L-asparaginase from human breast milk Lactobacillus reuteri induces apoptosis using therapeutic targets Caspase-8 and Caspase-9 in breast cancer cell line

Abstract

BACKGROUND:

Breast cancer is the most fatal type of cancer in women worldwide. Many chemotherapeutics targeted breast cancer however, they have frightening side effects. One method of controlling cancer cell growth is targeting apoptosis.

OBJECTIVE:

This study aimed to induce apoptosis in breast cancer cells by purifying L-asparaginase from human breast milk Lactobacillus reuteri isolates via inhibition of Caspases 8 and 9.

METHODS:

The best L. reuteri isolates producing L-asparagine with the highest enzyme activity were identified from human breast milk and chosen for L-asparaginase purification. The MTT cell viability assay used for measure the toxicity of the enzyme. Breast cancer cell line was used to study the effect of the enzyme on the caspase 8 and caspase 9 gene expression.

RESULTS:

The MTT cell viability assay showed the inhibition rates ranged between 30% and 80%, of cell death, occurred when 3.125, 6.25, 12.5, 25, 50, and 100 μg/ml of the enzyme used and IC50 was 4.305 μg/ml. The breast cell lines were treated with the enzyme at a concentration of IC50 value. The Cas8 and Cas9 genes expression in L-asparagine treated breast cancer cell line at a concentration of IC50 value were upregulated (the fold of gene expression are 2.071 and 1.197 respectively).

CONCLUSIONS:

Breast milk L. reuteri L-asparaginase induces apoptosis via Cas8 and Cas9 upregulation in the breast cancer cell line. L. reuteri L-asparaginase treatment may be the hopeful approach for the management of breast cancer. Furthermore, the results may highlight the fact that the presence of L-asparaginase-producing L. reuteri isolates in human breast milk may aid in breast cancer improvement or even prevention.

Keywords

Caspases 8,Caspases 9 L-asparagine breast cancer cell line

1. Introduction

Breast cancer is one of the main causes of mortality among women cancer. Moreover, the incidence of breast cancer is the highest among the cancers affecting women [1]. Many chemotherapeutic agents were introduced to breast cancer treatment approaches. However, these agents are having harsh side effects and are extremely costly. One method of controlling cancer cell growth is to target apoptosis, Apoptosis targeting is the best effective non-surgical approach. Interestingly, the apoptosis-targeting approach is affecting all cancer types [2]. Apoptosis is caused by the activation of caspases, which are aspartate-specific proteases having a tumor-suppressing function [3]. This could be because these proteins are required for development. The genetic deletion of many caspases results in abnormality or lethality [4].

Lactic acid bacteria (LAB) are the most thoroughly studied bacteria and are widely employed in the production of secondary metabolites around the world [5]. Many LAB like, Streptococci, Lactobacilli and Lactococci are expansively studied due to their beneficial effect and safe of LAB enzymes [6]. Several species of the genera Lactobacillus, produce Asparaginase [7]. Several types of cancers have been treated with LAB L-Asparaginase (including Lactobacillus L-Asparaginase) due to its biodegradable properties. L-Asparagine is hydrolyzed by L-Asparaginase into ammonia and aspartic acid affecting by that the L-Asparagine supplementation of cancer cells [8]. The purpose of this study is to induce apoptosis in breast cancer cells by L-asparaginase via inhibition of Caspases 8 and 9.

2. Material and methods

2.1. Sample collection and identification

Forty samples of mother milk were cultivated on de Mann–Rogosa–Sharpe (MRS) agar anaerobically. The distinct colonies are identified according to the phenotypic characteristics of lactobacilli. The isolates were then identified by API 50 to identify Lactobacillus reuteri isolates [5].

2.2. L-asparaginase-production screening

The identified Lactobacillus reuteri isolates were screened by semi-qualitative assay for activity of L-asparaginase using the modified M9 medium (3.0 g KH₂PO₄; 0.5 g MgSO₄⋅7H₂O; 5.0 g L-asparagine; 0.014 g CaCl₂⋅2H₂O; 0.5 g NaCl; 2.0% (w/v) glucose, 6.0 g Na₂HPO₄⋅2H₂O; 15.0 g agar, and 0.005% phenol red). The plats were inoculated with test organisms culture of bacteria and incubated at 37 °C for 48 h set of plat medium containing L-asparagine as the source of nitrogen.

L-asparagine is hydrolyzed by L-asparaginase hydrolyzes into ammonia and L-aspartic acid. The medium color changed to pink indicating ammonia production indicating positive L-asparaginase production. The L-asparaginase activity of each bacterium was noted by measuring a pink zone in the plat and subject to L-asparaginase activity [9]. The enzyme index was determined using the following equation: $\begin{eqnarray}\displaystyle \text{Hydrolysis zone}\text{} & =\text{} & \displaystyle \text{Diameter of zone/diameter}\nonumber\\ \displaystyle \text{} & \text{} & \displaystyle \text{of a bacterial colony}.\nonumber\end{eqnarray}$

2.3. Asparagine activity assay

The L-asparaginase activity was estimated by a modified method [10]. The release of ammonia due to L-asparagine hydrolysis by the L-asparaginase was determined by Nessler’s reaction to produce an orange-colored product. The mixture of the reaction comprises 0.04 M L-asparagine and 0.5 mL of the enzyme. The mixture was incubated for 30 min at 37 °C. A 1.5 M trichloroacetic acid (0.5 ml) was added to the mixture to stop the reaction followed by a 1000 rpm centrifugation step. The resultant supernatant was added to the Nessler’s reagent (1:2) and kept at room temperature for ten minutes. One optical density of the enzyme is defined as the amount of L-asparaginase that produces 1 μmol of ammonia at 37 °C per min.

2.4. Crude L-asparaginase preparation

Crude L-asparaginase purified from no. 24 L. reuteri isolate was prepared [11] with some modifications from the original method. The L. reuteri cells were centrifuged at 10000 rpm for 15 min at 4 °C to be sedimented. The supernatant was considered as the crude L-asparaginase extract.

2.5. L-asparaginase purification

Lactobacillus reuteri was purified by four steps: precipitation by 80% ammonium-sulfate, and DEAE-cellulose column-ion exchange gel filtration [12]. The Sephadex G-150 (Pharmacia Fine Chemicals Company). A Sephadex G-150 was suspended in 0.05 M phosphate buffer pH7 [13]. Enzyme activity was measured at each step and the absorbance of each fraction was measured at 280 nm.

2.6. Cell lines and culture conditions

Ahmed Murtada Jabria’s 2013 breast cancer cell line has been established by a 70-year-old Iraqi woman breast cancer patient [14]. Cell lines were maintained in 10% Fetal bovine supplemented RPMI. RPMI medium is also supplemented with 100 μg/mL streptomycin and 100 unit/mL penicillin [15]. The MTT test was used to determine the cytotoxic effect of the L-asparaginase [16]. Cell lines cells were treated with the L-asparaginase at a concentration of IC50 value (4.305 IU/ml). The cytotoxicity percentage or cell growth inhibition rate by was calculated by Al-Shammari et al., equation [17].

2.7. RNA isolation and cDNA synthesis

Total RNA was extracted from the cell lines using the TRIzol reagent following. A 250 g/ml glycogen was added before the separation of the phases, to the cell lysate to improve the precipitation of RNA from small amounts of cell samples. Complementary DNA (cDNA) was synthesized by SuperScript^{^TM} III First-Strand Synthesis System for RT-PCR. The resultant cDNA was deposited at −20 °C to be used for qPCR [18].

2.8. Quantitative real-time PCR (qRT–PCR)

The Quantitative Real-Time PCR (qRT–PCR) was carried out using the QIAGEN Rotor gene Q Real-time PCR System (Germany). The Cas8 primer was designed on accession no. ENST00000264274.13 while the Cas9 was designed on accession no. ENST00000333868.10 using primer3Plus software. The Cas8 forward primer is 5-GCAAAGGAAGCAAGAACCCA-3, and the reverse is 5-CCTGGTGT CTGAAGTTCCCT-3 (product size: 189 bp). The Cas9 forward primer is 5-AAGTGACC CTCCCAAGTAGC-3 and the reverse is 5-GTTCT GGCCAGGTCTCTTCT-3 (product size: 190 bp). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene serves as a calibrator.

The master mix consist of 2xTransStart^® Top Green qPCR Super Mix 10 μl, forward and reverse Primer (10 μM) 1 μl, and Nuclease-free water 6 μl, Then, 3 μl of cDNA was added to the reaction tube. The cycling protocol was programmed for the following optimized cycles and according to the thermal profile comprising the initial denaturation step (94 °C for 30 sec) followed by 40 cycles of denaturation at 94 °C for 10 sec, annealing at 58 °C for 15 sec, and extension at 72 °C for 20 sec. qRT–PCR was performed in a duplicate with non-amplification control (NAC), and a non-template control (NTC), as negative controls.

3. Results

3.1. Sample collection and Lactobacillus reuteri identification

To perform limited biochemical tests, 30 colonies were selected from MRS plates, and out of these Gram-positive, and catalase-negative isolates were identified as L. reuteri.

3.2. Semi-quantitative L-asparagine production screening

Lactobacillus reuteri. no. 12 was considered the highest L-asparagine producer showing a clear pink zone of hydrolysis around (7.6 mm). The isolates varied in their growth on this medium, 3 of the L. reuteri isolates appeared to give a clear pink zone of more than 6 mm but the other 2 isolates could not grow to indicate that the L. reuteri isolates varied in their ability to hydrolyze L-asparagine (Table 1).

Table 1
The specific activity of L. reuteri L-Asparagine

L. reuteri Isolate Pink zone (mm) Specific activity (U/mg) Intensity of color L. reuteri isolates Pink zone (mm) Specific activity (U/mg) Intensity of color

1 6 4.30 +++ 16 3.8 2.50 +

2 5.6 3.80 ++ 17 — — —

3 6.8 4.60 ++ 18 4.2 2.90 +

4 5 3.60 ++ 19 2.0 0.50 +

5 4 2.92 ++ 20 1.0 1.98 ++

6 2 1.80 + 21 5.0 0.22 ++

7 3 2.30 + 22 6.3 4.51 +++

8 4.4 3.10 ++ 23 6.0 4.0 +++

9 2 1.20 + 24 2.9 3.16 ++

10 5.1 3.70 ++ 25 5.0 4.1 +++

11 4.2 3.06 ++ 26 — — —

12 7.6 4.92 ++ 27 5.5 3.22 ++

13 6 4.20 +++ 28 4.2 2.59 +

14 3.9 2.90 + 29 3.0 3.22 +

15 6.3 4.0 ++ 30 3.30 2.80 +

L. reuteri Isolate	Pink zone (mm)	Specific activity (U/mg)	Intensity of color	L. reuteri isolates	Pink zone (mm)	Specific activity (U/mg)	Intensity of color
1	6	4.30	+++	16	3.8	2.50	+
2	5.6	3.80	++	17	—	—	—
3	6.8	4.60	++	18	4.2	2.90	+
4	5	3.60	++	19	2.0	0.50	+
5	4	2.92	++	20	1.0	1.98	++
6	2	1.80	+	21	5.0	0.22	++
7	3	2.30	+	22	6.3	4.51	+++
8	4.4	3.10	++	23	6.0	4.0	+++
9	2	1.20	+	24	2.9	3.16	++
10	5.1	3.70	++	25	5.0	4.1	+++
11	4.2	3.06	++	26	—	—	—
12	7.6	4.92	++	27	5.5	3.22	++
13	6	4.20	+++	28	4.2	2.59	+
14	3.9	2.90	+	29	3.0	3.22	+
15	6.3	4.0	++	30	3.30	2.80	+

L. reuteri: Lactobacillus reuteri, mm: millimeter, U: unit, mg: milligram.

3.3. Quantitative screening of L-asparagine production

Lactobacillus reuteri. isolates were cultivated in L-Asparagine broth. Lactobacillus reuteri. isolates revealed different specific activities (1.0–4.92 U/mg). The more active L-Asparagine productive isolates had different specific activities ranging between 4.60 to 4.92 (Table 1). L. reuteri no. 12 gave the highest specific activity (4.90 U/mg proteins).

3.4. Purification of L-asparaginase

After DEAE-cellulose purification, the total activity was 18 with a protein content of 0.09 mg/ml. The specific activity of the purified L-asparaginase was 111.1 U/mg showing a purification fold of 6.94. After ion exchange chromatography, the total activity was.16.8 U protein content of 0.05 mg/ml and the specific activity was 16 U/mg protein, showing a purification fold of 10 with the fractions collected after Gel filtration chromatography for L-asparaginase (Table 2).

Table 2
Purification steps of L. reuteri L-asparaginase

Purification step Volume (ml) Enzyme activity (U/ml) Protein concentration (mg/ml) Specific activity (U/mg) Total activity (U) Purification (folds) Yield (%)

Crude enzyme 75 0.6 0.36 1.6 45 1 100

Ammonium sulfate precipitation 70% 14 1.8 0.25 7.2 25.2 4.5 56

DEAE-cellulose 18 1 0.09 11.1 18 6.94 40

Sephadex-G150 21 0.8 0.05 16 16.8 10 37.3

Purification step	Volume (ml)	Enzyme activity (U/ml)	Protein concentration (mg/ml)	Specific activity (U/mg)	Total activity (U)	Purification (folds)	Yield (%)
Crude enzyme	75	0.6	0.36	1.6	45	1	100
Ammonium sulfate precipitation 70%	14	1.8	0.25	7.2	25.2	4.5	56
DEAE-cellulose	18	1	0.09	11.1	18	6.94	40
Sephadex-G150	21	0.8	0.05	16	16.8	10	37.3

L. reuteri: Lactobacillus reuteri, ml: milliliter, U: unit, mg: milligram.

3.5. Cytotoxicity of L. asparaginase to cell line

To test the antiproliferative effects of L. asparaginase a breast cancer cell line (AMJ) was treated at various doses (3.125–100 μg). A Sigma Plot was used to determine the dose-response curve and IC50 values. The results showed a variable effect of treatments on cell lines (AMJ) proliferation. The exposure cell line to the enzyme showed a slight effect on the viability of the normal cell line. The inhibition rates (IR%) are: 30%, %, 40%, 50%, 60%, 70.7%, and 80%, of cell death, occurred when 3.125, 6.25, 12.5, 25, 50 and 100 μg/ml of the enzyme used respectively and IC50 was 4.305 μg/ml.

3.6. The effect of L. asparaginase on gene expression of caspase 8 and 9

The enzyme at a concentration of IC50 value (4.305 IU/ml) upregulates the Caspase 8 gene (the fold of gene expression = 2.071) as shown in Table 3. The enzyme at the same concentration also upregulates the Caspase 9 gene (the fold of gene expression = 1.197—Table 4).

Table 3
Fold of Caspase 8 gene expression before and after treatment with IC50 of L. asparaginase

Groups Ct of Cas8 Means Ct of GAPDH ΔCt (Ct of Cas8 - Ct of GAPDH) 2-^ΔCt Experimental group/Control group The fold of gene expression

Test 21.350 16.020 5.330 0.0249 0.0249/0.0120 2.071

Control 22.370 15.990 6.380 0.0120 0.0120/0.0120 1.000

Groups	Ct of Cas8	Means Ct of GAPDH	ΔCt (Ct of Cas8 - Ct of GAPDH)	2-^ΔCt	Experimental group/Control group	The fold of gene expression
Test	21.350	16.020	5.330	0.0249	0.0249/0.0120	2.071
Control	22.370	15.990	6.380	0.0120	0.0120/0.0120	1.000

Ct: threshold cycle, Cas8: Caspase 8, IC50: half maximal inhibitory concentration.

Table 4

Fold of Caspase 9 gene expression before and after treatment with IC50 of L. asparaginase

Groups	Ct of Cas9	Means Ct of GAPDH	ΔCt (Ct of Cas9 - Ct of GAPDH)	2-^ΔCt	Experimental group/Control group	The fold of gene expression
Test	24.510	16.020	8.490	0.0028	0.0028/0.0023	1.197
Control	24.740	15.990	8.750	0.0023	0.0023/0.0023	1.000

Ct: threshold cycle, Cas9: Caspase 9, IC50: half maximal inhibitory concentration.

4. Discussion

The studying of L-Asparaginase has increased due to its anti-tumor potential [19]. In the present study, thirty bacterial L. reuteri isolated from breast milk have been hypothesized to be a continuous source of lactic acid bacteria [20,21]. Bacillus spp was found to be an asparaginase producer [22]. They found that the production of L-Asparagine from Lactobacillus plantarum impacts carbon and nitrogen sources on enzyme activity [23].

One of the strategies of cancer cell targeting is affecting the metabolism of an amino acid [24]. Cancer cells need a lot of asparagine to be grown and disseminated. Non-cancerous cells produce a sufficient amount of L-asparagine that covers their needs via L-asparagine synthetase [25,26]. When the L-asparaginase is present it will hydrolyze L-asparagine deplete by that an important nutrition factor for malignant cells without the asparagine these cells cannot survive. Human bodies use several stylish mechanisms to protect against malignant tumor survival after DNA mutations. These mechanisms are either repairing the DNA or cell death to prevent oncogenic cell development [27].

Apoptosis is programmed cell death that exhibits distraction of the cell without inflammation induction of the surrounding cells [28]. Enzymes called caspases are implicated in the inflammatory response regulation and death of the cell [29]. The caspases are proteases that have roles in apoptosis initiation and execution. The caspases are two groups: the initiator caspases such as caspase -2, -8, -9 and -10, two of these caspases are involved in breast cancer cell inhibition in this study (caspase -8, -9), which is the first to be activated in response to a signal. The second group of caspases is the executioner caspases like caspase -3, -6, and -7, which carry out the demolition phase of apoptosis. Many anti-cancer agents induce apoptosis by indirectly engaging the caspases. Individual caspase activation is a novel therapeutic strategy introduced in the last decade [30–32].

The extrinsic pathway and intrinsic pathway are the leading caspase pathways that lead to apoptosis. Cell stress like metabolic stress and damaged DNA and induces intrinsic pathways. This pathway was induced by most of the chemotherapeutic medicines that are used in cancer chemotherapy. The use of L-Asparaginase that depletes the asparagine in the breast cancer cell microenvironment is a promise metabolic stress intrinsic pathway inhibition, as utilized in this study. However, there are few published articles regarding caspase expression deregulation affecting the incidence of cancer and its progression [33].

There are two mechanisms by which the caspases are activated; initiator caspases can be activated by multimeric complexes like caspase-8 or 9. The death of the cells induced by DNA damage requires caspase 9 activation but there is no mitochondrial cytochrome c loss. This may be due to that the effector molecule of apoptosis is affected by caspase-9 directly. The researchers’ long-term objective in cancer management is apoptosis induction in malignant tumor cells [34]. Interestingly, induction of caspase-9 activation would be expected to kill the tumor cells [35,36]. Since the up-regulation of caspase-9 expressions in breast cancer cell lines induces apoptosis [37]. Whatever the agent that induces up-regulation of caspase-9, the breast cancer cell line will be affected. L-asparaginase does so in this study despite the mechanism by which this occurs is not fully been understood and needs clarification. Furthermore, the polymorphisms of the CASP9 promoter inhibit the caspase 9 gene expression in the cells leading to breast cancer growth [38].

The caspase-8-induced apoptosis could hypothetically be established for cancer cell death [39]. De Blasio et al. [40] found that the knockdown of the CASP8 gene augmented the metastasis of MDA-MB-231 cells via stimulation of the gene expression of genes involved in the metastasis. These genes are CXCR4, HMGA2, KLF4, CTNNB1, C-MYC, and VEGFA genes. In 2017 the first report of CASP8 gene expression was reduced in breast cancer cells as compared with the neighboring normal cells. Since the induction of apoptosis by purified L-asparaginase from human breast milk L. reuteri isolates via inhibition of Caspases 8 and 9. L-asparaginase was so successful in this study, and the caspase 8 and caspase 9 induction can be considered a new therapeutic and prevention line in breast cancer [41]. The results may highlight the fact that the presence of L-asparaginase-producing L. reuteri isolates in human breast milk may aid in breast cancer improvement or even prevention via the upregulation of Caspases 8 and 9.

5. Conclusions

Breast milk Lactobacillus reuteri L-asparaginase induces apoptosis via Caspases 8 and 9 upregulation in breast cancer cell line. L. reuteri L-asparaginase treatment may be the hopeful approach for the management of the treatment of breast cancer and can be potential for the prevention of breast cancer.

Footnotes

Acknowledgements

The authors like to express deep thanks to the Mustansiriyah University (), Baghdad, Iraq.

Conflict of interest

The authors have no conflict of interest to report.

Author contributions

Zaman Hussein Hassan performed the practical part of the work and shared in writing the manuscript.

Ibtesam Ghadban Auda wrote the manuscript and supervised.

Likaa Hamied Mahdi performed some practical work and supervised.

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