Reversal Effects of Bound Polyphenol from Foxtail Millet Bran on

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Reversal Effects of Bound Polyphenol from Foxtail Millet Bran on Multi-drug Resistance in Human HCT-8/Fu Colorectal Cancer Cell Yang Lu, Shuhua Shan, Hanqing Li, Jiangying Shi, Xiaoli Zhang, and Zhuoyu Li J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01659 • Publication Date (Web): 06 May 2018 Downloaded from http://pubs.acs.org on May 6, 2018

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Reversal Effects of Bound Polyphenol from Foxtail Millet Bran on Multi-drug Resistance in Human HCT-8/Fu Colorectal Cancer Cell

Yang Lu† #, Shuhua Shan † #, Hanqing Li ‡, Jiangying Shi †, Xiaoli Zhang †, Zhuoyu Li †, ‡ *

†

Institute of Biotechnology, Key Laboratory of Chemical Biology and Molecular Engineering

of National Ministry of Education , Shanxi University, Taiyuan, China ‡

School of Life Science and Technology, Shanxi University, Taiyuan, China

*Corresponding Author: Zhuoyu Li. Tel: +86 351 7018268. Fax: +86 351 7018268. Email: [email protected] #These authors contributed equally to this work.


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1

ABSTRACT:

2

Foxtail millet is the second-most widely planted species of millet and the most important

3

cereal food in China. Our previous study showed that bound polyphenol of inner shell (BPIS)

4

from foxtail millet bran displayed effective anti-tumor activities in vitro and in vivo. The

5

present research further implied that BPIS has the ability to reverse the multi-drug resistance

6

of colorectal cancer in human HCT-8/Fu cells, the IC50 values of 5-fluorouracil (5-Fu),

7

oxaliplatin (L-OHP) and vincristine (VCR) were decreased form 6593 ± 53.8 , 799 ± 48.9 ,

8

247 ± 10.3 μM to 5350 ± 22.3 (3261 ± 56.9) , 416 ± 16.6 (252 ± 15.6) and 144 ± 8.30 (83.8 ±

9

5.60) μM when HCT-8/Fu cells pretreated with 0.5 (1.0) mg/ml BPIS for 12 h. The 12

10

phenolic acid compounds of BPIS were identified by UPLC-Triple-TOF/MS method.

11

Especially, the fraction of molecular weight (MW) < 200 of BPIS reversed the multi-drug

12

resistance in HCT-8/Fu cells, and ferulic acid and p-coumaric acid were the main active

13

components, the IC50 values were 1.23 ± 0.195 and 2.68 ± 0.163 mg/ml , respectively. The

14

present data implied that BPIS significantly enhanced the sensitivity of chemotherapeutic

15

drugs through inhibiting cell proliferation, promoting cell apoptosis and increasing the

16

accumulation of rhodamine-123 (Rh-123) in HCT-8/Fu cells. RT-PCR and western blot data

17

indicated that BPIS also decreased the expression levels of multi-drug resistance protein 1

18

(MRP1), P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP). Collectively,

19

these results show that BPIS has potential ability to be used as a new drug-resistance reversal

20

agent in colorectal cancer.

21

KEY WORDS: Foxtail millet; BPIS; Active component analysis; Reverse multi-drug

22

resistance; Colorectal cancer cells



23

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1. Introduction

24

Human colorectal cancer is a kind of gastrointestinal cancer, which is the fourth most

25

common cancer and the fifth leading cause of cancer-related mortality in the China 1.

26

5-fluorouracil

(VCR)

are

common

27

chemotherapeutic agents, which are widely used to treat colorectal cancer

2, 3, 4

. Although

28

chemotherapeutic agents have improved the survival rate of colorectal cancer patients,

29

resistance to chemotherapy remains a major hurdle to obtaining a cure for this malignancy 5.

30

Traditional chemical reversal is limited in clinical application due to the single mechanism

31

and serious side effect or inconvenient in use. Therefore, exploring safe and effective reversal

32

agents of multi-drug resistance on human colorectal cancer has great influence for

33

improvement of the clinical effect.

(5-Fu),

oxaliplatin

(L-OHP)

and

vincristine

34

Polyphenols are widely present in foods and plant origin 6, 7. Epidemiological researches

35

have shown that polyphenols in diet can prevent or treat many human diseases, including

36

antimicrobial, anti-inflammatory, antiviral, immunomodulatory and anticancer activities 8, 9, 10,

37

11

38

drug resistance in human cancer cells 12. Limtrakul et al. reported that tetrahydrocurcumin, a

39

major metabolite of curcumin, could restore drug sensitivity in cancer cells

40

studies found favorable effects of flavonoids on modulating major ATP-binding cassette

41

transporters and reversing multidrug resistance in their studies

42

and his colleagues also showed that capsaicin could increase the intracellular P-gp substrates

43

by inhibiting chemotherapeutic drug efflux transporters

44

derived from foxtail millet has potential biological effects on human colorectal cancer, and

. Recently, natural phenolic extraction has drawn more and more attention in overcoming

14,

15,

. Leslie et al.

13

. Nabekura,

16

. In our previous study, BPIS

17


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45

could significantly promote tumor cell apoptosis 18. In the present study, we found BPIS could

46

reverse multi-drug resistance in colorectal cancer.

47

Analytical UPLC-Triple-TOF/MS system data showed that BPIS contained the

48

polyphenol compounds of 12 types, and ferulic acid and p-coumaric acid in BPIS are main

49

components that displayed reversal activities of drug resistance. Interestingly, the inhibitory

50

activities of either ferulic acid or p-coumaric acid only on HCT-8/Fu cells were far less than

51

BPIS. We therefore speculated that above two active components with other phenolic acids in

52

BPIS have synergistic effects on inhibiting cancer cell growth. Further, we found BPIS

53

significantly enhanced the sensitivity of chemotherapeutic drugs through inhibiting cell

54

proliferation, promoting cell apoptosis, increasing the accumulation of chemotherapy drug

55

and inhibiting drug resistance proteins in HCT-8/Fu cells. Hence, BPIS as a natural product in

56

cereal food have a potential ability to be used as a safe and efficient reversal agent for clinic.

57

2. Materials and methods

58

2.1. Chemicals

59

RPMI-1640 medium and fetal bovine serum (FBS) were obtained from GIBCO (Grand

60

Island, NY, USA). Hoechst 33342, MTT and dimethyl sulphoxide (DMSO) were obtained

61

from Sigma (St. Louis, MO, USA). Annexin V-FITC apoptosis detection kit was obtained

62

from Oncogene (San Diego, CA, USA). 5-Fu (99%), L-OHP (99%), VCR (98%), p-coumaric

63

acid (≧ 99%), ferulic acid (≧ 99.5%), 4-hydroxybenzoic acid (> 99.5%), vanillic acid (98%),

64

syringic acid (98%) and isoferulic acid (≧ 98%) standard samples were purchased from

65

Aladdin (Shanghai, China). The antibody for BCRP was obtained from Sangon

66

Biotechnology (Shanghai, China); Antibodies for MRP1, P-gp, Bax, caspase-8, Bcl-2, and



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67

caspase-9 were obtained from Bioworld Technology (Minneapolis, MN, USA); Antibody for

68

GAPDH was purchased from Abmart (Arlington, MA, USA).

69

2.2 Determination of BPIS composition

70

The method for preparation of foxtail millet bran and extraction of the bound

71

polyphenols were used as described earlier 18. To analyze phenolic acids composition of BPIS,

72

the sample (0.78g of BPIS-fraction) was mixed with 20 ml methyl alcohol, followed by

73

ultrasonication for 30 min. The supernatant was collected and evaporated to dryness.

74

Subsequently, the mixture was dissolved in 1 ml methyl alcohol, followed by centrifugation

75

for 30 min at 10000 rmp/min. The supernatant was collected and detected using analytical

76

UPLC-Triple-TOF/MS system (Waters Corp., Milford, MA). The separation was performed at

77

30°C using an Acquity ZORBAX-SB C18 (100 mm × 4.6 mm i.d., 1.8 µm). The mobile phase

78

used were solvent A (0.1% aqueous formic acid) and solvent B (0.1% formic acid in

79

acetonitrile). Moreover, the flow rate was 0.8 ml/min, and the injection volume was 5 μl. The

80

phenolic acids composition of BPIS were identified based on the retention time and MS

81

spectra. Percent recoveries were determined by spiking a known amount of pure compound

82

into a sample by using an RP-HPLC method reported previously

83

acquired and processed using Waters Empower software (Waters Corp., Milford, MA).

84

2.3 Cell line and cell culture

. Data signals were

19

85

Human colon epithelial cell line FHC and colorectal cancer cell lines HCT-8, HCT-8/Fu

86

were obtained from the Chinese Type Culture Collection (Shanghai China). The cells were

87

grown in RPMI-1640 medium supplemented with 10% (v/v) heat-inactivated fetal calf serum,

88

2 mM glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin (Sigma; St. Louis, MO,


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89

USA) at 37°C in a 5% CO2 humdified atmosphere. HCT-8/Fu cells were routinely maintained

90

in the medium containing 1,000 ng/ml 5-Fu.

91

2.4 Cell viability assays

92

The inhibitory effect of polyphenol components, BPIS alone, chemotherapy drugs, BPIS

93

plus chemotherapy drugs for colorectal cancer cell lines HCT-8 and HCT-8/Fu were measured

94

by MTT assay. HCT-8 and HCT-8/Fu cells were plated in 96-well culture plates at the density

95

of 5 × 103/well. Cells were allowed to attach to the bottom overnight, and then treated with

96

different concentrations of BPIS, chemotherapy drugs and polyphenol components of BPIS

97

for 24 h. Then 20 μl of MTT (5 mg/ml) was added to each well and incubated for 4 h at 37°C

98

in the dark. After removing the supernatant, formazan crystals formed were dissolved in

99

DMSO. The optical density was measured at 570 nm. The cell survival rate was calculated

100

with the formula: cell survival rate (%) = (OD570 treated / OD570 control) × 100%.

101

2.5 Clonogenic survival assay

102

The cell colony formation experiment was carried out as described earlier with some

103

modifications 20. HCT-8/Fu cells were trypsinized and seeded in a new 12-well tissue culture

104

plate (7 × 103 cells/well) for 24 h. Subsequently, the cells were treated with different

105

concentrations of BPIS for a week. Then, HCT-8/Fu cells were fixed with 6% glutaraldehyde

106

and stained with 0.1% crystal violet. Finally, the crystals were dissolved with 150 μl of 1%

107

SDS and the absorbance was measured at 570 nm.

108

2.6 Morphological changes of cell nucleus

109

Assays of morphological changes of cell nucleus were performed as previously

110

described 18. Briefly, HCT-8/Fu cells were incubated with different concentrations of BPIS for



111

24 h in laser confocal Petri dishes (1 × 104 cells/dish). After treatment, cells were washed

112

three times and stained with 1 μl Hoechst 33342 (10 μg/ml) and incubated in darkness for 10

113

min. Then morphology of cell nucleus were observed through laser confocal scanning

114

microscope (LCSM).

115

2.7 Rhodamine-123 intracellular accumulation and cell apoptosis assay

116

The Rh-123 intracellular accumulation and cells apoptosis rate were detected by flow

117

cytometry (BD Bioscience, San Jose, CA, USA). HCT-8/Fu cells (2 × 104 cells/well) were

118

plated into 6-well plates. After attachment, the cells treated with BPIS for 24 h. Then, they

119

were digested with 0.25% trypsin, seeded in new centrifuge tubes (1 × 106 cells/ ml), and

120

stained with Rh-123 (1 mM) 2 μl in a 5% CO2 humidified atmosphere at 37°C for 20 min.

121

Subsequently, the cells were centrifuged at 1100 rpm/min for 5min, discard the supernatant

122

and washed the cells twice with PBS before test. The apoptosis in HCT-8/Fu cells were

123

measured by the annexin v and propidium iodide assay. Briefly, after treated with different

124

doses of BPIS for 24 h, cells were stained with 200 μl Annexin V and 300 μl PI solution in the

125

dark at room temperature for 20 min. Then, cell apoptosis was detected by flow cytometry.

126

2.8 Western blot analysis

127

To investigate the expression changes of the drug resistance proteins and apoptosis

128

proteins with BPIS treatment. HCT-8/Fu cells were intubated with 0.5, 1.0, 2.0 mg/ml BPIS

129

for 24 h at 37°C. The total protein extract for each sample was dissolved in lysis buffer and

130

equal amounts of protein (60 ng) were separated by SDS-PAGE electrophoresisv (10 %).

131

Subsequently, the protein was transferred to polyvinylidene fluoride membranes. Antibodies

132

against MRP1, P-gp, BCRP, Bcl-2, Bax, caspase-8 and caspase-9 were 1500-fold diluted with


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133

TBST solution including 5% BSA, and antibody against GAPDH was 2000-fold diluted. The

134

PVDF membranes were incubated overnight at 4℃ with primary antibodies. Then, the

135

membranes were washed three times and incubated with HRP-conjugated anti-rabbit

136

secondary

137

chemoluminescence.

138

2.9 Quantitative RT-PCR analysis

antibody.

Finally,

the

targets

band

recorded

by

using

enchanced

139

Total RNA was extracted from cells using Trizol Reagent (Takara, Japan) according to

140

the manufacturer’s protocol. For cDNA synthesis, RNA was reverse-transcribed using the

141

reagent Kit with gDNA Eraser (Takara, Japan) and the concentraion of RNA used for the

142

synthesis of cDNA was 500 ng/μl. Primer synthesis were completed by Sangon Biotech

143

(shanghai, China) and were listed in Table 1. Quantitative RT-PCR was using Tip Green

144

qPCR SuperMix (TransStart, China). The PCR reaction in the following conditions: one cycle

145

at 94°C for 30 sec, followed by 40 cycles at 94°C for 5 sec, 60°C for 15 sec, and 72°C for 10

146

sec. The cDNA level for each gene was normalized to GAPDH mRNA levels.

147

2.10 Statistical analysis

148

Student's t-test was used for single variable comparisons. Comparisons of means of ≥ 3

149

groups were performed by analysis of variance (ANOVA), followed by Tukey’s post-hoc test.

150

The data were represented as the mean ± standard deviation (±SD) from at least three

151

independent experiments. The p values of less than 0.05 and 0.01 were considered that the

152

difference was significant and highly significant compared with control.

153

3. Results

154

3.1 Analysis and identification of phenolic acid in BPIS



155

The phenolic acid compositions of BPIS are presented in Table 3. In this part, we first

156

measured the effcets of BPIS on the cell survival of HCT-8/Fu and FHC, the results showed

157

that BPIS significantly inhibited HCT-8/Fu cell survival rate, but also no inhibitory effect on

158

FHC (Figure 1A, B). Subsequently, BPIS was dialyzed with a molecular weight cut off 200.

159

The anti-tumor activity of each fraction was examined by MTT assay. These results indicated

160

that the fraction of MW < 200 in BPIS exhibited inhibitory activity on HCT-8/Fu cells (Figure

161

1C) and IC50 was 1.82 ± 0.215 mg/ml in Table 2. Interestingly, we further found that

162

inhibiting effects of BPIS on HCT-8/Fu cells were far more than the fraction of MW < 200

163

(Figure 1C), and the IC50 value was 0.842 ± 0.091 mg/ml (Table 2). Then, six phenolic acids

164

contents in the fraction of MW < 200 were tested and data were presented in Table 3.

165

However, only the ferulic acid and p-coumaric acid had been identified with inhibitory

166

activity on HCT-8/Fu cells (Figure 1D). The IC50 values were 1.23 ± 0.195 and 2.68 ± 0.163

167

mg/ml, respectively (Table 3). Accroding to the content ratio of p-coumaric acid and ferulic

168

acid in BPIS (1:1.13), the standards of p-coumaric acid and ferulic acid were artifically mixed.

169

Finally, the effects of BPIS, BPIS (MW < 200), BPIS (MW > 200) and artifical mixture on

170

cell growth were tested. The results showed that artifical mixture and BPIS (MW < 200) had

171

the similar results in inhibiting proliferation on HCT-8/Fu cells, the IC50 values were 1.79 ±

172

0.173 and 1.82 ± 0.215, respectively (Table 2). Together, these results showed that ferulic acid

173

and p-coumaric acid were the main active phenolic acids in BPIS.

174

3.2 Multi-drug resistance test of HCT-8/Fu cells

175

In order to detect the multi-drug resistance of HCT-8/Fu cells, the human colorectal

176

cancer HCT-8 cell lines and their corresponding drug resistant sublines HCT-8/Fu were used


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177

as cell models. Two human colorectal cancer cell lines were treated with increasing

178

concentration of 5-Fu, L-OHP and VCR. The inhibitory rate was determined by MTT assay

179

(Figure 2A, B and C). As shown in Table 4, the IC50 values of 5-Fu, L-OHP, VCR were 922 ±

180

16.9, 128 ± 10.1, and 63.3 ± 2.80 μM for 24 h in HCT-8 cells, respectively. However, the IC50

181

values of 5-Fu, L-OHP, VCR were 6593 ± 53.8, 799 ± 48.9 and 247 ± 10.3 μM for 24 h in

182

HCT-8/Fu cells, respectively. The drug resistance indexs were 7.15, 6.24 and 3.9 in Table 4. It

183

is reported that the ATP-binding cassette (ABC) transporters have been identified to be

184

associated with many human diseases

185

including P-glycoprotein (P-gp; ABCB1), multi-drug resistance protein 1 (MRP1; ABCC1)

186

and breast cancer resistance protein (BCRP; ABCG2), are believed to severely affect cancer

187

chemotherapy

188

stilbenes could reverse chemotherapy resistance by inhibiting the activities of P-gp, MRP1

189

and BCRP

190

were determined by quantitative RT-PCR and western blot assays. As shown in Figure 2D, E

191

and F, three resistance genes were significantly up-regulated in mRNA or protein level in

192

HCT-8/Fu cells. These data suggest that HCT-8/Fu cells have strong resistance to these

193

chemotherapy drugs, especially to 5-Fu.

194

3.3 BPIS reverses the multi-drug resistance in HCT-8/Fu cells

. Only three important ABC drug transporters,

21, 22

. More importantly, many plant polyphenols such as flavonoids and

23, 24, 25, 26

. Thus, the expressions of multi-drug resistance genes P-gp, MRP1, BCRP

27, 28

195

To confirm whether BPIS could reverse drug resistance in HCT-8/Fu cells, the

196

clonogenic survival assay was implemented. The ability of individual cells to grow into

197

survival colonies was measured by a colony formation experiment. As shown in Figure 3A,

198

BPIS could strongly inhibited the clonogenic survival of HCT-8/Fu cells. With concentration



199

of 1.0 mg/ml BPIS treatment, cell colony formation were inhibited by approximately 50%

200

(Figure 3B). To further confirm the reversal effect of BPIS, HCT-8/Fu cells were treated with

201

0.5 mg/ml, 1.0 mg/ml BPIS for 12 h, respectively. Then, added fresh RPMI 1640 medium to

202

the cells and treated with increasing concentrations of 5-Fu, L-OHP and VCR for 12 h. The

203

results show that different concentrations of BPIS pretreatment significantly enhance the

204

chemotherapy sensitivity of HCT-8/Fu compared with the group treated with 5-Fu, L-OHP,

205

VCR alone (Figure 3C-H), and the drug resistance reversal folds were listed in Table 5.

206

3.4 BPIS induces cell apoptosis in HCT-8/Fu cells

207

In order to identify whether BPIS reverse drug resistance by promoting cell apoptosis in

208

HCT-8/Fu cells, the cell apoptosis ratio was determined with the different concentrations of

209

BPIS treatment. The typical morphological changes of cell nuclear in HCT-8/Fu cells were

210

observed through Hoechst 33342 staining analysis. As shown in Figure 4A, after BPIS

211

treatment, the stereotypical apoptotic feature was observed in HCT-8/Fu cells, including

212

nuclear condensation and stronger fluorescent signals. In the Figure 4B, the apoptotic cells

213

were increased form 35.4 ± 1.45 to 78.39 ± 1.91% with 0.5 and 2.0 mg/ml BPIS. To further

214

quantitatively evaluate the cell apoptosis ability of BPIS-induced, cells were stained with

215

AnnexinV/propidium iodide and analyzed by flow cytometry. The results showed that the

216

early and late cell apoptosis rates were 14.4 ± 4.36%, 17.18 ± 1.73%, 32.76 ± 5.15% and

217

80.87 ± 8.66% with 0, 0.5, 1.0 and 2.0 mg/ml BPIS treatment for 24 h (Figure 4C and D). On

218

the other hand, it has been known that the B-cell lymphoma-2 (Bcl-2) protein family are

219

critical regulators of apoptosis, because this family includes inhibitors and inducers of cell

220

apoptosis, like Bcl-2 and Bax.

. The caspase-cascade system also play a central role as

29, 30


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221

executioners in apoptosis and 15 caspases in mammals have been identified. Among these,

222

caspase-8 (cysteine-aspartic acid protease 8) and caspase-9 (cysteine-aspartic acid protease 9)

223

as key initiators are carrying apoptosis signals by triggering caspase-cascade system to active

224

effector in apoptosis

225

expression was reduced, while Bax, caspase-8 and caspase-9 proteins levels were increased in

226

HCT-8/Fu cells after treated with 0, 0.5, 1.0, 2.0 mg/ml BPIS (Figure 4E and F). These results

227

indicate that apoptosis was strongly induced by BPIS.

228

3.5 BPIS increased the degree of intracellular accumulation of Rhodamine-123 in

229

HCT-8/Fu cells

31, 32

. Moreover, the western blot results indicated that Bcl-2 preotein

230

Drug-resistance mechanisms that include drug efflux, inhibition of cell death,

231

detoxifcation, DNA damage repair, alternation of drug targets, stem cells, and epithelial to

232

mesenchymal transition

233

transporter, is considered as a drug pump that mediated cellular uptake and the efflux of

234

anticancer drugs from cancer cells

235

evaluating the activities of P-gp, and P-gp inhibition results in a large intracellular

236

accumulation of Rh-123 38. Our data showed that BPIS treatment could markedly increase the

237

mean fluorescence intensity of Rh-123 in a dose dependent manner, and the mean

238

fluorescence intensity increased from 22.05 ± 4.43 to 70.03 ± 7.15, 144.68 ± 5.75 and 152.19

239

± 6.72, respectively (Figure 5A and B). As expectedly, the expression of P-gp in mRNA and

240

protein levels were down-regulated after BPIS treatment in a dose-dependent manner (Figure

241

6A and B). Additionally, we observed that the expression of MRP1 and BCRP in mRNA and

242

protein levels also dramatically reduced after BPIS treatment. The results indicate that BPIS

. P-gp, a member of ATP-binding cassette (ABC) family

33, 34

35, 36, 37

. The Rh-123 is commonly used as an indicator for



243

significantly enhances the accumulation of Rh-123 in HCT-8/Fu cells, which attributes to the

244

decrease of MRP1, P-gp and BCRP expressions of BPIS-induced (Figure 6A, B and C).

245

4. Discussion

246

Colorectal cancer is the fifth most common malignancy in Chinese males and females

247

with a poor long time survival 39. It is known that 5-Fu, L-OHP and VCR have clinically been

248

used as a chemotherapeutic agents for colorectal cancer. However, the extensive application

249

of chemotherapy drugs inevitably causes drug resistance, which is a major obstacle to cancer

250

chemotherapy. In present study, we found that BPIS could effectively reverse chemotherapy

251

drugs resistance in HCT-8/Fu cell model. As shown in Table 5, BPIS treatment significantly

252

increased the sensitivity of HCT-8/Fu cells for 5-Fu, L-OHP and VCR.

253

Further, we investigate the active components with drug resistance reversal function in

254

BPIS using UPLC-Triple-TOF/MS system. The phenolic acids of 12 types were identified

255

(Table 3). Through dialyzing with a molecular weight cutoff 200, we confirmed that the

256

fraction of MW < 200 exhibited reversal activity in HCT-8/Fu cells, but MW > 200 is not

257

(Figure 1C, Table 2). Based on these, the content and reversal activities of phenolic acids in

258

the MW < 200 were analyzed (Table 3). The results showed that ferulic acid and p-coumaric

259

acid inhibited proliferation of HCT-8/Fu cells (Figure 1D). And we found that artifical

260

mixture and BPIS (MW < 200) have the similar IC50 values in Table 2. These results indicated

261

that ferulic acid and p-coumaric acid were the main active compounds in BPIS. Interestingly,

262

the reversal activities of only above two components were not as effective as BPIS, which

263

shows other non-active phenolic acids with ferulic acids and p-coumaric may have synergistic

264

effects on reversing the multi-drug resistance in human colon cancer cells.


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Drug resistance can be reversed through inhibiting proliferation, enhancing apoptosis

265 266

and decreasing related drug resistance genes expression

. Tomohiro Nabekura study

267

reported that (-)-Epigallocateachin gallate (EGCG), one of the main components of tea, could

268

inhibit cells proliferation, down-regulate the expression of P-glycoprotein and reverse

269

multidrug resistance in human carcinoma KB-C2 cells

270

could effectively reverse 5-Fu, L-OHP and VCR resistance in HCT-8/Fu cells by

271

inhibiting the proliferation and promoting apoptosis in HCT-8/Fu cells (Figure 3 and 4).

40, 41, 42

. In present, we found that BPIS

43

P-gp, a drug efflux protein with a molecular weight of 170-kD, is expressed in almost all

272 273

the tissues. And it is also a transmembrane glycoprotein encoded by MDR1 gene

.

274

Meanwhile, Rh-123 is considered to be an important indicator for detecting the P-gp activities

275

38

276

with different concentrations of BPIS treatment (Figure 5), which attributes to inhibiting the

277

mRNA and protein expressions of P-gp.

44

. In this study, the results show that intracellular Rh-123 fluorescence is gradually increased

278

According to reports, cancer patients who do not respond to chemotherapy drugs usually

279

have a high expression of various ATP-binding cassette (ABC) efflux transporters, which are

280

ubiquitous and identified in both prokaryotes and eukaryotes, resulting in decreasing drug

281

accumulation in cancer cells

282

categorized into seven subfamilies, and fifteen of these members have been associated with

283

multidrug resistance

284

drug resistance cancers. Our results reveal that BPIS markedly restrain the expressions of

285

MRP1 and BCRP in HCT-8/Fu cells, and in particular, the inhibitory effects of BPIS on

286

BCRP expression are most effective among these resistance proteins (Figure 6), which

22

45, 46

. As transmembrane proteins, ABC transporters were

. Moreover, MRP1, and BCRP are commonly associated with many



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287

suggests that BPIS reverses the multi-drug resistance of human HCT-8/Fu cell line by

288

inhibiting the expression of drug resistance-associated proteins.

289

Overall, BPIS can significantly enhance the sensitivity of chemotherapeutic drugs

290

through inhibiting cell proliferation, promoting cell apoptosis and increasing the accumulation

291

of chemotherapy drug by inhibiting the expression of drug resistance proteins in HCT-8/Fu

292

cells. This study highlights the potential therapeutic usefulness of BPIS as a new

293

drug-resistance reversal reagent in colorectal cancer.

294 295

Fundings

296

This study was supported by the National Natural Science Foundation of China (No.

297

31770382, 31500630,81603020), Shanxi Scholarship Council of China (No. 2015-2), Shanxi

298

Province Science Foundation for Youths (No. 2015021200), Scientific and Technologial

299

Innovation Programs of Higher Education Institutions in Shanxi (No. 2015175), National

300

Training

301

201610119005), “1331 project” Collaborative Innovation Center (1331 CIC).

Program

of

Innovation

and

Entrepreneurship

302 303 304

Notes The authors affirm that there is no conflict of interest.

305 306 307 308


for

Undergraduates

(No.

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Figure legends

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Figure 1. Effect of BPIS and related polyphenols on HCT-8/Fu and FHC cells were

443

determined by the MTT assay (24 h). (A, B) HCT-8/Fu and FHC cells (5 × 103/well) were

444

treated for 24 h with different concentrations of BPIS. The same letter in two columns

445

indicated that there is no significant difference between the two groups, p

Reversal Effects of Bound Polyphenol from Foxtail Millet Bran on

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