Anticancer and Antimigration Effects of a Combinatorial Treatment of

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Bioactive Constituents, Metabolites, and Functions

Anti-cancer and anti-migration effects of combinatorial treatment with 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., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01445 • Publication Date (Web): 08 May 2018 Downloaded from http://pubs.acs.org on May 9, 2018

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Journal of Agricultural and Food Chemistry

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Anti-cancer and anti-migration effects of combinatorial treatment

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with 5-fluorouracil and Lactobacillus paracasei subsp. paracasei NTU

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101-fermented skim milk extracts on colorectal cancer cells

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Chia-Yuan Chang, Tzu-Ming Pan*

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Department of Biochemical Science & Technology, National Taiwan University,

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Taipei, Taiwan.

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* Corresponding author:

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Dr. Tzu-Ming Pan, Professor

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Department of Biochemical Science and Technology, College of Life Science,

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National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei 10617, Taiwan

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Tel: +886-2-3366-4519 ext 10; Fax: +886-2-3366-3838; E-mail: [email protected]

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Keywords: Lactobacillus paracasei subsp. paracasei NTU 101, fermented skim milk

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extract, colorectal cancer, 5-fluorouracil, anticancer property

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Abstract

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Colorectal cancer (CRC) is one of the most prevalent cancers worldwide.

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Metabolites of lactic acid bacteria (LAB) have anti-cancer and anti-metastasis

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capacity. This study aimed to investigate the chemotherapeutic combination effect of

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Lactobacillus

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(NTU101-FM) extracts and the chemotherapeutic drug 5-fluorouracil (5-FU) in a

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cellular CRC model. NTU101-FM extracts effectively reduced CRC cell viability but

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was not cytotoxic to colon epithelial cells. Moreover, they increased RAW 264.7 cell

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viability. Notably, the cell viability of CRCs was decreased by 5-FU in combination

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with NTU101-FM extracts; combinatorial treatment inhibited cell viability

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significantly more than 5-FU alone (p < 0.05). An ethanol extract of NTU101-FM

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effectively attenuated CT26 cell migration. In conclusion, the ethanol extract prepared

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from NTU101-FM has potential application as an anti-cancer agent in CRC.

paracasei

subsp.

paracasei NTU

101-fermented skim

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milk

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Introduction

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Colorectal cancer (CRC) is one of the most prevalent forms of cancer

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worldwide.1 Statistically, the estimated cancer-related mortalities in North America in

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2017, CRC-related mortality proceeds that of lung cancer in men and lung and breast

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cancers in women.2 Recently, various treatments for CRC, such as surgery,

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chemotherapy, radiation therapy, immunotherapy, etc., have been reported.3 Surgery

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combined with chemotherapy is currently one of the most common and effective CRC

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treatment modalities. Although chemotherapy is effective for CRC, current

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chemotherapeutic drugs indiscriminately target both healthy and malignant cells,4

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thereby resulting in numerous deleterious side effects including cardiotoxicities,5

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hematopoietic suppression,6 vomiting, and fatigue, all of which affect the quality of

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life and even lead to death.7 Thus, numerous studies have focused on the extraction

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and isolation of antitumor compounds from natural foods.8 The usage of which can

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reduce the excessive usage of chemotherapeutic drugs, thereby indirectly reducing the

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possibility of these side effects to arise.

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In addition, numerous studies have indicated the colonic microflora to be

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involved in the etiology of CRC,8 and that maintenance of a balance in the colonic

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microflora would prevent CRC through supplementation of probiotics.9 Probiotics are

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live microorganisms, which maintain the balance of colonic microflora and have 3

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positive effects on the host. Lactobacilli are one of the most illustrious probiotic

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bacteria10 and are generally recognized as safe (GRAS) and commonly used in food

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industry. The components from Lactobacillus strains include the live bacteria,10

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heat-killed cells,11 cell wall,12 cytoplasmic fraction,11 and Lactobacillus-fermented

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products,13 which attenuate the effects of CRC by inducing apoptosis14 and promoting

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immunomodulatory effects.15

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In our laboratory, we have screened a Lactobacillus strain, Lactobacillus

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paracasei subsp. paracasei NTU 101 (NTU 101), isolated from human neonatal

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feces.16 The components of NTU 101 have been reported to have ameliorative effects

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on hypercholesterolemia,17 obesity,18 and diabetes,19 prevent hypertension-induced

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vascular dementia,20 play a key role in immunomodulation,21-22 present an anti-cancer

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ability,11, 23 and other benefits.

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However, few studies have investigated whether similar lactic acid bacteria

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(LAB)-fermented extracts have any adjuvant chemotherapeutic activity. Furthermore,

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the effects of NTU 101-fermented extracts on chemotherapy are unknown.

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Materials and methods

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Materials

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CT26, HT-29, Caco-2, and RAW 264.7 cells were purchased from the Bioresource

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Collection and Research Center (BCRC, Hsinchu, Taiwan). Dimethyl sulfoxide

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(DMSO) and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)

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were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Fetal bovine serum

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(FBS), Roswell Park Memorial Institute (RPMI) 1640 medium, and Dulbecco's

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modified Eagle's medium (DMEM) were purchased from HyClone Laboratories

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(Logan, UT, USA). Two well silicone cell culture-inserts were purchased from

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Blossom Biotechnologies (Taipei, Taiwan).

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Fermentation of skim milk with NTU 101 and extraction

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For fermentation, 25% (w/v) skim milk (Anchor, Auckland, New Zealand) was

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pasteurized in a water bath at 90ºC for 1 h, cooled to 37ºC, and inoculated with a 1% v/v

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suspension of NTU 101. Thereafter, it was incubated at 37ºC for 72 h before being

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freeze-dried using an SDF-25 freeze dryer (Chang Jung Business Co., Feng-Jen,

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Taiwan). Further, 95% ethanol or deionized water was used to extract the freeze-dried

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NTU 101-fermented skim milk (NTU101-FM) powders after sonication and incubation

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at 37ºC for 1 h. The extracts were then centrifuged at 4ºC and 10,000 × g for 30 min and

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the supernatants were dried in a rotary evaporator and stored at -20ºC until use.

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Cell culture

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Cell lines used in our study included RAW 264.7 mouse leukemic monocyte

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macrophages and Caco-2 human colon epithelial cells, which were cultured in

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DMEM. HT-29 human colorectal cancer cells and CT26 murine colon carcinoma

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cells were cultured in RPMI-1640 medium. All cell culture media were supplemented

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with 10% FBS and incubated at 37ºC, 95% humidity, and 5% CO2. The cells grew as

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monolayers in 10-cm2 cell culture dishes. Media were replenished every 48 to 72 h.

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Cells were passaged by dislodging them from the dish, using 0.25% trypsin-EDTA

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(the RAW 264.7 cells were dislodged using a cell scraper) and transferred to new

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dishes when required.

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Cell viability To evaluate the cell viability of NTU 101-FM extracts on RAW 264.7, Caco-2, HT-29, and CT26, cells were assessed via the MTT assay

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with certain

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modifications. Briefly, the aforementioned cells were seeded at a density of 5 × 104

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cells/well in a 24-well plate. For cell adherence, the plate was pre-incubated for 24 h.

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The cells were then incubated with NTU101-FM extracts (water or ethanol extracts)

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at various concentrations or chemotherapeutic drugs 5-FU or UFUR® at 37°C for 24 h.

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Thereafter, MTT assays were performed and cell viability was calculated using the

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following formula: cell viability (% of control) = (ODsample/ODcontrol) × 100%.

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Wound healing assay

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CT26 cells were seeded in 24-well cell culture plates with silicon culture-insert.

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The culture-insert provides two cell culture reservoirs, separated by a 500-µm wall.

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Density of the cell suspension was adjusted to 5 × 105 cells/mL, and 70 µL of cell

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suspensions were seeded into each culture reservoir. After 24 h of incubation, the

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Culture-Insert was gently removed, thereby creating a 500-µm-wide wound. After

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removing the insert, cell debris and non-attached cells were eliminated by washing the

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cell layer with phosphate-buffered saline (PBS). Thereafter, the sample or cell-free

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medium was re-added to fill the 24-well cell culture plates. The plates were then

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photographed at indicated time points, wherein the wound area and migrating cell

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number were analyzed using ImageJ software (NIH, Bethesda, MD, USA).

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Statistical analysis

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All experiments were performed in triplicate, and the data are presented as mean

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± standard deviation (SD) values. Data were analyzed using a statistical software

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(SPSS) (IBM Software, Armonk, NY, USA) and Duncan's multiple range test was 7

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performed to compare each group. The threshold for statistical significant difference

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was set as p < 0.05.

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Results and Discussion

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Anti-proliferative effects of NTU101-FM extracts against colon carcinoma cell

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Probiotic bacteria and their fermented products are reported to have anti-tumor

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activity.25-26 Shahani and Ayebo (1980) previously reported that consuming marked

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amounts of Lactobacilli- or Bifidobacteria-fermented milk products could reduce the

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incidence of CRC.27 These may decelerate CRC progression by affecting metabolism

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and the immune system, thereby protecting the colon. Furthermore, they may inhibit

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colon tumor growth through production of anti-tumorigenic or anti-mutagenic

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compounds.25,

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Furthermore, Deepak et al. (2016) reported that L. acidophilus exhibits distinct

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anticancer activity against colon cancer. 30 In this study, we used HT-29 and CT26 as

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the cell in vitro model of colon adenocarcinoma to evaluate the potential of

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anti-proliferative activity of water extract (WE) and ethanol extract (EE) of

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NTU101-FM. The inhibitory activity of EE and WE at different concentrations

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(250-1000 µg/mL) against HT-29 and CT26 cells are shown in Figures 1A and 1B.

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EE and WE at 500 µg/mL significantly inhibited cell viability in HT-29 cells (p