Anticancer and Antimigration Effects of a Combinatorial Treatment of 5

This study aimed to investigate the chemotherapeutic effects of Lactobacillus paracasei subsp. paracasei NTU 101-fermented skim milk (NTU101-FM) extra...
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Cite This: J. Agric. Food Chem. 2018, 66, 5549−5555

Anticancer and Antimigration Effects of a Combinatorial Treatment of 5‑Fluorouracil and Lactobacillus paracasei subsp. paracasei NTU 101 Fermented Skim Milk Extracts on Colorectal Cancer Cells Chia-Yuan Chang and Tzu-Ming Pan*

J. Agric. Food Chem. 2018.66:5549-5555. Downloaded from pubs.acs.org by UNIV OF TOLEDO on 09/27/18. For personal use only.

Department of Biochemical Science & Technology, National Taiwan University, Number 1, Section 4, Roosevelt Road, Taipei 10617, Taiwan ABSTRACT: Colorectal cancer (CRC) is one of the most prevalent cancers worldwide. Metabolites of lactic acid bacteria (LAB) have anticancer and antimetastasis capacities. This study aimed to investigate the chemotherapeutic effects of Lactobacillus paracasei subsp. paracasei NTU 101-fermented skim milk (NTU101-FM) extracts in combination with the chemotherapeutic drug 5-fluorouracil (5-FU) in a cellular CRC model. The NTU101-FM extracts effectively reduced CRC cell viability but were not cytotoxic to colon epithelial cells. Moreover, they increased RAW 264.7 cell viability. Notably, the cell viability of CRC cells was decreased by 5-FU in combination with the NTU101-FM extracts; the combinatorial treatment inhibited cell viability significantly more than 5-FU alone (p < 0.05). An ethanol extract of NTU101-FM effectively attenuated CT26 cell migration. In conclusion, the ethanol extract prepared from NTU101-FM has a potential application as an anticancer agent in CRC. KEYWORDS: Lactobacillus paracasei subsp. paracasei NTU 101, fermented skim milk extract, colorectal cancer, 5-fluorouracil, anticancer properties

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isolated from human neonatal feces.16 The components of NTU 101 have been reported to have ameliorative effects on hypercholesterolemia,17 obesity,18 and diabetes;19 prevent hypertension-induced vascular dementia;20 play a key role in immunomodulation;21,22 present anticancer abilities;11,23 and have other benefits. However, few studies have investigated whether similar lactic acid bacteria (LAB)-fermented extracts have any adjuvant chemotherapeutic activity. Furthermore, the effects of NTU 101-fermented extracts on chemotherapy are unknown.

INTRODUCTION Colorectal cancer (CRC) is one of the most prevalent forms of cancer worldwide.1 Statistically, for the estimated cancer-related mortalities in North America in 2017, CRC-related mortality exceeded that of lung cancer in men and those of lung and breast cancer in women.2 Recently, various treatments for CRC, such as surgery, chemotherapy, radiation therapy, immunotherapy, and others, have been reported.3 Surgery combined with chemotherapy is currently one of the most common and effective CRCtreatment modalities. Although chemotherapy is effective for CRC, current chemotherapeutic drugs indiscriminately target both healthy and malignant cells,4 thereby resulting in numerous deleterious side effects, including cardiotoxicity,5 hematopoietic suppression,6 vomiting, and fatigue, all of which affect quality of life and can even lead to death.7 Thus, numerous studies have focused on the extraction and isolation of antitumor compounds from natural foods,8 the usage of which can reduce the excessive usage of chemotherapeutic drugs, thereby indirectly reducing the possibility of these side effects arising. In addition, numerous studies have indicated that the colonic microflora are involved in the etiology of CRC8 and that the maintenance of a balance in the colonic microflora, through supplementation with probiotics, could prevent CRC.9 Probiotics are live microorganisms that maintain the balance of colonic microflora and have positive effects on the host. Lactobacilli are one of the most illustrious probiotic bacteria,10 are generally recognized as safe (GRAS), and are commonly used in food industry. The components from Lactobacillus strains include the live bacteria,10 heat-killed cells,11 cell wall,12 cytoplasmic fractions,11 and Lactobacillus-fermented products,13 which attenuate the effects of CRC by inducing apoptosis14 and promoting immunomodulatory effects.15 In our laboratory, we have screened a Lactobacillus strain, Lactobacillus paracasei subsp. paracasei NTU 101 (NTU 101), © 2018 American Chemical Society

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MATERIALS AND METHODS

Materials. CT26, HT-29, Caco-2, and RAW 264.7 cells were purchased from the Bioresource Collection and Research Center (BCRC, Hsinchu, Taiwan). Dimethyl sulfoxide (DMSO) and 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich Corporation (St. Louis, MO). Fetal bovine serum (FBS), Roswell Park Memorial Institute (RPMI) 1640 medium, and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from HyClone Laboratories (Logan, UT). Two-well silicone cell-culture inserts were purchased from Blossom Biotechnologies (Taipei, Taiwan). Fermentation of Skim Milk with NTU 101 and Subsequent Extraction. For the fermentation, 25% (w/v) skim milk (Anchor, Auckland, New Zealand) was pasteurized in a water bath at 90 °C for 1 h, cooled to 37 °C, and inoculated with a 1% (v/v) suspension of NTU 101. Thereafter, it was incubated at 37 °C for 72 h before being freezedried using an SDF-25 freeze-dryer (Chang Jung Business Company, Feng-Jen, Taiwan). Furthermore, 95% ethanol or deionized water was used to extract the freeze-dried to NTU 101-fermented skim milk Received: Revised: Accepted: Published: 5549

March 23, 2018 May 7, 2018 May 8, 2018 May 8, 2018 DOI: 10.1021/acs.jafc.8b01445 J. Agric. Food Chem. 2018, 66, 5549−5555

Article

Journal of Agricultural and Food Chemistry (NTU101-FM) powders after their sonication and incubation at 37 °C for 1 h. The extracts were then centrifuged at 4 °C and 10,000 × g for 30 min, and the supernatants were dried in a rotary evaporator and stored at −20 °C until use. Cell Culture. The cell lines used in our study included RAW 264.7 mouse leukemic monocyte macrophages and Caco-2 human colon epithelial cells, which were cultured in DMEM. HT-29 human colorectal cancer cells and CT26 murine colon carcinoma cells were cultured in RPMI-1640 medium. All cell culture media were supplemented with 10% FBS and incubated at 37 °C, 95% humidity, and 5% CO2. The cells grew as monolayers in 10 cm2 cell-culture dishes. Media were replenished every 48 to 72 h. Cells were passaged by being dislodged from the dishes using 0.25% trypsin-EDTA (the RAW 264.7 cells were dislodged using a cell scraper) and transferred to new dishes when required. Cell Viability. To evaluate the cell-viability effects of the NTU 101FM extracts on the RAW 264.7, Caco-2, HT-29, and CT26 cells, the MTT assay24 with certain modifications was used. Briefly, the aforementioned cells were seeded at a density of 5 × 104 cells/well in a 24-well plate. For cell adherence, the plate was preincubated for 24 h. The cells were then incubated with either NTU101-FM extracts (water or ethanol extracts) at various concentrations or a chemotherapeutic drug (5-FU or UFUR) at 37 °C for 24 h. Thereafter, MTT assays were performed, and cell viability was calculated using the following formula: cell viability (% of control) = (ODsample/ODcontrol) × 100%. Wound-Healing Assay. CT26 cells were seeded in 24-well cellculture plates with silicon culture inserts. Each culture insert provides two cell-culture reservoirs separated by a 500 μm wall. The density of the cell suspension was adjusted to 5 × 105 cells/mL, and 70 μL of the cell suspension was seeded into each culture reservoir. After 24 h of incubation, the culture insert was gently removed, thereby creating a 500 μm wide wound. After the removal of the insert, cell debris and nonattached cells were eliminated by washing the cell layer with phosphate-buffered saline (PBS). Thereafter, the sample or cell-free medium was readded to fill the 24-well cell-culture plates. The plates were then photographed at indicated time points, wherein the wound areas and migrating-cell numbers were analyzed using ImageJ software (NIH, Bethesda, MD). Statistical Analysis. All experiments were performed in triplicate, and the data are presented as means ± standard-deviation (SD) values. Data were analyzed using a statistical software (SPSS, IBM Software, Armonk, NY), and Duncan’s multiple-range test was performed to compare each group. The threshold for statistically significant differences was set as p < 0.05.

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RESULTS AND DISCUSSION Antiproliferative Effects of NTU101-FM Extracts against Colon Carcinoma Cells. Probiotic bacteria and their fermented products are reported to have antitumor activities.25,26 Shahani and Ayebo27 previously reported that consuming marked amounts of lactobacilli- or bifidobacteria-fermented milk products could reduce the incidence of CRC. These may decelerate CRC progression by affecting metabolism and the immune system, thereby protecting the colon. Furthermore, they may inhibit colon-tumor growth through the production of antitumorigenic or antimutagenic compounds.25,28 LAB notably decreased the cell viability of HT-29 cells.29 Furthermore, Deepak et al.30 reported that L. acidophilus exhibits distinct anticancer activity against colon cancer. In this study, we used HT-29 and CT26 cells as in vitro models of colon adenocarcinoma to evaluate the potential antiproliferative activities of water extracts (WE) and ethanol extracts (EE) of NTU101-FM. The inhibitory activities of EE and WE at different concentrations (250−1000 μg/mL) against HT-29 and CT26 cells are shown in Figure 1A,B. The EE and WE at 500 μg/mL significantly inhibited cell viability in HT-29 cells (p < 0.05). The EE and WE significantly inhibited CT26 cell viability by 18.9 and

Figure 1. Effects of NTU101-FM extracts on the viability of HT-29 (A), CT26 (B), Caco-2 (C), and macrophage RAW 264.7 (D) cells. The above cells were treated with the extracts of NTU101-FM for 24 h. The amounts of viable cells were determined by MTT assays. Data are presented as means ± SD (n = 3). Values bearing different lowercase and uppercase letters within each group are significantly different (p < 0.05) according to Duncan’s multiple-range test. WE, water extracts of NTU 101-fermented skim milk; EE, ethanol extracts of NTU 101-fermented skim milk.

13.7%, respectively, at 250 μg/mL (p < 0.05); as the concentration of EE increased to 1000 μg/mL, the inhibition increased to 23.5%. Previously, NTU 101 was shown to have immunomodulatory21,22 and anticancer effects.11,23 Therefore, we concluded that our extracts of NTU101-FM contained specific anticancer components that act against CRC. Inhibitory Effects of the NTU101-FM Extract on Caco-2 and RAW 264.7 Cell Viability. Having established that NTU101-FM extracts significantly inhibit cancer cell viability, 5550

DOI: 10.1021/acs.jafc.8b01445 J. Agric. Food Chem. 2018, 66, 5549−5555

Article

Journal of Agricultural and Food Chemistry

Figure 2. Effects of chemotherapy drugs on the viability of CT26 cells (A,B) and HT-29 cells (C,D). The CT26 cells and HT-29 cells were treated with the different chemotherapy drugs for 24 h. The amounts of viable cells were determined by MTT assays. Data are presented as means ± SD (n = 3). Values bearing different uppercase letters within each group are significantly different (p < 0.05) according to Duncan’s multiple-range test. 5-FU, 5-fluorouracil.

we attempted to confirm a safe dose of NTU101-FM extracts for normal cells. We used Caco-2 and RAW 264.7 cells as controls to confirm the inhibitory effects of the NTU101-FM extracts. Caco2 cells, although derived from a colon carcinoma, differentiate in a manner similar to normal cells of the small intestine when cultured in a particular way.31 In 1993, Caco-2 cells were used to investigate the potential toxic effects of drugs or foods on the intestinal mucosa.32 As shown in Figure 1C, when Caco-2 cells were treated with 250−1000 μg/mL NTU101-FM extracts, no

Figure 3. Inhibitory effects of the extracts of NTU101-FM in combination with 5-FU on CT26 (A,B) and HT-29 (C,D) cell growth. Data are presented as means ± SD (n = 3). Values bearing different uppercase letters within each group are significantly different (p < 0.05) according to Duncan’s multiple-range test. WE, water extracts of NTU 101-fermented skim milk; EE, ethanol extracts of NTU 101-fermented skim milk; 5-FU, 5-fluorouracil. 5551

DOI: 10.1021/acs.jafc.8b01445 J. Agric. Food Chem. 2018, 66, 5549−5555

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Journal of Agricultural and Food Chemistry

Figure 4. Suppression of migration of colon cancer cells by WE, EE, or 5-FU in vitro. WE, water extracts of NTU 101-fermented skim milk; EE, ethanol extracts of NTU 101-fermented skim milk; 5-FU, 5-fluorouracil.

Table 1. Inhibitory Effects of the Water Extracts of NTU101-FM and 5-FU on CT26 Cell Migrationa wound area (%)

cell numbers

group (μg/mL)

0h

24 h

48 h

0h

24 h

48 h

control 5-FU (1.25) 5-FU (2.5) WE (500) WE (1000)

100.0 ± 2.5 A 100.0 ± 2.9 A 100.0 ± 3.8 A 100.0 ± 1.6 A 100.0 ± 2.6 A

68.4 ± 11.1 B 93.3 ± 1.9 A 94.8 ± 8.5 A 85.4 ± 14.6 AB 74.3 ± 6.9 B

35.7 ± 7.9 C 103.9 ± 11.8 A 100.7 ± 9.3 A 66.5 ± 14.8 B 77.4 ± 9.0 B

6.7 ± 4.0 A 5.7 ± 3.1 A 5.0 ± 1.0 A 3.7 ± 2.1 A 3.3 ± 1.5 A

155.3 ± 48.3 A 157.3 ± 8.3 A 115.7 ± 29.3 A 161.0 ± 30.0 A 155.7 ± 13.2 A

292.3 ± 57.8 A 107.0 ± 23.4 B 112.3 ± 45.1 B 293.7 ± 94.4 A 218.3 ± 91.5 AB

Data are presented as means ± SD (n = 3). Values with different uppercase letters within each column were significantly different according to Duncan’s multiple-range tests (p < 0.05). 5-FU, 5-fluorouracil; WE, water extracts of NTU 101-fermented skim milk. a

Table 2. Inhibitory Effects of the Ethanol Extracts of NTU101-FM and 5-FU on CT26 Cell Migrationa wound area (%)

cell numbers

group (μg/mL)

0h

24 h

48 h

0h

24 h

48 h

control 5-FU (1.25) 5-FU (2.5) EE (500) EE (1000)

100.0 ± 2.5 A 100.0 ± 2.9 A 100.0 ± 3.8 A 100.0 ± 1.6 A 100.0 ± 0.9 A

68.4 ± 11.1 B 93.3 ± 1.9 A 94.8 ± 8.5 A 92.5 ± 10.4 A 96.1 ± 2.3 A

35.7 ± 7.9 C 103.9 ± 11.8 A 100.7 ± 9.3 A 82.5 ± 7.2 B 97.8 ± 0.8 A

6.7 ± 4.0 A 5.7 ± 3.1 A 5.0 ± 1.0 A 4.7 ± 3.2 A 6.3 ± 3.1 A

155.3 ± 48.3 A 157.3 ± 29.3 A 115.7 ± 30.0 A 143.3 ± 8.7 A 42.7 ± 20.5 C

292.3 ± 57.8 A 107.0 ± 23.4 B 112.3 ± 45.1 B 190.3 ± 40.5 AB 95.0 ± 9.5 B

a Data are presented as means ± SD (n = 3). Values with different uppercase letters within each column were significantly different according to Duncan’s multiple-range tests (p < 0.05). 5-FU, 5-fluorouracil; EE, ethanol extracts of NTU 101-fermented skim milk.

evaluated using MTT assays in HT-29 and CT26 cells. Figure 2 indicates the effects of increasing concentrations of 5-FU or UFUR on the viability of CT26 and HT-29 cancer cell lines. 5FU significantly inhibited HT-29 and CT26 cell growth compared with UFUR (p < 0.05). The inhibition of CT26 cell growth by 5-FU at 10 μg/mL was >2-fold that of UFUR; furthermore, 5-FU was 18.9% more effective than UFUR in inhibiting HT-29 cell growth. These results indicate that 5-FU has prominent cytotoxic effects on CRC cells. Using these data, we could calculate the IC50 values of 5-FU on CT26 and HT-29 cells, which were estimated to be 8.3 and 20.6 μg/mL, respectively. We therefore decided to use 5-FU at concentrations below its IC50 values in subsequent experiments. Antiproliferative Effects of the Combinatorial Treatment of NTU101-FM Extracts with 5-FU. Although chemo-

cytotoxic effects were observed. Similarly, no cytotoxic effects were observed for RAW 264.7 cells (Figure 1D). This concentration range (250−1000 μg/mL) promoted the proliferation of RAW 264.7 cells. The activation or proliferation of RAW 264.7 cells regulate immune mediators, thereby resulting in potentially protective effects on immune-related diseases.33,34 These results indicate that NTU101-FM extracts have potential immunomodulatory effects and do not damage the intestinal mucosa. Effects of Chemotherapeutic Agents on the Cell Viability of HT-29 and CT26 Colon Cancer Cells. Chemotherapy is presently one of the most general and widely used treatments for CRC. 5-FU35 and UFUR36 have been diffusely used as first-line chemotherapeutic drugs for CRC for many years. The effects of 5-FU or UFUR on cell viability were 5552

DOI: 10.1021/acs.jafc.8b01445 J. Agric. Food Chem. 2018, 66, 5549−5555

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inhibition of cell proliferation when 5-FU was simultaneously administrated with NTU101-FM EE compared with that due to the 5-FU monotherapy. Suppression of the Migration of CT26 Colon Cancer Cells in Vitro by NTU101-FM Extracts. Although chemotherapy and surgery effectively control many types of cancers at the primary site and cancer metastasis and proliferation, they result in poor prognoses.43 Furthermore, tumor metastasis is a major factor contributing to cancer-related mortalities. The primary metastatic sites for colon cancer are the liver, lung, and peritoneum.44−46 Metastasis is a complex process including migration and cancer-cell invasion.47 Friedl and Wolf48 reported that increasing migration potentials of cancer cells underlie tumor invasion. Therefore, inhibiting cancer-cell migration could be a potential target for antimetastatic therapy. In this context, we investigated the potential antimigratory effects of NTU101-FM extracts on CT26 colon cancer cells in vitro through a woundhealing assay. As shown in Figure 4, the wound-healing assay for the CT26 cells revealed that the 5-FU and NTU101-FM extracts significantly suppressed cell motility compared with that of the control group (p < 0.05). Cell-migration rates were quantified using ImageJ software (NIH, Bethesda, MD; Tables 1 and 2). As shown in Table 1, CT26 cells treated with the WE of NTU101FM (500 and 1000 μg/mL) after 48 h displayed a significant inhibition of cell migration compared with that of the control group (p < 0.05). The wound areas of the 500 and 1000 μg/mL NTU101-FM WE treatments were 66.5 and 77.4%, respectively. However, Table 2 indicates that CT26 cells treated with the EE of NTU101-FM (500 and 1000 μg/mL) only needed 24 h to present a significant inhibition in cell migration, and their wound areas were 92.5 and 96.1%, respectively (p < 0.05). Nonetheless, upon treatment with the EE of NTU101-FM after 48 h, the wound areas were still above 80%. Furthermore, the EE of NTU101-FM (1000 μg/mL) displayed antimigration effects compared with 5-FU alone; however, the difference was not significant. These results indicate that EE exhibited a better antimigration effect than WE on the CT26 cells. Attenuation of the Migration of CT26 Colon Cancer Cells in Vitro by NTU101-FM Extracts Combined with 5FU. Having established that the EE of the NTU101-FM extracts has the same inhibitory ability as 5-FU, we evaluated the antimigration effects of 5-FU combined with the EE of NTU101FM on CT26 cells. However, as shown in Table 3, comparing the antimigration ability of combined treatment with that of only 5FU did not yield any significant differences. Our results clarify that LAB and their fermentation products are important in the treatment of CRC. NTU101-FM extracts displayed significant antiproliferative activities against HT-29 and CT26 cells. Furthermore, the effective dosage of the NTU101-FM extracts did not display any cytotoxic effects on Caco-2 and RAW 264.7 cells. Notably, when the HT-29 and CT26 cells were treated with a combination of the NTU101-FM extracts and 5-FU, the antiproliferative activity was significantly better than that of the monotherapy with 5-FU. In addition, the dosage of 5-FU can be reduced, which thereby reduces chemotherapeutic-drug-induced cytotoxicity. On the basis of these results, we infer that NTU101-FM extracts have the potential to reduce side effects caused by chemotherapeutic drugs. Furthermore, NTU101-FM extracts could enhance immune responses by increasing the proliferation of RAW 264.7 cells. Finally, we showed that NTU101-FM extracts inhibited the migration of CT26 cells. Furthermore, the migration-inhibitory effect of the EE of NTU101-FM was better

Table 3. Inhibitory Effects of the Ethanol Extracts of NTU101FM in Combination with 5-FU on CT26 Cell Migrationa wound area (%) group (μg/mL)

0h

24 h

48 h

control 5-FU (1.25) 5-FU (2.5) EE (500) EE (1000) 5-FU (1.25) + EE (500) 5-FU (1.25) + EE (1000) 5-FU (2.5) + EE (500) 5-FU (2.5) + EE (1000)

100.0 ± 4.6 A 100.0 ± 3.8 A 100.0 ± 1.6 A 100.0 ± 0.9 A 100.0 ± 0.9 A 100.0 ± 9.5 A

70.4 ± 9.1 C 100.1 ± 5.3 AB 98.4 ± 5.9 AB 87.2 ± 2.9 B 97.2 ± 3.4 AB 102.7 ± 13.4 A

43.2 ± 8.7 D 101.4 ± 7.0 BC 107.9 ± 10.2 B 91.6 ± 3.8 C 105.1 ± 3.1 BC 100.0 ± 3.5 BC

100.0 ± 5.5 A

87.8 ± 10.4 AB

101.2 ± 7.9 BC

100.0 ± 3.4 A

101.4 ± 7.7 AB

109.5 ± 7.1 B

100.0 ± 4.8 A

95.0 ± 3.0 AB

152.1 ± 7.5 A

Data are presented as means ± SD (n = 3). Values with different uppercase letters within each column were significantly different according to Duncan’s multiple-range tests (p < 0.05). 5-FU, 5fluorouracil; EE, ethanol extracts of NTU 101-fermented skim milk.

a

therapeutic drugs are widely used for treating CRC, 5-FU and UFUR have two limitations. First, there are several side effects, which may affect the quality of life of the patients.37 Second, the therapeutic effects of single uses of 5-FU in advanced colon cancer are low.38,39 Therefore, it was important to identify appropriate substances that can be combined with 5-FU or UFUR to reduce their side effects and enhance their therapeutic effects. Increasing evidence suggests that oxaliplatin in combination with 5-FU significantly increased the effectiveness of its treatment of colon cancer.40 In addition, many commonly used Chinese herbs, such as Panax notoginseng41 and curcumin,42 have synergistic antiproliferative effects on colon cancer cells. However, few studies have investigated whether LAB-fermented products have any effects on colon cancer when administered in combination with chemotherapeutic drugs. Thus, we investigated the antiproliferative effects of combinatorial treatments of NTU101-FM extracts with 5-FU. Eight treatment groups were designed, as shown in Figure 3. In the previous experiment, we examined the effects of 5-FU and the NTU101-FM extracts alone on CT26 and HT-29 cell viability. Figure 3A,B show the combined antiproliferative effects of the NTU101-FM extracts and 5-FU on CT26 cells. The results suggest that NTU101-FM WE combined with 5-FU could not effectively inhibit cell proliferation; the inhibitory effects were equipotent to those of the 5-FU monotherapy group (Figure 3A). However, as seen in Figure 3B, compared with the single administration of 5-FU (2.5 μg/mL), the group of NTU101-FM EE combined with 5-FU (2.5 μg/mL of 5-FU combined with 500 or 1000 μg/mL of EE) yielded an inhibitory rate increases by 18.9 and 24.3%, respectively. Figure 3C,D represents the combined antiproliferative effects of the NTU101-FM extracts and 5-FU on HT-29 cells. In this part, compared with the single-administration-5-FU group (2.5 μg/mL), NTU101-FM WE combined with 5-FU (1.25 μg/mL 5-FU combined with 1000 μg/mL WE and 2.5 μg/ mL 5-FU combined with 500 or 1000 μg/mL WE) increased the inhibitory rate of proliferation by 9.8, 10.8, and 21.3%, respectively. The rate of inhibition of NTU101-FM EE combined with 5-FU (1.25 or 2.5 μg/mL 5-FU combined with 500 or 1000 μg/mL EE) also increased to at least 21.4%. These results suggest that EE enhances the sensitivity of the CT26 and HT-29 cells to the 5-FU treatment, leading to a more effective 5553

DOI: 10.1021/acs.jafc.8b01445 J. Agric. Food Chem. 2018, 66, 5549−5555

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(12) Tuo, Y. F.; Zhang, L. W.; Yi, H. X.; Zhang, Y. C.; Zhang, W. Q.; Han, X.; Du, M.; Jiao, Y. H.; Wang, S. M. Short communication: antiproliferative effect of wild Lactobacillus strains isolated from fermented foods on HT-29 cells. J. Dairy Sci. 2010, 93, 2362−2366. (13) Wang, S. M.; Zhang, L. W.; Fan, R. B.; Han, X.; Yi, H. X.; Zhang, L. L.; Xue, C. H.; Li, H. B.; Zhang, Y. H.; Shigwedha, N. Induction of HT29 cells apoptosis by lactobacilli isolated from fermented products. Res. Microbiol. 2014, 165, 202−214. (14) Choi, S. S.; Kim, Y.; Han, K. S.; You, S.; Oh, S.; Kim, S. H. Effects of Lactobacillus strains on cancer cell proliferation and oxidative stress in vitro. Lett. Appl. Microbiol. 2006, 42, 452−458. (15) Tuo, Y. F.; Zhang, L. W.; Han, X.; Du, M.; Zhang, Y. C.; Yi, H. X.; Zhang, W. Q.; Jiao, Y. H. In vitro assessment of immunomodulating activity of the two Lactobacillus strains isolated from traditional fermented milk. World J. Microbiol. Biotechnol. 2011, 27, 505−511. (16) Lin, F. M.; Chiu, C. H.; Pan, T. M. Fermentation of a milk-soymilk and Lycium chinense Miller mixture using a new isolate of Lactobacillus paracasei subsp. paracasei NTU 101 and Bif idobacterium longum. J. Ind. Microbiol. Biotechnol. 2004, 31, 559−564. (17) Lin, C. H.; Chen, Y. H.; Tsai, T. Y.; Pan, T. M. Effects of deep sea water and Lactobacillus paracasei subsp. paracasei NTU 101 on hypercholesterolemia hamsters gut microbiota. Appl. Microbiol. Biotechnol. 2017, 101, 321−329. (18) Cheng, M. C.; Tsai, T. Y.; Pan, T. M. Anti-obesity activity of the water extract of Lactobacillus paracasei subsp. paracasei NTU 101 fermented soy milk products. Food Funct. 2015, 6, 3522−3530. (19) Hung, S. C.; Tseng, W. T.; Pan, T. M. Lactobacillus paracasei subsp paracasei NTU 101 ameliorates impaired glucose tolerance induced by a high-fat, high-fructose diet in Sprague-Dawley rats. J. Funct. Foods 2016, 24, 472−481. (20) Cheng, M. C.; Pan, T. M. Prevention of hypertension-induced vascular dementia by Lactobacillus paracasei subsp. paracasei NTU 101fermented products. Pharm. Biol. 2017, 55, 487−496. (21) Tsai, Y. T.; Cheng, P. C.; Pan, T. M. Immunomodulating activity of Lactobacillus paracasei subsp. paracasei NTU 101 in enterohemorrhagic Escherichia coli O157H7-infected mice. J. Agric. Food Chem. 2010, 58, 11265−11272. (22) Tsai, Y. T.; Cheng, P. C.; Fan, C. K.; Pan, T. M. Time-dependent persistence of enhanced immune response by a potential probiotic strain Lactobacillus paracasei subsp. paracasei NTU 101. Int. J. Food Microbiol. 2008, 128, 219−225. (23) Chiang, S. S.; Pan, T. M. Beneficial effects of Lactobacillus paracasei subsp. paracasei NTU 101 and its fermented products. Appl. Microbiol. Biotechnol. 2012, 93, 903−916. (24) Mosmann, T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 1983, 65, 55−63. (25) Fotiadis, C. I.; Stoidis, C. N.; Spyropoulos, B. G.; Zografos, E. D. Role of probiotics, prebiotics and synbiotics in chemoprevention for colorectal cancer. World J. Gastroenterol. 2008, 14, 6453−6457. (26) Schiffrin, E. J.; Rochat, F.; Link-Amster, H.; Aeschlimann, J. M.; Donnet-Hughes, A. Immunomodulation of human blood cells following the ingestion of lactic acid bacteria. J. Dairy Sci. 1995, 78, 491−497. (27) Shahani, K. M.; Ayebo, A. D. Role of dietary lactobacilli in gastrointestinal microecology. Am. J. Clin. Nutr. 1980, 33, 2448−2457. (28) Butler, L. M.; Hewett, P. J.; Fitridge, R. A.; Cowled, P. A. Deregulation of apoptosis in colorectal carcinoma: theoretical and therapeutic implications. Aust. N. Z. J. Surg. 1999, 69, 88−94. (29) Baricault, L.; Denariaz, G.; Houri, J. J.; Bouley, C.; Sapin, C.; Trugnan, G. Use of HT-29, a cultured human colon cancer cell line, to study the effect of fermented milks on colon cancer cell growth and differentiation. Carcinogenesis 1995, 16, 245−252. (30) Deepak, V.; Ramachandran, S.; Balahmar, R. M.; Pandian, S. R.; Sivasubramaniam, S. D.; Nellaiah, H.; Sundar, K. In vitro evaluation of anticancer properties of exopolysaccharides from Lactobacillus acidophilus in colon cancer cell lines. In Vitro Cell. Dev. Biol.: Anim. 2016, 52, 163−173.

than that of the WE. Hence, the EE of NTU101-FM was confirmed to have antimetastatic potential. However, one major limitation of this study is that it only used an in vitro model. Therefore, in our preliminary animal experiments using an orthotopic mouse model of CRC, it was revealed that NTU101FM can reduce the side effects of chemotherapeutic drugs. Our data strongly suggest that NTU101-FM extracts may have the potential to serve as a combinatorial therapeutic supplements with 5-FU or UFUR to improve the quality of life of patients and the efficiency of chemotherapy in the treatment of CRC.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +886-2-3366-4519 ext. 10. Fax: +886-2-3366-3838. Email: [email protected]. ORCID

Tzu-Ming Pan: 0000-0002-9865-1893 Funding

This study did not receive any specific grants from funding agencies. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED 5-FU, 5-fluorouracil; CRC, colorectal cancer; DMEM, Dulbecco’s modified Eagle’s medium; DMSO, dimethyl sulfoxide; EE, ethanol extracts of NTU 101-fermented skim milk; FBS, fetal bovine serum; LAB, lactic acid bacteria; MTT, 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; NTU101-FM, NTU 101-fermented skim milk; PBS, phosphate-buffered saline; RPMI, Roswell Park Memorial Institute; WE, water extracts of NTU 101-fermented skim milk

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DOI: 10.1021/acs.jafc.8b01445 J. Agric. Food Chem. 2018, 66, 5549−5555