Anticancer Activity Exerted by Novel Bioactive Protein from Bacillus Endophyticus Strain
DR92 Isolated from Cow Urine
Sheetal Pardeshi1,2, Raghavendra Ramappa1*, Aravinda Subrayashastry1, Shankarling Mavinamar3, Kiranmayee4 and Mary Shobha Rani Inala4
1Talent Development Centre, Indian Institute of Science, Challakere Campus at Kudapura, India
2Department of Microbiology, Modern College of Arts, Science and Commerce, Shivajinagar, India
3Department of PG studies in Botany, Karnataka State Akkamahadevi Wmen’s University, Vijayapura, India
4Department of Cell Biology and Molecular Genetics, Sri Devaraj Urs Academy of Higher Education and Research, India
Submission:February 18, 2021; Published: September 13, 2021
*Corresponding author: Raghavendra Ramappa, Talent Development centre, Indian institute of Science Challakere Campus at Kudapura, Challakere-577536 Chitradurga District, Karnataka, India
How to cite this article: Sheetal P, Raghavendra R, Aravinda S, Shankarling M, Kiranmayee, et al . Anticancer Activity Exerted by Novel Bioactive
Protein from Bacillus Endophyticus Strain DR92 Isolated from Cow Urine. Adv Biotech & Micro. 2021; 16(4): 555943 DOI:10.19080/AIBM.2021.16.555943
Breast cancer is a complex and most frequently diagnosed cancer among other types. Increasing prevalence necessitates search for new anticancer substances and medicines. Among them, bacterial proteins are a promising group of bioactive proteins and potential anticancer drugs. In this study, we attempted to isolate bacteria from cow urine. The useful bacterium was identified as Bacillus endophyticus strain DR92 by 16S rRNA gene sequence comparison. Low molecular weight proteins have been extracted, purified from B. endophyticus, confirmed and characterized by SDS-PAGE and MALDI-TOF MS. The observed molecular weights were around 8531 and 8731 daltons. The purified protein was screened for anti-proliferative activity by MTT assay against breast cancer cell line Zr-75-1 which showed 13% viability. To conclude from the study, the purified protein from B. endophyticus could be a promising anti-proliferative drug for cancer. Further work has to be done to find out the mode of action.
Global scenario with respect to occurrence of cancer in individuals of all ages is alarming and mortality due to the same is increasing rapidly. cancer occurrence in an individual depends on many factors such as diet, lack of exercise, exposure to carcinogenic pollutants through inhalation, ingestion [1,2]. oncogenic pathogen infections  and most importantly genetic predisposition [4-6]. Among all, breast cancer is the leading type of cancer occurring in females causing around 25% morbidity and 15% mortality worldwide. Statistical analysis of data for the past 30 years of Indian population shows that cancer occurrence has increased from 0.6 million in 1991 to 1.4 million in 2015, of which 21% cases are of breast cancer .
The present treatment of breast cancer uses a combination of chemotherapy, hormonal therapy, immunoglobulin administration and radiation. Unfortunately, 100% removal of cancer cells may
not be possible in all cases using these treatments and thus recurrence is inevitable in such patients. Many reports from different parts of the world are coming up describing alarming development of acquired resistance in secondary cancer cells towards anticancer agents used in treatment of primary cancer in the patient with incidence of recurrence [8-10]. Due to developing resistance among cancer cells for existing anticancer drugs, there is imperative need of bio-prospecting possible resources for new anticancer candidates.
Microorganisms have been employed in numerous processes such as alcohol fermentation and cheese production since ancient times but with the advent of technologies in study of microorganisms especially with respect to the primary and secondary metabolites they produce, their applicability for medicinal purposes has increased. Microorganisms have been reported for production of metabolites with myriad of
bioactivities and are of choice for large scale production of such
bioactive compounds due to ease in cultivation, maintenance,
genetic manipulations and downstream processing.  have
described anticancer potential of azurin protein and peptide
p28 isolated from Pseudomonas aeruginosa, whereas Bacillus
thuringiensis extracellular polysaccharide has been reported to
show anticancer activity . Use of Brevibacillus choshinensis
transformant as bacterial cancer therapy to deliver anticancer
proteins in the body efficiently is also reported by . Thus,
bacteria as a potential resource for anticancer peptides have
been validated through various reports. In present study, we
aimed at isolation of microorganisms with potential to produce
anticancer proteins that may serve as drug candidates for future
As per ancient Indian literature, cow urine is described to have
multiple medicinal properties and is recommended to be used to
treat various ailments including cancer . Reports coming from
different parts of India and some other countries have illustrated
scientific proof of the same [15,16]. However, bioactive potential
of microflora of cow urine has been less explored and thus was
undertaken in present study.
Cow urine sample were collected inasterile container from
Kudapura, Challakere taluk, Chitradurga district. Karnataka.
India. Sample was brought to laboratory and used immediately
for isolation of bacteria.
One ml of cow urine sample was serially diluted0.1 ml of each
aliquot was spread on Nutrient agar with pH 7.2 ± 0.2. Plates
were incubated at 370C for 24 hr. The colony showing iridescence
was selected and purified on Nutrient agar plate. Culture was
maintained and preserved on the same media slant at 40C until
further use .
Preliminary identification of isolate was carried out by
morphological studies which included Gram staining, motility,
endospore staining and cultural characteristics on agar plates
such as colony morphology and pigmentation. Biochemical
characterization of isolate was performed for catalase, oxidase,
carbohydrate fermentation, H2S production, starch hydrolysis,
urease production and nitrate reduction .
Genomic DNA was isolated according to the manufacturer’s
protocol given in DNA extraction kit (Chromgene, Bangalore,
India). Universal primers 27F and 1492R were used to amplify 10
to 12 ng of DNA template by PCR. The amplified 1.5 kb product
was purified by QI quickgel extraction kit (Qiagen, Germany) and
directly sequenced on ABI (Applied Biosystem, Sigma, Mumbai)
automated sequencer as recommended by manufacturer. 16S
rRNA gene sequence was aligned with submitted sequences
available in the NCBI database using clustalX software. Phylogeny
was established using maximum likelihood method.
The pure culture of bacterium was inoculated into enrichment
medium containing peptone 5; sodium chloride 5; yeast extract
1.5; starch 2% followed by incubation at 37°C under shaking
conditions (150 rpm) for 2 days at initial pH 7.4. Sample was
centrifuged at 10,000 rpm for 20 min at 40 C and the supernatant
was collected and subjected to Ammonium sulphate precipitation.
Initial 20% and final 65% precipitation was collected and dialyzed
using cut off 3kDa membrane with 10mM -Tris -HCl, 50mM NaCl,
and 20mM Imidazole. Dialysate was concentrated using 3kDa cut
off centricons; protein was subjected to further purification using
FPLC (AkTA START) Sephadex G-75 column (Sigma chemicals Co.
St. Louis, MO, USA) matrix and the flow rate of eluate as maintained
at 0.5ml /minute using 10mM Tris-HCl; 50mM NaCl. Each 2ml
fraction was collected and optical density (OD) was recorded at
a wavelength of 280 nm. Purified protein peak fractions were
collected and concentrated using 3kDa cut offcentricons which
were further characterized using SDS-PAGE and MALDI-TOF.
The purified protein was subjected to SDS-PAGE for molecular
mass determination according to the procedure of Laemmli and
Favre 1973. By using 10 % separating gel, 4 % stacking gel under
non reducing conditions. The molecular mass of the purified
protein was estimated by comparison of its electrophoretic
mobility with those of molecular mass marker proteins (GE
Healthcare, Bangalore, India). The gel was stained with Coomassie
brilliant blue R-250 and de-stained with solution containing 15 %
methanol and 5 % acetic acid.
Gel-filtration chromatography was performed using a
Sephadex G-75 column (GE Healthcare, Bangalore, India) with
a conventional FPLC system equilibrated with 10 mM Tris–HCl
buffer containing 50 mM NaCl (pH 7.5) at a flow rate of 0.5 mL/
min and monitored at 280 nm.
Matrix-assisted laser desorption ionization time-of-flightmass
spectrometer (MALDI-TOF-MS) was used to determine the molecular weight of the protein. Analysis was performed using
a Reflex IV MALDI-TOF-MS system (Bruker Daltonics, Bremen,
Germany). The purified protein 0.5 μl was spotted on an Anchor
chip target plate with 0.5 μl of freshly prepared matrix solution2,5-
Dihydroxybenzoic acid. The above mixture was loaded on the
Anchor chip target plate and the sample was allowed to dry at
room temperature for 10 min. The dried target plate was inserted
into the mass spectrometer and analysis was performed. The mass
spectrum was acquired on a MALDI-TOF/TOF mass spectrometer
(Autoflex II; Bruker Daltonics) equipped with a pulsed nitrogen
laser (337 nm) in positive reflection mode.
The obtained breast cancer cell lines (Zr 75-1) from
National Centre for Cell Science (NCCS) Pune were sub-culturedcryopreserved in liquid nitrogen / - 800Cdeep freezer
until use. Prior to the analysis, the cells were thawed and were
grown for required cell density by using RPMI 1640 along with
10% foetal bovine serum. All the cultures were incubated at 37oC
in an atmosphere of 5% CO2 and 95 % air.
The cytotoxic activity of protein sample was measured by
bromide (MTT). Cells of 200 μl (1 x 10 5 cells/ml density) were
seeded in a 96 well plates overnight. The Concentrated purified
protein stock of 1mg/ml solution was made and 100 μg/ml was
added to the plate containing cell lines and incubated for 24h.
Rinsed the cells with 1X PBS and incubated with 20 μl of 5 mg/
ml MTT at 370C for 3-4 h. After incubation; dissolved the dark
blue crystals of formazan were dissolved with 100 μl of DMSO and
continued incubation for about 10 minutes. The level of reduced
MTT was determined by measuring the absorbance at 570 nm.
Cell viability was calculated by using the formula given below.
The isolate was Gram positive, rod shaped, approximately 3μm
in length showing flat circular colonies with iridescence, round
margin and mucoid consistency. The biochemical feature of the
isolate is described in table 1. The partial 16S rRNA gene sequence
of 445bp length was deposited in NCBI with the accession number
MH604592. Based on the results of the phenotypic and genotypic
analyses, we concluded that the isolate belongs to Bacillus
endophyticus (Figure 1).
Protein purification was performed by using Sephadex G-75
desalting column by using system FPLC (Akta start). Protein peak
fractions were collected (Figure 2), concentrated to 1mg /ml
using 3kDa cut-off centricons. The resultant protein was used for
The molecular weight of purified novel bioactive protein was
determined by comparison with Rf values of standards in 10%
SDS-PAGE. The observed protein band was corresponding to the
molecular weight of 8 kDa (Figure 3). Further the protein was
characterized using MALDI-TOF-MS (Figure 4).
The results obtained demonstrated that the purified protein
from cow urine has a significant anticancer potential on top ranked
cancer i.e; breast cancer cell line (Zr-75-1). After calculating the
viability, the test protein exhibited 14.25 % viability and 85.75 %
dead or non-viability. Below given table 2 describes the percentage
viable and non - viable cells up on treatment.
Biochemical composition of cow urine has been studied to a
great extent and is reported to contain essential elements, enzymes
and hormones that can exert health benefits to humans Satyapal Singh, 2019, Joshi and Adhikari, 2019. Different researches
have also validated the therapeutic potential of cow urine as
antioxidant, anticarcinogenic, antidiabetic and antimicrobial
agent [18-21] Recently, patents have been granted for use of cow
urine distillate along with antibiotic quinolone or fluoroquinolone
and antifungal agents such as azoles, clotrimazolesmystatin or
amphotericin which enhance antimicrobial effect of these agents
, as well as, for the use of bioactive fraction from cow urine
distillate as a bioavailability facilitator and pharmaceutically
acceptable additives for anti-infective and anticancer agents
[23,24]. It will be interesting to know if any of these bioactive
compounds are metabolites of microflora of cow urine imparting
medicinal properties to cow urine which was one of the objectives
of this study. Discovery of new anticancer agents and especially
of anticancer peptides is of significance due to rising resistance
of cancer cells to chemotherapeutic agents [25-28]. In coherence
with this, in present study, we report novel bioactive proteins
from Bacillus endophyticus strain DR92 isolated from cow urine
against breast cancer cell lines (Zr 75-1). Previously, Bacillus
endophyticus JUPR15 isolated from environmental samples
has been presented to produce protease inhibitors exhibiting
promising activity and to induce apoptosis in HeLa, HepG2 and
MCF-7 cancer cell lines [29,30]. which is in consistency with our
results. Apart from anticancer activities, metabolites from genus
Bacillus are described to display variety of bioactivities .
Bacteria such as Streptomyces and other genera of actinobacteria
are reported to be able to produce anticancer metabolites .
Production of anticancer agents has also been attributed to
endophytic fungi for which comprehensive review can be found
elsewhere [33-35] have reported potential of <3kDa peptide
fraction from Spirullina platensis to significantly reduce viability
of Human colon adenocarcinoma cell line (SW480).
Anticaner agents such asgenistein, lycopene, resveratrol
(currently under clinical trials) and taxene paclitaxel (approved
for human use in 1992) from plants have also been reported ,
but mass production of such compounds is a problem due to their
plant origin. Peptides from hydrolysates of food derived proteins
such as milk, eggs, oyster, soybean, peas, Spirulina are reported
to have anticancer and immunomodulatory properties . but
production of these peptides requires hydrolysis of proteins using
commercial proteases, fermentation with bacterial strains and
gastrointestinal digestion which increases the cost of process and
makes purification protocols laborious. The present study findings
about the functionality of the protein are significant and bacterial
protein production is easy in large scale. Anticancer peptides
have been known to act directly by binding or indirectly to kill
cancer cells Hilchie, et al.  have published a broad review on
mechanisms of anticancer peptides. Some reports elaborate on
use of nano-carriers and liposomes for delivery of anticancer
peptides to increase their half-life in experimental animals . A
detailed study will be needed in order to elucidate exact mode of
action of the novel protein reported here.
Bacterial proteins or peptides already used in various cancer
treatments include actinomycin D from Actinomycesantibioticus,
bleomycin from Streptomyces verticillus, doxorubicin from
Streptomyces peucetius var. caesius, mitomycin C from
Streptomyces caespitosus and diphtheria toxin, while p28,
arginine deiminase ADI are in clinical trials. Therapeutic peptides
that are GnRH agonists such as buserelin, gonadorelin, leuprolide,
nafarelin, goserelin, triptorelin, and histrelin, cetrorelix, abarelix,
degarelix are in clinical use for treatment of breast and prostate
cancer . This signifies our work of presenting more options
for the established successful treatment protocols of cancer.
Cancer prevalence is increasing alarmingly and multi drug
resistance in cancer cells is of utmost concern. Present study
signifies the potential of tiny life forms of holding a tremendous
potential to produce bioactive compounds that can be used in a
variety of applications including cancer treatment. In this study,
we report purification of novel anticancer protein from bacteria
isolated from cow urine. The bacterial isolate was found to be
Gram positive short rod and was characterized using various
biochemical tests. The isolate was identified to be Bacillus
endophyticus by 16S rRNA sequencing (Accession number
MH604592). Protein fraction from isolate was obtained by
ammonium sulphate precipitation and was purified by SDS PAGE
and FPLC. Further we report that the novel bioactive protein thus purified is having molecular weight of 8kDa using MALDI TOF MS
analysis and exhibits anticancer activity against breast cancer cell
line Zr 75-1 which showed only 13% cell viability after treatment
with novel protein. The findings of present study are significant
in the wake of increasing anticancer drug resistance where
alternatives to current therapies are needed. Studies like this
can help identify potential anticancer candidates for future use.
Moreover, ease of handling, mass production, purity and safety of
bioactive compounds makes this approach striking and economic.
Authors are grateful to Prof M S Hegde Convener Talent
development centre; Indian institute of science, Challakere,
Chitradurga district, Karnataka state, India, for his support in
present work. We are greatly thankful to Sri Devaraj Urs Academy
of Higher Education and Research centre Kolar district, Karnataka
state, India, for their aid in conducting anti-proliferative studies.
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