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Inhibitory effect of Bowman–Birk protease inhibitor on autophagy in MDAMB231 breast cancer cell line


1 Department of Biology, Faculty of Science, Razi University, Kermanshah, Iran
2 Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran

Date of Submission24-Sep-2018
Date of Decision17-Apr-2019
Date of Acceptance09-Jun-2019
Date of Web Publication13-May-2020

Correspondence Address:
Hassan Akrami,
Department of Biology, Faculty of Science, Razi University, Kermanshah
Iran
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jcrt.JCRT_622_18

 > Abstract 


Background: Autophagy has an essential role in cellular energetic balance, cell cycle, and cell death, so the change in autophagy level is crucial in many human diseases such as cancer. Herbal medicine has been widely used to treat cancer. Bowman–Birk protease inhibitor (BBI), a protease inhibitor extracted from soybean, has antitumorigenic, anti-inflammatory, and anti-angiogenic activities. In this study, we evaluated the effect of BBI on the growth of breast cancer cell line and transcript level of autophagy and apoptosis-related genes.
Materials and Methods: BBI was purified from soybean by ion-exchange chromatography method. The viability of MDA-MB-231 cells that were treated with BBI was measured by MTT assay, and the transcript level of genes involved in autophagy and apoptosis was measured by real-time–polymerase chain reaction (PCR) technique.
Results: The results of BBI purification showed that 100 g of the ethanolic fraction yielded 300-mg BBI with more than 95% purity. MTT results revealed that BBI inhibited the cell growth of MDA-MB-231 cell line in a dose-dependent manner, with an IC50 of 200 μg/mL. The results of real-time reverse transcription-PCR exhibited that BBI altered the expression of Atg5, Beclin1, light chain 3-II, and sequestosome1 and increased the Bax/Bcl2 ratio in MDA-MB-231 cell line.
Conclusion: According to our results, BBI could inhibit autophagy and induce apoptosis in MDA-MB-231 cell line. Thus, BBI may be used as a therapeutic drug in the treatment of breast cancer whether alone or with chemotherapeutic drugs.

Keywords: Apoptosis, autophagy, autophagy inhibitor, Bowman–Birk protease inhibitor, breast cancer



How to cite this URL:
Kyani S, Akrami H, Mostafaei A, Akbari S, Salehi Z. Inhibitory effect of Bowman–Birk protease inhibitor on autophagy in MDAMB231 breast cancer cell line. J Can Res Ther [Epub ahead of print] [cited 2020 Aug 10]. Available from: http://www.cancerjournal.net/preprintarticle.asp?id=284263




 > Introduction Top


Autophagy is a conserved catabolic process that involves in the capture of long-lived cytoplasmic components and organelles and delivers them to vacuolar compartments for degradation.[1] Autophagy is a multistep process that includes autophagy initiation, nucleation, elongation, and maturation–degradation–recycling.[2] To accomplish these steps, autophagy-related genes (ATGs) such as Beclin-1 (Atg6) and Atg5 are required. Beclin-1 is a part of PI3KCIII complex and is necessary for the autophagosome formation.[3]Atg5 is a gene required for autophagy elongation. The Atg5 protein is involved as a part of E3 ligase for light chain 3 (LC3)-phosphatidylethanolamine conjugation.[3] Furthermore, LC3-II (microtubule-associated protein LC3) and sequestosome 1 (SQSTM1) genes act as completion indicators of autophagy process.[4] The role of autophagy in cancer is complicated; it has binary roles on cancer cells such as inhibitor or inducer of apoptosis. Hence, it is believed that autophagy acts like a double-edged sword.[5] In recent decades, researchers used autophagy for cancer treatment.[3] Furthermore, autophagy may cause drug resistance in cancer cells against chemotherapy or radiotherapy.[3],[6]

Breast cancer is the second-most commonly diagnosed cancer type and the major cause of death among women.[7] Variety of risk factors, include genetic characteristics involves in breast cancer etiology, diagnosis of late-stage disease, lack of adequate adjuvant and systemic treatment induced increases breast cancer mortality.[8]

Some studies have proven an inverse association between fruits, vegetables, and the risk of developing breast cancer.[9] Plants and their extracts have fewer side effects and can be introduced as alternative medicines.[10] Bowman–Birk protease inhibitor (BBI) is extracted from soybean seeds and is a small polypeptide with 8 kD and 71 amino acids and has seven disulfide bonds in its structure. BBI inhibits the trypsin and chymotrypsin activity.[11],[12] Recent studies have shown that BBI possess significant antitumor potential bothin vitro and invivo. BBI has a powerful effect on gastric, colon, and prostate cancers.[13],[14] In addition, BBI has anti-inflammatory properties.[15] However, the effect and action mechanism of BBI remain uncovered on human breast cancer.

In this study, we evaluated the effect of BBI on the expression of genes related to autophagy including Atg5, Beclin1, LC3-II, and SQSTM1 and two apoptotic genes (Bax and Bcl2) in human breast cancer cell line, MDA-MB-231.


 > Materials and Methods Top


Materials

Human breast cancer cell line (MDA-MB-231) was obtained from the National Cell Bank (Pasteur Institute, Tehran, Iran). Dulbecco's Modified Eagle Medium (DMEM) and fetal bovine serum (FBS) were supplied by (GIBCO Invitrogen Carlsbad, CA, USA). DEAE-Sephacel™ was purchased from Amersham Pharmacia Biotech, Uppsala, Sweden. 3-(4, 5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide (MTT) was supplied by Sigma-Aldrich Corp. - St Louis, USA

Soybean Bowman–Birk protease inhibitor extraction

Atfirst, soybean was ground into powder and defatted and depigmented by adding four volumes of methanol to one weight of the powder for overnight. Then, the defatted powder was centrifuged at 5000 × g for 30 min at 25°C, and the pellet was air-dried. For obtaining the alcoholic extract, four volumes of ethanol 60% (v/v) was added to one weight of the dried powder in sealed Erlenmeyer flask and mixed for 8 h at 50°C. The mixture was centrifuged at 5000 × g for 30 min at 25°C, and the supernatant was evaporated under reduced pressure to dryness. After drying, the powder was dissolved in 20 mM sodium acetate buffer (pH 4.5) and precipitated by saturated ammonium sulfate (70%) at 4°C. The pellet was obtained by centrifugation at 5000 × g for 30 min at 4°C.

Bowman–Birk protease inhibitor purification by anion exchange chromatography

For the purification of BBI, a DEAE-Sephacel column (15 cm × 2.6 cm) was equilibrated with 50 mM Tris-HCl buffer (pH 8) containing 1 mM ethylenediaminetetraacetic acid. The solution containing BBI was loaded into the column at a flow rate of buffer by 70 mL/h. After passing the solution, the column was washed with ten column volumes of 50 mM Tris-HCl buffer (pH 8) and eluted with two column volumes of linear gradient (0–0.5 M) of NaCl in the buffer. Then, the column eluate was precipitated with 70% saturated ammonium sulfate for an overnight at 4°C, and the pellet was recovered by centrifugation in 5000 g for 30 min at 4°C. Finally, the pellet was dissolved and dialyzed against deionized water to remove buffer salts. The purity of BBI was analyzed by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Total protein assay

The concentration of total protein in each stage of purification was determined by the Bradford protein assay. The assay reagent included 10 volumes of the dye reagent (0.1% Coomassie Brilliant Blue G-250, 20% ethanol, and 8.5% phosphoric acid) that was mixed with 1 volume of the sample. After 3 min read the standards and samples at 595 nm. Bovine serum albumin was used as the protein standard.

Quantification of trypsin inhibitory activity of Bowman–Birk protease inhibitor

The trypsin inhibitory activity (TIA) of the purified BBI was measured at 25°C in 67-mM phosphate buffer (pH 7) and 5-mM HCl with N-alpha benzoyl L-arginine ethyl ester (BAEE9) as a specific substrate.[16] The TIA of BBI is based on the inhibition of the dye development from BAEE degradation by trypsin. To determine the trypsin activity of BBI (trypsin inhibitor), BBI was added to a substrate solution. The esterase effect of Trypsin was supplied by Sigma-Aldrich Corp. -St Louis, USA on the substrate was accompanied by increased absorption. In addition, the increased absorption was inhibited when BBI is added, and the esterase effect of trypsin was blocked by BBI. One trypsin inhibitory unit (TIU) was defined as the amount of BBI that gives a reduction in absorbance at 253 nm of 0.001 in 5 min in the defined assay volume.[16] Absorbance was recorded in a ultraviolet-visible (UV/VIS) Cecil BioAquarius CE 7250 doublebeam spectrophotometer (SelectScience, Science House Church Farm Business Park, Corston Bath BA2 9AP UK). Specific TIA of BBI was expressed as inhibitor units (IU) per milligram of protein.

Quantification of chymotrypsin inhibitory activity of Bowman–Birk protease inhibitor

N-acetyl-L-tyrosine ethyl ester monohydrate is a substrate of α-chymotrypsin, and has an absorbance at 237 nm. Chymo TIA of the purified BBI was measured by measuring the esterolytic activity of the enzyme. A decrease in absorbance at 237 nm occurred after adding BBI. One chymotrypsin IU (CIU) was defined as the amount of BBI that gives 0.0075 reduction in absorbance at 237 nm in a minute.[17] Readings were recorded in UV/VIS Cecil BioAquarius CE 7250 double-beam spectrophotometer. The amount of inhibitory unit is calculated by the following formula:



where ε = Molar extinction coefficient = 0.0075, ΔA = Change in absorbance.

MDA-MB-231 cell culture

MDA-MB-231, a human breast cancer cell line, was cultured in DMEM medium, supplemented with 10% (v/v) heat-inactivated FBS at 37°C in a humidified atmosphere with 5% CO2. Culture media were changed twice per week.

MTT assay

3-(4,5-dimethylthiazol-2-yl)-2.5-diphenyltetrazoliumbromide (MTT) assay was used for measuring cell viability. For this purpose, 3.0 × 10[3] cells were seeded in a 96-well plate and treated with different increasing concentrations of BBI and analyzed.[18] The experiment was repeated for three times.

RNA extraction and cDNA synthesis

RNA extraction of MDA-MB-231 cells was done by RNeasy plus Mini kit (PARS TOUS, Mashhad, Iran) according to the manufacturer's protocol. For confidence of RNA quality, RNA yield and purity were estimated by 1% agarose gel electrophoresis with optical density at 260/280 nm ratio. The complementary DNA (cDNA) was synthesized from RNA with Quanti Tect™ Revers Transcription Kit (PARS TOUS, Iran) through the protocol of the manufacturer. Primers were designed using Beacon Designer 7.0, PREMIER Biosoft International 3786 Corina Way, USA. and Primer-BLAST. The sequences of primers are shown in [Table 1].
Table 1: Sequence details of primers and products

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Quantitative reverse transcription-polymerase chain reaction

The amplification efficiency of experiments was determined by drawing a standard curve through an initial dilution of 1:1 of cDNA samples and two-fold serial dilution series. The transcript levels of Atg5, Beclin1, LC3-II, SQSTM1, Bax, and Bcl2 were measured using Rotor-Gene 3000 System (Corbett Research, Mortlake NSW 2137, Australia) and Quanti Tect™ SYBER Green PCR kit (PARS TOUS, Iran) in accordance with the manufacturer's protocol. For analyzing data, the 2−△△Ct method was used based on the threshold cycle (Ct) values for target genes and β-actin as an endogenous control gene. Each experiment was performed in duplicates.

Statistical analysis

One-way ANOVA was used for statistical analysis, and posthoc test (Tukey) was used to analyze the significance between groups. Results from each assay were presented as the mean ± standard error of mean, and P < 0.05 was considered statistically significant.


 > Results Top


Purification of Bowman–Birk protease inhibitor

The results of BBI purification showed that 100 g of the ethanolic fraction yielded 300-mg BBI with >95% purity [Figure 1]. In addition, 4 g of ethanolic extract contained 90-mg protein, but after precipitation by ammonium sulfate, 70-mg protein was obtained. On evaluating trypsin or chymo TIA of BBI, the results showed 560 TIUs per mg protein (560 TIU/mg) and 700 chymo TIUs per mg protein (700 CIU/mg) for purified BBI.
Figure 1: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of soybean protein. (a) Total extracted protein, (b) purified Bowman–Birk protease inhibitor after DEAE-Sephacel column and (c) pure Bowman–Birk protease inhibitor after dialysis. Gel was stained by Coomassie brilliant blue R-250

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Bowman–Birk protease inhibitor inhibits the growth of MDA-MB-231 cell line

The cells were treated with different concentrations of BBI from 25 μg/mL to 1000 μg/mL [Figure 2]. BBI at a concentration below 50 μg/mL had no significant inhibitory effect on cell growth. BBI at 200 μg/mL inhibited >50% growth of MDA-MB-231 cell line after 24 h (P < 0.001). The inhibitory effects of BBI on cell growth were estimated by the MTT assay. Our results showed that BBI inhibited the cell growth of MDA-MB-231 cell line in a dose-dependent manner with an IC50 of 100 μg/mL.
Figure 2: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MTT assay: Viability of MDA-MB-231 cells treated with the different concentrations of Bowman–Birk protease inhibitor using MTT assay. *P = 0.05 versus control group, Student's t-test analysis

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Bowman–Birk protease inhibitor altered the expression of autophagy-related genes in MDA-MB-231 cell line

We analyzed the effect of BBI on autophagy in MDA-MB-231 breast cancer cell line by analyzing the quantitative expression of Beclin-1, Atg-5, LC3-II, and SQSTM1 genes that were involved in autophagy process. The data analysis of real-time reverse transcription-polymerase chain reaction results by normalizing to β-actin gene in MDA-MB-231 treated with 200-μg/mL BBI showed a decrease in the transcript level of Beclin-1 and Atg-5 genes to 0.26 and 0.06, respectively, but an increase in the transcription of LC3-II and SQSTM1 genes to 3.22 and 4.01 folds, respectively [Figure 3].
Figure 3: The effect of Bowman–Birk protease inhibitor on mRNA levels of Beclin-1, Atg-5, LC3-II, p62, and Bax/Bcl-2 ratio. Bowman– Birk protease inhibitor at 500 μg/mL upregulated the transcription of LC3-II, p62, and Bax, while downregulated the transcription of Beclin-1, Atg-5, and Bcl-2 in MDA-MB-231 cells. *P = 0.05 versus control group, Student's t-test analysis

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Bowman–Birk protease inhibitor induced apoptosis by changing the Bax/Bcl2 expression ratio

For determining the apoptotic activity of BBI, we assessed the Bax/Bcl2 expression ratio. The result showed that the expression level of Bax and Bcl2 genes in MDA-MB-231 cells treated with 200-μg/mL BBI against untreated MDA-MB-231 cells showed an increase in Bax/Bcl2 ratio, so BBI has an anti-apoptotic activity in MDA-MB-231 breast cancer cell line [Figure 3].


 > Discussion Top


Tumor cells require energy and nutrient for growth and survival, and autophagy is the main process that provides energy and nutrient for cancer cells' survival.[19] When tumor cells are undergoing chemotherapy or radiation therapy, the level of autophagy is increased.[19] Thus, autophagy leads to drug resistance in cancer cells against the therapies.[20] Recent data suggested that suppression of autophagy inhibited the growth of cancer cell and improved the efficiency of chemotherapy or radiotherapy.[21],[22] Until now, many autophagy inhibitors such as antimalarial drugs, i.e., chloroquine and its analogs hydroxychloroquine and mefloquine, have been presented.[4],[23],[24]

Previous studies have shown that, in breast, ovarian, and prostate cancers, mono allelic loss of essential autophagy gene, Beclin-1, has been observed.[24] Deletion of Beclin-1 is related to the tumor suppressive role of autophagy in these cancers.[24] After a while, lack of energy and nutrient deficiency in cancer cells decreased cell growth.[22] For this reason, it is reported that cancer cells need autophagy to obtain energy for growth and survival. Beclin-1 gene produces a low level protein due to monoallelic loss of Beclin-1 gene of the Beclin-1 protein for triggering autophagy to overcome the stressful condition.[22] In a study, the lack of a single copy of Beclin-1 caused increasing tumor in mice.[25] Therefore, in this study, we tried to provide more validated data on whether BBI could inhibit autophagy in breast cancer cell line. For this purpose, MDA-MB-231 breast cancer cells were treated with BBI, and the effect of BBI on the transcript of genes involved in autophagy and apoptosis processes was measured. BBI decreased the expression of Beclin 1 and Atg5 genes in MDAMB231 cells that was treated with BBI, in comparison with the untreated MDA-MB-231 cell as control group. The decrease expression of these genes caused suppression of autophagy and decreased survival of cancer cell.

The study of Chang et al. showed that suppression of autophagy by pharmacological agents and small interfering RNAs (siRNA) inhibited cell proliferation and also increased apoptosis in hepatoblastoma cells.[26]

A study done by Sharma et al. on MDA-MB-231 cells that were treated with mefloquine showed an increase in the expression level of LC3-II and p62 genes.

Our study indicated an increase in LC3-II expression compared to control group and suggested that LC3-II was not degraded in autolysosome. LC3-II monitoring is a good candidate for autophagy flux because LC-II is degraded in autolysosomes.[27],[28] Our result showed that the expression of SQSTM1 was increased compared to control group, and these results suggested that autophagy was complicated and the whole process was finished and SQSTM1 was intact in autolysosome.[29],[30] Interestingly, to escape the hypoxia in cancer cells, SQSTM1 was degraded in the autophagosomes.[31],[32]

Apoptosis or programmed cell death is a biological pathway that plays an essential role in the development, tissue homeostasis, differentiation, disease progression, and removal of defective cells. Thus, up- or downregulation of apoptosis leads to cancer or other diseases such as autoimmune diseases.[33]

When an antitumor drug had a good efficiency, it should result an increase in cancer cells' death.[34] On the other hand, when Bax/Bcl2 expression ratio is increased, the apoptosis is induced which leads to the release of cytochrome c in the intrinsic pathway of apoptosis.[35] Human melanoma cells are susceptible to CD95/Fas-induced apoptosis when Bax/Bcl2 ratio >1.[36] Because BBI-treated MDA-MB-231 cells showed an increase in Bax to Bcl-2 ratio >1, it is possible that BBI can induce the intrinsic pathway of apoptosis.


 > Conclusion Top


Our data showed that BBI inhibits autophagy and also induces apoptosis in MDA-MB-231 cell line. Thus, BBI could be used as a therapeutic agent in the treatment of breast cancer.

Acknowledgment

We would like to acknowledge and extend our gratitude to Kamran Mansouri and Diba Borzabadi Farahani for their contribution in completing this work.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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