Metastatic colorectal cancer (mCRC) is one of the four major cancer types and is among the leading causes of death in industrialized countries [1
]. Colon cancer develops from normal tissue, which transforms to a dysplastic lesion, adenoma, adenocarcinoma, and finally to an invasive adenocarcinoma [2
]. During this transformation, the epidermal growth factor receptor (EGFR) pathway plays a major role since it is able to regulate cell growth. EGFR is a member of the Human epidermal growth factor receptor (HER) family of receptor tyrosine kinases (RTKs), and its activation stimulates several signaling cascades, leading to RAS
gene mutation. Mutant RAS protein activates the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway which mediatescell proliferation, metastasis and survival. RAS protein activates other downstream signaling cascades such as the phosphatidylinositol-3-kinase (PI3K)/AKT or c-Jun N terminal kinase (JNK) pathways [3
]. Targeting EGFR has been extensively studied in oncology, and monoclonal antibodies (e.g., cetuximab) against the extracellular domain of the EGFR have been developed [4
] as treatments against cancers, including colorectal cancer [6
]. However, this promising therapy was ineffective against v-ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS)-mutated cancers [7
]. Unfortunately, it is estimated that 30–40% of colon cancer patients have a KRAS mutation, giving them very few therapeutic options [5
]. Therefore, developing therapies against KRAS-mutated colon cancer is an important, unmet need [10
In this study, we prepared Phellinuslinteus
grown on germinated brown rice (PBR) extracts to increase the sensitivity of KRAS-mutated colon cancers to cetuximab. P. linteus
(Mesima), a fungus of the family Hymenochaetaceae, is a medicinal mushroom, used widely as a traditional Asian medicine to treat stomachache, inflammation, and tumors. Recent studies have shown that theextract of P. linteus
has anti-inflammatory and antitumor activities [11
]. Furthermore, proteoglycanpurified from P. linteus
suppressed colon cancer by protecting T cells and disrupting the EGFR/AKT pathway [13
], and inhibited SW480 colon cancer cell growth by G2/M phase arrest and suppressed tumor growth in a xenografted model by altering the Wnt/β-catenin pathway [15
]. However, the availability of P. linteus
is limited because of supply shortages and high costs. In this study, we grew P. linteus
on germinated brown rice as an ideal growth medium to address the supply shortage issue [17
]. The G12V KRAS-mutated cell line, SW480, was co-treated with PBR extract and cetuximab, and proliferation and clonogenic features were evaluated. The cause of cell death was investigated using annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining, and effects on the RAS/MAPK pathway were investigated using Western blotting. In addition, PBR extract and cetuximab were administered to mice xenografted with colon cancer cells to investigate their suppressive effect on colon cancer growth.
Approximately 30–40% of patients with colon cancer have a KRAS mutation, and they cannot benefit from cetuximab therapy [5
]. In this study, we demonstrated that PBR extract treatment could sensitize KRAS mutant cells to cetuximab therapy in vitro and in vivo. In Figure 1
, G12V KRAS mutant SW480 cell line growth was evaluated with cetuximab, PBR extract, or acombined treatment of the two. When cetuximab and PBR extract were combined, enhanced antitumor activity (as assessed by cell viability and clonogenic properties) in G12V KRAS mutant colon cancer cells was observed, which was caused by apoptosis. From the RAS/MAPK pathway study, it was concluded that PBR extract can overcome cetuximab resistance via the modulation of KRAS expression and the induction of apoptosis by altering the phosphorylation of MAPK. In a mouse model, the growth of KRAS-mutant xenografted tumors was suppressed when PBR extract was added to cetuximab during treatment.
Several studies have tried to overcome the cetuximab resistance of KRAS-mutated colon cancers, including those using microRNA [19
], L-ascorbic acid, lauric acid, cisplatin, dasatinib, and simvastatin. Cetuximab sensitization was achieved with dasatinib and simvastatin [20
]; however, the requirement of adding more drugs limited the clinical application. Recently, the induction of sensitivity with natural products, such as ascorbic acid and lauric acid, was studied. Weng et al. demonstrated that lauric acid significantly improved sensitivity to cetuximabwith KRAS and BRAF mutants [22
]. Lauric acid can be easily obtained from natural food, and it is reportedly harmless to the cardiovascular system. Ascorbic acid can abrogate cetuximab resistance in mutant KRAS human colon cancers [23
]. This enhanced antitumor activity of cetuximaboccurred via the inhibition of cellular proliferation or the induction of apoptosis in various cancer cell types.
In the present study, adding PBR extract increased cell sensitivity to cetuximab, achieving an equivalent suppressive effect with lower cetuximab concentrations. That might result from functional compounds in the PBR extract. Compared to the levels in whole P. linteus
, γ-aminobutyricacid (GABA) and β-glucan concentrations are high and ergosterol peroxide is present in the PBR extract. The GABA and β-glucan concentrations were found to be 141.14 and 29.20 mg/g on a dry weight basis, respectively, which are four-fold and 7.5-fold higher, respectively, than those found in whole P. linteus
(CARI homepage, http://www.cellacti.com/sub03_01.php
). The ergosterol peroxide content in PBR extract was found to be 1200 ppm, but it wasnot found in whole P. linteus
]. GABA is the main inhibitory neurotransmitter in the vertebrate brain and is involved in the proliferation, migration, and survival of neurons in fetal and newborn mammalian brains [25
]. Interestingly, GABA inhibits the migration of colon cancer cells, which delays cancer invasion and metastasis. GABA has an inhibitory action on cancer cell proliferation and stimulates cancer cell apoptosis [26
]. Ergosterol peroxide is an antitumor sterol produced by mushrooms that shows inhibitory activity against human colon cancer cells [27
]. Further, β-glucan provides beneficial health effects that are derived from its antitumor, anti-infectious disease, and anti-atherosclerosis properties [28
]. Interestingly, β-glucan was able to enhance antitumor effects by synergizing with monoclonal antibodies against various antigens, tumor types, or tumor sites [29
]. Since β-glucan showed synergistic effects with EGFR [30
], the increased sensitivity of cetuximab combined with PBR extract could result from the high concentrations of β-glucan in the PBR extract. Investigating the role of β-glucan as a sensitizer of cetuximab resistance deserves further study.
To our knowledge, this is the first report to show the potential role of PBR extract to overcome cetuximab resistance in KRAS-mutant colon cancer. Our current study combined the antitumor monoclonal antibody drug, cetuximab, and the functional food, PBR extract, to act synergistically against KRAS-mutant colon cancer cells. This synergistic effect could result from β-glucan in the PBR extract. From the data provided, it appears that PBR extract can sensitize KRAS mutant tumors to cetuximab toxicity. PBR is a food that is consumed in daily life. Therefore, these findings highlight a straightforward and practical therapeutic for CRC patients with KRAS mutations, which is currently an unmet need.
4. Materials and Methods
4.1. Preparation of PBR Extract
PBR was provided by Cell Activation Research Institute (CARI; Kyungji-Do, Korea). Dried, 6-week-old PBR was ground into a fine powder using a grinder. The powder (2 kg) was extracted with ethanol and ethyl acetate at 20–25 °C. After filtration, the ethanolic extracts were dried with a rotary evaporator under vacuum, and the dried extract was stored at −20 °C. Authenticated voucher specimens of PBR (Kucari 0905) were deposited in the CARI (Kyungji-Do, Korea).
4.2. Cell Viability by MTS Proliferation Assay and DAPI/PI Staining
The SW480and HT-29cell line was purchased from American Type Culture Collection (ATCC, Rockville, MD, USA) and were cultured in regular media consisting of RPMI-1640 (Invitrogen, Carlsbad, CA, USA), 10% fetal bovine serum (FBS; Invitrogen), and 100 U/mL penicillin-streptomycin (Sigma, St. Louis, MO, USA). For the cell viability assay, cells were seeded into 96-well plates (20,000 cells/well) in triplicate. Twenty-four hours later cells were treated with samples (10 and 30 µg/mL cetuximab, 100 µg/mL PBR extract, or their combination) and incubated for 3 days. At the end of the incubation, cell viability was determined by adding (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) reagent for 1 h at 37 °C, according to manufacturer’s instructions (MTS Assay, Promega, Fitchburg, WI, USA). Absorbance readings were acquired at 490 nm (Model 550; Bio-Rad, Hercules, CA, USA), and the reduction in cell growth was calculated as a percentage of absorbance in the absence of treatment.
4.3. Live/Dead Staining
For microscopic observation, cells were fixed with 4% paraformaldehyde (Sigma) in phosphate-buffered saline (PBS for 10 min at room temperature, and were then washed with PBS three times. Fixed cells were stained with 1 µg/mL of DAPI (Sigma) for 10 min at room temperature. The stained cells were then observed under an inverted fluorescence microscope (EVOS®, Thermo Fisher Scientific, Waltham, MA, USA).
4.4. Clonogenic Assay
Cells were seeded in 6-well plates (2000 cells/well) for the colony formation assay. Following 24h incubation, cells were treated with cetuximab, PBR extract, or their combination. After 10 days, cells were washed with PBS and fixed with 4% paraformaldehyde for 5 min. The colonies were stained with 1% crystal violet (Sigma) for 30 s, and were rinsed three times with PBS followed by air-drying.
4.5. Flow Cytometry
The extent of SW480 apoptosis after treatment was evaluated using an Annexin V-FITC/PI apoptosis detection kit (BD Biosciences, San Jose, CA, USA), according to the manufacturer’s instructions. SW480 colon cancer cells were seeded in a six-well plate (5 × 105 cells/well) overnight, and were then treated with cetuximab (10, 30 or 100 μg/mL), PBR extract (100 μg/mL), or a combination of cetuximab and PBR extract. After 48 h, cells were harvested, washed in ice-cold PBS, and collected by centrifugation at 500× g for 10 min. Cells were stained simultaneously with FITC-labeled annexin V (5 µL) and PI (5 µL) at room temperature for 10 min, protected from light. Stained cells were analyzed using a fluorescence-activated cell sorter flow cytometer (FC 500 Series Flow Cytometry, Beckman Coulter, Indianapolis, IN, USA). At least 10,000 cells were used for each analysis, and experiments were performed in triplicate.
4.6. Western Blotting
Cells were washed three times in cold PBS, and protein lysates were obtained by extraction with RIPA buffer containing protease inhibitors (Sigma). Proteins were separated on 10% pre-cast SDS-PAGE gels (Bolt™ 4–12% Bis-Tris Plus Gels) (Invitrogen), and were then transferred using the iBlot system (Life Technology). Membranes were incubated with the following primary antibodies: rabbit anti-KRAS, anti-phospho-p42/44 MAPK (ERK1/2), anti-p42/44 (ERK1/2), anti-phospho-Akt, anti-phospho-p38 MAPK, and anti-p38 (all from Cell Signaling, Danvers, MA; used at 1:1000 dilution); rabbit anti-Akt (1:1000, Santa Cruz Biotechnology, Dallas, TX, USA); and mouse anti-β-actin (1:4000, Sigma). Membranes were then incubated with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse antibodies (Pierce Biotechnology Inc., Rockford, IL, USA), and detected using a SuperSignal® West Femto enhancer kit (Pierce). Protein intensity was analyzed by Image J (NIH, Bethesda, MD, USA) in triplicate. The ratios of KRAS/β-actin, phosphor-AKT/AKT, phosphor-p42/44/p42/44 andphosphor-p38/p38 were calculated. The expression level was compared to no treatment group.
4.7. Animal Experiment
The inhibitory effect of cetuximab, PBR extract, and their combination on colon cancer growth was investigated in an animal model. Athymic nude mice (sixweeks, male) were purchased from BioToxTech animal center (Cheongju, Korea). All animals were handled following the guidelines of the Institutional Animal Care and Use Committee (IACUC) at Silla University. Animal protocol was reviewed and approved by IACUC at 01 December 2016 (#160782). Mice were injected bilaterally in the dorsal flank with SW480 cells (5 × 106
]. The mice were treated twice per week with an intraperitoneal injection of 10 mg/kg cetuximab in PBS. PBR extract was administered by oral gavage (400 mg/kg/day) daily. Once tumors reached 100 mm3
, mice were started on their respective treatments (cetuximab, PBR extract, combination of cetuximab and PBR extract, or saline control, n
= 7/group). Tumor volumes were calculated using the following formula: V
)/2, where V
= volume, L
= length and W
= width. The mice were euthanized on day 23, and tumor viscera were imaged with a digital camera.
4.8. Statistical Analysis
Data are expressed as the means ± standard error of the mean (SEM). Statistical analyses were performed by one-way analysis of variance (ANOVA), using the SPSS software, version 12 (SPSS Inc., Chicago, IL, USA). Differences were considered significant at p < 0.05.