM1 (Fig. 3B). These results indicate that urushiol V might inhibit FoxM1 protein expression at the post-transcriptional level.
In order to further understand the mechanisms by which urushiol V decreased FoxM1 expression, we examined the effect of urushiol V on FoxM1 stabilization using cycloheximide (CHX) to prevent protein synthesis. SW480 cells were pre-treated with CHX and then exposed to urushiol V for 16 h. As shown in Fig. 4A, protein level of FoxM1 was further reduced by the co-treatment of CHX and urushiol V compared to that by CHX treatment alone, indicating that urushiol V could affect FoxM1 protein stability.
To confirm the effect of urushiol V on FoxM1 protein degradation, SW480 cells were pre-treated with MG132, a proteasome inhibitor, and chloroquine (CQ), a lysosomal inhibitor, and then treated with urushiol V for 16 h. As shown in Fig. 4B, even in the presence of MG132 and CQ, urushiol V decreased FoxM1 protein levels. Proteasome inhibitors such as MG115, MG132, and bortezomib was known to inhibit FoxM1 transcriptional activity and expression (Bhat
It has reported that
Upregulation of FoxM1 has recently been reported to be closely related to 5-FU resistance in CRC (Xie
We also evaluated levels of drug resistance-related proteins in parental colon cancer cells and their resistant cell lines by western blot. Expression levels of FoxM1 (1.65-fold), free TS (1.64-fold), and complex TS (3.25-fold) in SW480/5-FUR cells were significantly higher than those in parental SW480 cells. Higher TS expression in CRC decreases the efficacy of 5-FU and this is one of the main reasons of resistance of CRC cells to 5-FU (Peters
5-FU is converted to its active metabolite fluoro-deoxyuridine monophosphate (FdUMP) through nucleotide metabolic pathways for thymidine monophosphate (dTMP). It forms a ternary complex with TS and 5,10-methylenetetrahydro-folate (5,10-CH2THF), leading to inhibition of TS (Drake
In summary, we confirmed that upregulation of FoxM1 and TS was associated with resistance to 5-FU. Therefore, inhibition of FoxM1 or TS is a therapeutic strategy for enhancing 5-FU cytotoxicity and antitumor efficacy in SW480/5-FUR cells.
To elucidate whether urushiol V could affect levels of FoxM1 and TS in SW480/5-FUR cells, we treated SW480/5-FUR cells with urushiol V at indicated concentrations for 24 h and then performed western blot assays. Results showed that urushiol V dose-dependently inhibited FoxM1 protein expression in SW480/5-FUR cells. The protein levels of free TS and complex TS were also reduced by urushiol V treatment (Fig. 6A). To further examine the effect of urushiol V in the presence of 5-FU, SW480/5-FUR cells were treated with 30 µM urushiol V and 1 µM 5-FU for 24 h. The protein levels of FoxM1, free TS, and complex TS were increased by treatment of 1 µM 5-FU compared with the control group. However, levels of FoxM1 and free TS (but not complex TS) were significantly reduced by combination treatment of 5-FU with urushiol V (30 µM) compared to those by single 5-FU treatment (Fig. 6B). To evaluate whether urushiol V enhanced the cytotoxicity of 5-FU, SW480/5-FUR cells were treated with indicated concentrations of urushiol V and 1 µM 5-FU for 72 h. Treatment with 1 µM 5-FU did not show any significant effect on proliferation of SW480/5-FUR cells. However, co-treatment of 1 µM 5-FU and 10 µM urushiol V significantly reduced proliferation (Fig. 6C) and clonogenic growth (Fig. 6D) of SW480/5-FUR cells as compared with 10 µM urushiol V only. These findings suggest that urushiol V can restore the cytotoxicity of 5-FU in SW480/5-FUR cells by reducing the expression of FoxM1.
Colorectal cancer (CRC) is the third-most common cancer in the world (Sung
AMPK plays an essential role in cellular energy homeostasis. It controls processes related to tumor development, including cell cycle regulation, cell proliferation, protein synthesis, and survival (Motoshima
mTOR is one of downstream targets of AMPK. It regulates cell growth, cell survival, metabolism, protein synthesis, and transcription (Tian
Increased level of FoxM1 is correlated with resistance to 5-FU in many cancers, including CRC. A recent study has shown that FoxM1 can enhance 5-FU resistance through the regulation of TS, a cellular target of 5-FU chemotherapy in CRC (Varghese
Collectively, our findings showed that urushiol V could inhibit the proliferation of SW480 colon cancer cells by downregulating FoxM1. Urushiol V also enhanced anti-proliferative activity of 5-FU by suppressing the levels of FoxM1 and TS in 5-FU resistant colon cancer cells (SW480/5-FUR) (Fig. 7). These results demonstrate that urushiol V has potential therapeutic and/or adjuvant applications for CRC chemotherapy.
This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (grant number 2021R1A6A3A01086698).
There are no conflicts of interest.