Parasporal Cry Protein Parasporin-2 Produced by Bacillus

thuringiensis Has in Vitro Toxicity on Human Cancer Cells (HepG2)

under the Action of Proteinase K

Xuan Zhou

School of Biochemistry and molecular biology, University of Western Australia, Perth, WA6009, Australia

Keywords: Parasporal Cry Protein, Bacillus thuringiensis, Cancer Cells, Proteinase K.

Abstract: Liver cancer is one of the malignant tumors with the fastest increasing morbidity and mortality and the greatest

threat to people's health and life. This article investigates whether parasporin-2 produced by the hydrolytic

non-hemolytic Bacillus thuriensis can recognize liver cancer cells and have cytotoxicity to them. In this paper,

Bacillus thurinensis parasporal Cry protein—parasporin genotypes were determined in this strain using the

PCR amplification. Then, parasporin-2 was separated by SDS-PAGE and purified. The same concentration of

cultured HepG2 (human liver cancer cells) and L-O2 (normal human liver fibroblasts) was added to the plate

and divided into two groups. Then, 3 solutions contain parasporin-2 and protease K was added to the plate.

The damage to the cells was observed under a microscope and graded the degree of cell damage (CPE), and

MTT then determined cytotoxicity (CT). Analysis the SDS-page and following conclusions may be drawn by

comparing CPE and CT in each group. First, through horizontal comparison, the data of HepG2 and L-O2

cells in each group were compared, to determine whether parasporin-2 is toxic to liver cancer cells but not to

ordinary liver fibroblasts. Besides, longitudinal comparison is the situation of CPE and CT in different groups,

whether Parasporin-2 plus Protease K can produce toxicity on HepG2 of liver cancer cells. Bacillus

thuringiensis can produce parasporin-2, and after the decomposition of protease K protein, parasporin-2 can

produce recognition and cytotoxicity to liver cancer cells. analysis the degree of cell destruction and toxicity,

after dealing with the proteinase k, cut parasporin – 2 toxicity is activated. Conclude that parasporin - 2 which

hydrolyzed by protease k has a recognition on the liver cancer cells and it's toxic to cancer cells but will not

produce toxicity to normal liver cells.

1 INTRODUCTION

Shigetane Ishiwata, a Japanese, first discovered

Bacillus thuringiensis in Japan in 1901 and described

the dying state of the larvae in Bacillus thuringiensis

(E. Hough, 1989). In 1905, from his experiments, he

realized that the poisoning appeared to be caused by a

sort of poison and occurred before the multiplication

of bacillus. His incomplete identification led to the

German Ernst Berliner's first morphologically valid

description and the successful isolation and naming of

Bacillus thuringiensis from Anagasta Kuehniella in

the Mediterranean (Sansinenea, 2012).

Bacillus thuringiensis is an aerobic, gram-positive,

spore-forming facultative bacterial pathogen. Under

conditions of adequate nutrition and environment,

spores germinate and then produce vegetative cells,

which grow and reproduce by fission and produce a

variety of nutrients. Bacterial spores consist mainly of

one or more insecticidal proteins in the form of

crystalline inclusions, known as insecticidal crystal

proteins (ICP) or 𝛿 -endotoxin (Kim, 2000). The

insecticidal crystal proteins of Bacillus thuringiensis

mainly consist of CRY crystal protein and CYT

cytolytic protein.

Bacillus thuringiensis, as a soil bacterium, is

known to be used as a biological insecticide and

mosquito control in agriculture and forestry. Today,

the use of biological insecticides is one of the most

important components of integrated pest management

and has been recognized by countries around the

world. However, bacillus thuringiensis was shown in

1999 to have a new cytotoxic effect on human cancer

cells (Akao, Mizuki, Yamashita, Saitoh, and Ohba,

1999).

Parasporin-2 is a new Cry crystal protein that can

be isolated from Bacillus thuringiensis. Through

research, the n-terminal region of Parasporin-2 can be

Zhou, X.

Parasporal Cry Protein Parasporin-2 Produced by Bacillus thuringiensis Has in Vitro Toxicity on Human Cancer Cells (HepG2) under the Action of Proteinase K.

DOI: 10.5220/0011264400003443

In Proceedings of the 4th International Conference on Biomedical Engineering and Bioinformatics (ICBEB 2022), pages 723-728

ISBN: 978-989-758-595-1

723

cleared to effectively activate the toxin activity, while

C-terminal digestion can also lead to cell damage

(Akiba, 2009); (Kitada, 2006). This paper investigates

whether the human liver cells HepG2 can be killed by

Parasporin-2— a kind of crystal protein of Bacillus

thurinensis in vitro. This study predict that parasporin-

2 can induce toxicity on liver cancer cells (HepG2)

under the action of protease K.

2 METHODS AND MATERIALS

Bacteria cultivation- The soil isolates of Bacillus

subtilis HepG2 were grown on AGAR at 28 ° C for 8

days, and an appropriate amount of beef powder and

polypeptide were added, along with a small amount of

NaCl, and the PH was maintained at 7.6.

Human cells cultivation- In this study, we used

HepG2 (human liver cancer cells) and L-O2 (human

normal liver fibroblasts) cell lines and purchased

normal T cells in the blood center to separate from

lymphocytes. Cells were maintained in RPMI 1640

and 10% fetal BSA and 30uL kanamycin were added

at 37℃, and prepare normal human red blood cells

(Mizuki, Ohba, Akao, Yamashita, Saitoh, and Park,

1999).

DNA isolation and PCR amplification- PCR was

used to test the gene of parasporin-2. Total genomic

DNA was used as PCR template by parasporin pure

bacterial DNA purification equipment isolated from

parasporal protein. Heat circulator was used to prepare

a reaction mixture containing 50-100 ng total genome

DNA of Bacillus thuringiensis with 19× L PCR buffer

(10 mM TRis-HCl). PH value 9.0, 50mm KCl, 1.5mm

MgCl

), dNTPs 75×M each, primers 0.2×M each

(Table 1), Taq DNA polymerase 1.5U. Template DNA

preheated at 94℃ for 2 minutes. Denaturation at 94℃

for 1 min, primer annealing for 45 s, PCR

amplification at 72℃ for 1 min. PCR detection was

performed for 30 cycles. PCR was analyzed under

1.2% agarose gel, and then stained with UV irradiation

(Sansinenea, 2012).

DNA sequencing- PCR products were purified

using PCR purification kit (Bolotin, 2016).

Isolation Parasporin from parasporal crystals-

Separation spores of Bacillus thuringiensis strains

cells with distilled water three times, and broken in

distilled water, using two-way separation purification

spores by parasposal crystal, the parasposal Cry

protein in the 50 mM, ph10 NaCO

dissolve 1 h, then

add 1 mM phenylmethylsulfonyl fluoride and EDTA

to stop. The protein solution of PH10.0 was treated

with protease K and incubated at 37°C for 90min, and

PMSF is used to stop protease digestion. Combining

on ion exchange column, with 50 mm NaCl elution

toxin protein parasporin 20 mM Tris HCl buffer, in pH

8.0. The active component is then treated and gel

filtration (Ito, 2004).

SDS-PAGE AND WESTERN BLOTTING-

Membrane-enriched or cytosolic fractions were

suspended in SDS sample buffer for SDS-PAGE. The

separated proteins were transferred

electrophoretically to nitrocellulose membranes, and

immunodetection was carried out using antibodies

against parasporin-2. Binding of the primary antibody

was visualized using a horseradish peroxidase-labeled

anti-rabbit parasporin-2 secondary antibody and

Lumilight plus. Chemiluminescence need to use

FluorS-MultImager.

Cytotoxicity assay and Hemolytic assay- prepare

some microplates each containing the same amount

and concentration of HEPG2 and L-O2 cell solutions

in each well. The measured solution was divided into

six groups as below (Table 2), group 1&2 were the

negative control group, and the solution was 100%

absorbent cell suspension. Group 3&4 was

parasporin-2 solution actioned with protease K. Group

4&5 are parasporin-2 solution after separation and

purification. At the same time, Drop three solutions,

each solution into an HEPG2 plate and an L-O2 plate.

Then mark microplates. Measure their absorbance and

repeat the experiment more than three times. The

degree of CPE was graded based on the proportion of

damaged cells (Table 3).

Table 1: Solution and cell composition tables for each group of microplates. Contain (✓).

Buffer Parasporin-2 Protease

HepG2 L-O2

Group 1 ✓ ✓ ✓

Group 2 ✓ ✓ ✓

Group 3

✓ ✓ ✓ ✓

Group 4

✓ ✓ ✓

✓

Group 5 ✓ ✓ ✓

Group 6

✓ ✓

✓

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Table 2: The expression of percentage of cells destroyed.

𝟓%

5%-10% 10%-30% 30% -60% 60%-90%

The proportion of damaged cells (CPE) - + ++ +++ ++++

The experiment was repeated for more than three

times. Cytotoxicity was determined by MTT [3-(4, 5-

Dimethyl-2-thiazolyl)-2, 5-diphenyl-2h Tetrazolium

Bromide] (Mizuki, 2000). Each of the microplates

contained 90 microliters of cell suspension, each

containing a certain number of cells. Measurements

were made using a nonradioactive cell test system.

The number of cell proliferations was measured after

incubation for 16 hours at 37 ° c and 24h after

administration. Calculate survival rate. The average

absorption properties of the sample had 100% cell

survival as a negative control. The degree of

cytotoxicity (CT) was graded on the basis of the

relative value of absorbance. (Table 4)

Table 3: The expression of the degree of cytotoxicity.

0.70

0.60-0.90 0.30-0.60 0.10-0.30

0.10

The degree of cytotoxicity (CT) - + ++ +++ ++++

Statistical analysis- All of the numerical data

were analyzed through one-way ANOVA method All

of the mean multiple comparisons were conducted

using Tukey’s Post Hoc test. P≤0.05 was considered

a significant difference. Graphs were prepared using

Microsoft Office Excel.

RESULTS

3.1 Analyze the PCR Results

PCR is a rapid and highly sensitive method for

detecting and identifying Bt genes (Carozzi, Kramer,

Warren, Evola, and Koziel, 1991). The efficacy of

PCR for cry genes and ps genes identification relies

on the alternation of conserved and variable

nucleotide regions. Through comparison and inquiry,

the existence of parasporin-2 genes in the parasporal

protein produced by Bacillus thuringiensis strain was

confirmed (Figure 1).

Figure 1: Prime sequences of gene capA, ps1, ps2, ps3, ps4

(Moazamian, Bahador, Azarpira, and Rasouli, 2018).

3.2 SDS-PAGE and Western Blot

Analysis

Protein gel electrophoresis provides an obvious

display of complex protein collections from a

biological sample. The gels can be compared with

each other to evaluate the similarities and differences

between samples. The two-dimensional gel provides

separation and information on two important physical

properties of protein components in the sample,

namely, apparent molecular mass. Western blot was

used to isolate and purify parasporin-2 in

combination with anti-Parasporin-2 (which can be

obtained by injecting antibodies into rabbits).

Through SDS analysis, the paracrystal protein

produced by Bacillus thuringiensis HepG2 was

hydrolyzed after being hydrolyzed by protease K, and

the reference strains of SDS-PAGE swimming lane 3-

Parasporal Cry Protein Parasporin-2 Produced by Bacillus thuringiensis Has in Vitro Toxicity on Human Cancer Cells (HepG2) under the

Action of Proteinase K

725

6 were parasporin-1,2,3,4. (Figure 2) Primary and

secondary of parasporin-2 antibodies were detected

and imaged by Western Blot using

immunofluorescence (Lenina, Naveenkumar,

Sozhavendan, Balakrishnan, Balasubramani, and

Udayasuriyan, 2014).

Figure 2. Lanes 3–6 reference strains of parasporin (PS4, PS3, PS2 and PS1) (Lenina, Naveenkumar, Sozhavendan,

Balakrishnan, Balasubramani, and Udayasuriyan, 2014).

3.3 Cytotoxicity Analysis

Detect the proportion of HegG2 and L-O2 cells

distraction and the degree of cytotoxicity. Each group

for possible results corresponds to the groups in Table

Table 5 shows all the possible outcomes, but since

many of the outcomes were not possible, by making

a separate table of the following possible outcomes.

Table 4: All outcomes of cytotoxicity assay and Hemolytic assay.

The possible results are as follows:(1) As the

Table 6 shows, it means group 3&4 have high

proportion on cell damage and high degree of

cytotoxicity. Group 1,2,5&6 have low proportion on

cell damage and low degree of cytotoxicity. (2) As the

Table 7 shows, it means all groups have low

proportion on cell damage and low degree of

cytotoxicity. (3) As the Table 8 shows, it means group

1&2 have high proportion on cell damage and high

degree of cytotoxicity. Group 3,4,5&6 have low

proportion on cell damage and low degree of

cytotoxicity, etc.

OUT COM ES

CPE - CT ++++ OUTCOME 1

CPE ++++ CT - OUTCOME 2

CPE - CT ++++ OUTCOME 3

CPE ++++ CT - OUTCOME 4

CPE - CT ++++ OUTCOME 5

CPE ++++ CT - OUTCOME 6

CPE - CT ++++ OUTCOME 7

CPE ++++ CT - OUTCOME 8

CPE - CT ++++ OUTCOME 9

CPE ++++ CT - OUTCOME 10

CPE - CT ++++ OUTCOME 11

CPE ++++ CT - OUTCOME 12

CPE - CT ++++ OUTCOME 13

CPE ++++ CT - OUTCOME 14

CPE - CT ++++ OUTCOME 15

CPE ++++ CT - OUTCOME 16

++++

THE CPR

CT OF GROUP 3

CPE CT

++++

CPE

++++

CPE

THE CPR

CT OF GROUP

THE CPR

CT OF GROUP

CPE

THE CPR

CT OF GROUP

CPE

++++

ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics

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Table 5-12. The possible results 5-12. The above table

reflects the results of Cytotoxicity assay and Hemolytic

assay. An increase in the number of + indicates an increase

in the value. For example, ++++ indicates that the value is

too large, and + indicates that the value is small. And-means

that the value is close to 0. CEP stands for proportion of

damaged cells, and CT stands for the relative value of

absorbance. The step of Cytotoxicity assay and Hemolytic

assay should be repeated at least 3 times.

4 CONCLUSION

The morphology of Bacillus thuringiensis spores

(cubic, spherical, rhomboid and irregular) was

observed by isolation. It indicates the parasporal

crystal protein. Through SDS-PAGE analysis and

Western Blot, which can determine the existence of

parasporin-2. Through the observation of brightfield

confocal microscopy and MTT results, the

absorbance of solution (which contain parasporin and

protease K) were analyzed.

The cells observed under the microscope were

compared with Figure 3 and 4, and the damage

degree of the cells was calculated. The higher the

degree of cell destruction, the higher the

transmittance measured by MTT, indicating the

stronger the cytotoxicity of the solution. The next step

is to analyze possible results (Table 5-12). For

possible result 1, it means parasporin-2 after action

with protease K is toxic, and it don’t have specific

identification of cancer cells. Possible result 2 reflects

neither parasporin-2 with protease K nor solutions

containing only parasporin-2 are not toxic to cancer

cells. In possible result 3, it means parasporin-2 is

toxic to liver cells. As to possible result 4, which is

closest to my prediction, reflects only after reaction

with protease K, parasporin-2 can identify the cancer

cells and kill them, but it is no toxic to normal liver

cells. Besides, the solution contains only parasporin-

2 are not toxic to HepG2 and L-O2. In possible result

5, contrarily with the possible result 4, the group with

the solution contains only parasporin-2 has high

damaged cells and high cytotoxicity, which means

parasporin-2 is toxic and has identification of cancer

cells. Possible result 6 reflects that parasporin-2 is

toxic to cancer liver cells, whether it reacts with

protease K. In possible result 7, which is contrary to

the possible result 6. It means parasporin-2 is toxic to

normal liver cells, whether it reacts with protease K.

As for the possible result 8, parasporin-2 reacts with

protease K isn’t toxic to HepG2 but is toxic to L-O2,

and parasporin-2 is toxic to not only live cancer cells

and normal liver cells.

Protease K acts on the C terminal and activates

parasporin-2 to identify and produce cytotoxicity on

liver cancer cells. Parasporin-2 can specifically

bound to the plasma membrane of liver cancer cells.

It rapidly increases the membrane permeability, and

that it dramatically alters the cytoskeleton and

organelle morphologies. Thus, parasporin-2 is a cell

discriminating, membrane-targeting, and pore-

inducing toxin that subsequently causes irreversible

intracellular decay in liver cancer cells. It can be

found from other studies that the insecticidal spectra

of many strains of Bacillus thuringiensis currently

studied are very narrow. For example, cry crystals

produced by a hemolytic bacillus thuringiensis

studied in Japan produce toxins in only a few genera

of the beetle family (Kaur, 2006). Therefore, Bacillus

thuringiensis may have tremendous potential for non-

insecticidal applications, such as the treatment of

human cancers.

Tabl e 5 Tabl e 6

CPE CT CEP CT

Group 1 - ++++ Gr oup 1 - ++++

Group 2 - ++++ Gr oup 2 - ++++

Group 3 ++++ - Gr oup 3 - ++++

Group 4 ++++ - Gr oup 4 - ++++

Group 5 - ++++ Gr oup 5 - ++++

Grouo 6 - ++++ Gr ouo 6 - ++++

Tabl e 7 Tabl e 8

CEP CT CEP CT

Group 1 - ++++ Gr oup 1 - ++++

Group 2 - ++++ Gr oup 2 - ++++

Group 3 ++++ - Gr oup 3 ++++ -

Group 4 ++++ - Gr oup 4 - ++++

Group 5 ++++ - Gr oup 5 - ++++

Grouo 6 ++++ - Gr ouo 6 - ++++

Tabl e 9 Tabl e 10

CEP CT CEP CT

Group 1 - ++++ Gr oup 1 - ++++

Group 2 - ++++ Gr oup 2 - ++++

Group 3 - ++++ Gr oup 3 ++++ -

Group 4 - ++++ Gr oup 4 - ++++

Group 5 ++++ - Group 5 ++++ -

Grouo 6 - ++++ Gr ouo 6 - ++++

Tabl e 11 Tabl e 12

CEP CT CEP CT

Group 1 - ++++ Gr oup 1 - ++++

Group 2 - ++++ Gr oup 2 - ++++

Group 3 - ++++ Gr oup 3 - ++++

Group 4 ++++ - Group 4 ++++ -

Group 5 - ++++ Gr oup 5 ++++ -

Grouo 6 ++++ - Grouo 6 ++++ -

Parasporal Cry Protein Parasporin-2 Produced by Bacillus thuringiensis Has in Vitro Toxicity on Human Cancer Cells (HepG2) under the

Action of Proteinase K

727

Figure 3: HepG2 cells with parasporin-2 under the brightfield confocal microscopy (S. Kitadam 2006).

Figure 4: L-O2 cells under the brightfield confocal microscopy (X. Liang, G. Xu, Q. Gao, and X. Tao, 2016).

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