Original Article
Combination of Ononis hirta and
Bifidobacterium longum decreases
syngeneic mouse mammary tumor burden
and enhances immune response
ABSTRACT
Background: The resistance of solid tumors to conventional therapies has prompted the need for alternative therapies
Aim: To evaluate in vitro and in vivo effect of extracts from Ononis hirta against resistant mouse mammary gland cell line (66 cl-4-GFP)
and to use a combination of Ononis hirta extract with Bifidobacterium longum to target resistant solid tumors in mice.
Materials and Methods: Different solvent extracts of Ononis hirta were prepared and their in vitro antiproliferative activity was
tested against 66 cl-4-GFP cell line using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Thin layer
chromatography (TLC) and high-performance liquid chromatography (HPLC) were used to identify the active extracts. Balb/C mice
were transplanted with 66 cl-4-GFP cell line and in vivo antitumor activity was assessed for the plant extract, Bifidobacterium longum,
and a combination of plant extract and Bifidobacterium longum. Histological examination of tumors was performed using standard
hematoxylin/eosin staining protocol while gram stain was used to detect the presence of anaerobic bacteria in these sections.
Results: A combination of Ononis hirta methanol extract and Bifidobacterium longum showed high ability in targeting solid mammary
gland tumors in mice. It also induced extensive necrosis in these tumors. Thirty percent of mice treated with such combination
were cured of their cancers. The mechanism underlying this anticancer activity involves immune system activation exemplified by
the observed rejection of reinoculated tumors by cured mice. Chemical TLC analysis of the active methanol extract showed the
presence of flavonoids, terpenoids, and alkaloids. HPLC analysis confirmed the presence of flavonoids and alkaloids in Ononis hirta
methanol extract.
Conclusion: The complete regression of the tumor is encouraging and shows that plant extracts in combination with Bifidobacterium
longum is an inviting option to treat solid tumors.
Wamidh H. Talib,
Adel M.
Mahasneh1
Department of
Clinical Pharmacy and
Therapeutics, Applied
Science University,
1
Department of
Biological Sciences,
University of Jordan,
Amman, Jordan
For correspondence:
Dr. Wamidh H. Talib,
Department of
Clinical Pharmacy and
Therapeutics, Applied
Science University,
Amman, Jordan.
E-mail: altaei_
wamidh@yahoo.com
KEY WORDS: Anticancer activity, bacteriolytic therapy, Bifidobacterium longum, Ononis hirta methanol extract
INTRODUCTION
Ononis hirta (L.) (Family: Fabaceae) is an annual,
nonclimbing herb distributed in different regions
of the Middle East including Jordan. In Jordanian,
traditional medicine, Ononis hirta, is used to treat
different ailments including cancer, necrosis, and
cold sores.[1]
Cancer development is a multistep process
including induction of genetic instability, abnormal
expression of genes, abnormal signal transduction,
angiogenesis, metastasis, and immune evasion.[2]
Continuous cell division of cancer cells lead to the
formation of tumors. In solid tumors, blood vessels
become structurally and functionally abnormal;
this abnormality leads to heterogeneous blood
flow which creates chronically hypoxic and acidic
regions in the core of the solid tumor.[3] The two
traditional therapies (chemotherapy and radiation)
are not greatly efficient in treating hypoxic cancer
cells and showed limited selectivity. The killing
effect of ionizing radiation depends on the presence
of oxygen which is absent or very low in the tumor
core and the poor vascularization minimizes the
delivery of chemotherapeutic agents.[4]
One of the approaches to selectively target the
hypoxic region of the tumor is the use of anaerobic
bacteria alone or in combination with other
agents.[5-7]
Access this article online
Website: www.cancerjournal.net
DOI: 10.4103/0973-1482.103523
PMID: ***
Over the past 50 years, several strains of facultative
and obligate anaerobic bacteria have been used to
localize and cause lysis in transplanted tumors in
animals.[8] Many researches focused on Clostridium
or Clostridium spores as anticancer agents alone
or in combination with conventional therapies.[9]
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Talib and Mahasneh: Combined bacteriolytic therapy of solid tumors
Compared with Clostridium, Bifidobacterium is nontoxic,
nonspore forming, and naturally found in the alimentary
canal of human digestive system.[10] Many phytochemicals
were proved to target cancer development.[6] Such natural
products can be combined with anaerobic bacteria to
increase the therapeutic stress applied on solid tumors.
Previous work in our laboratory reported high and selective
antiproliferative activity of the methanol extract of Ononis
hirta aerial parts against MCF-7 (mammary gland tumor)
cell line.[11]
The objective of this study was to test a new anticancer
combination therapy composed of Bifidobacterium longum
and Ononis hirta methanol extracts against resistant mammary
gland tumor inoculated in mice.
MATERIALS AND METHODS
Plant material
Ononis hirta aerial parts were collected from Ajloun area
in the north of Jordan. The taxonomic identity of the plant
was authenticated by Prof. Ahmad EL-Oqlah (Department of
Biological Sciences, Yarmouk University, Irbid, Jordan).
Bacterial strain and culture conditions
Bifidobacterium longum subsp. infantis (DSMZ 20090) was
used in this study. The bacterium was cultured using MRS
media (Oxoid, UK) supplemented with 0.05% cysteine. Bacterial
culture was incubated anaerobically (anaerogen bags, Oxoid,
UK) for 12 h at 37°C before testing day.
Experimental animals
Six to eight weeks old female Balb/C mice were used in
this study. Mice were kept in separate cages with wooden
shavings as bedding. The environmental parameters were
temperature around 25°C, 50%–60% humidity, and continuous
air ventilation. The research followed the International Ethical
Standards for the care and use of laboratory animal.
Tumor cell line and culture conditions
The mouse mammary cancer cell line (66CL-4-GFP) was kindly
provided by Dr. Bob Sanders (Department of Genetics and
Microbiology, University of Texas, Austin, USA). The cell line
was derived from spontaneous mammary tumor in Balb/C
mice and isolated as 6-thioguanine resistant clone. These cells
were transfected with green florescence protein (GFP). It was
maintained using DMEM-F12 supplemented with 10% FBS,
29 g/ml L-glutamine, 40 g/ml gentamicin, and 2.4 mg/ml
HEPES buffer.
Preparation of plant extracts
Plant aerial parts were dried at room temperature and were
finely ground. Suitable amounts of the powdered plant materials
were soaked in 95% ethanol (1 l per 100 g) for 2 weeks. The
crude ethanol extract (extract 1) was obtained after the solvent
was evaporated at 40°C to dryness under reduced pressure
418
using rotary evaporator (Buchi R-215, Switzerland). The
residues were further subjected to solvent-solvent partitioning
between chloroform (extract 2) and water (extract 3). The dried
chloroform extract was also partitioned between n-hexane
(extract 4) and 10% aqueous methanol (extract 5), while butanol
extract (extract 6) was fractionated from aqueous extract.[12] All
solvents were evaporated to dryness under reduced pressure
to produce the crude extracts, which were collected and stored
at −20°C for further testing.
In vitro antiproliferative activity assay
The antiproliferative activity of Ononis hirta extracts
was measured using MTT [3-(4, 5-dimethylthiazol-2-yl)-2,
5-diphenyltetrazolium bromide] assay (Promega, USA).
The assay detects the reduction of MTT by mitochondrial
dehydrogenase to blue formazan product, which reflects
the normal function of mitochondria and cell viability.[13]
Exponentially growing 66CL-4-GFP cells were washed and
seeded at 17000 cells/well (in 200 l of DMEM-F12) in 96-well
microplates (Nunc, Denmark). After 24 h incubation at 37°C, a
partial monolayer was formed then the medium was removed
using micropipette and 200 l of the medium containing the
plant extract (initially dissolved in DMSO) were added and
reincubated at 37°C for 48 h. A volume of 100 l of the medium
were aspirated and 15 l of the MTT solution were added to
the remaining medium (100 l) in each well. After 4 h contact
with the MTT solution, blue crystals were formed. A volume of
100 l of the stop solution were added and incubated further
for 1 h. Reduced MTT was assayed at 550 nm using a microplate
reader (Das, Italy). Control groups received the same amount
of DMSO (0.1%) and untreated cells were used as a negative
control, whereas cells treated with vincristine sulfate were used
as a positive control (0.05, 0.1, 0.5, 1, 5, 10, 25, 50, and 100 nM).
Acute toxicity of Ononis hirta methanol extract
In order to select the dose ranges for actual LD50 (median lethal
dose) determination, a pilot study was conducted on a small
group of mice. Plant extract was dissolved in PBS containing
5% tween 20. Two female mice (6 weeks old, 20–23 g weight)
were injected intraperitoneally with a specific dose of plant
extract and were observed for 24 h for any mortality. The
next doses were increased by 1.5 if the dose was tolerated,
or decreased by 0.7 if it was lethal using new animals. The
maximum nonlethal and the minimum lethal doses were used
as the lower and upper limits to prepare LD50 doses.[14]
For LD50 determination, five groups (n = 6) of mice were
injected intraperitoneally with different concentrations (310,
330, 350, 390, and 450 mg/kg) of plant extracts within the
upper and lower limits determined in the pilot study. The
untreated sixth group (n = 6) was used as a negative control.
Mice were monitored for 24 h for mortality and general
behavior. The concentration that showed 50% mortality was
recorded as the LD50. The LD50 value was derived using the
arithmetical method of Karber.[14]
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Talib and Mahasneh: Combined bacteriolytic therapy of solid tumors
Tumor inoculation
The mouse mammary tumor cells (66 cl-4-GFP) were harvested
by trypsinization, centrifuged, washed, and resuspended in
MEM-F12 media at a density of 1 × 106/100 l. Cell viability
was assessed using trypan blue exclusion method. Mice
(6 week old, 20–25 g weight) were injected subcutaneously
in the abdominal area using a 23-gauge needle syringe with
1 × 106 cells suspended in 100 l phosphate buffer saline (PBS).
Antitumor activity testing
Tumor-bearing mice (N = 10) were placed in four groups so
that the average tumor volume for all groups was closely
matched. Treatments begun 9 days following tumor cell
inoculation. Group 1 served as a negative control and received
daily intraperitoneal injection (100 l) of the vehicle (5%
tween 20 in PBS). Group 2 was exposed to daily intraperitoneal
injection (100 l) of Ononis hirta methanol extract for 14 days
at concentration =28.5 mg/kg (10% of the determined LD50
value). Group 3 received an intratumoral injection (100 l,
1.5 × 107 bacterial cells) of Bifidobacterium longum that
was grown for 12 h anaerobically and were washed twice
in PBS before injection at day zero. Group 4 received a
combined treatment consisting of the same concentration
of Bifidobacterium longum at day zero in addition to daily
intraperitoneal plant extract injection (100 l, 28.5 mg/kg).
Mice were monitored during the 2 weeks treatment period
and the tumor size was measured every 2 days using the
equation: length × width2 × 0.5.[15] After the last dose,
tumor-bearing mice in all groups were sacrificed and their
tumors were dissected and stored in 10% salined formalin
for further testing.
Histological examination of tumor sections
Dissected tumors (5 × 5 ×4 mm) fixed in 10% salined formalin
were gradually dehydrated using serial ethanol concentrations
80%, 95%, and 100%. Dehydrated tumors were cleared two
times using xylene (2 h each). Infiltration was performed
by exposing tumors to wax two times for 90 min each.
Dehydration, clearing, and infiltration were performed using
tissue processor (Thermo Shandon, UK). Paraffin sections
(4 m thick) were prepared using rotary microtome (Reichert,
Germany). Sections were attached to clean slides using egg
albumin. Standard hematoxylin- eosin procedure was used to
stain tumor sections for histological examination.
Gram stain of tumor sections
Paraffin sections of tumors were subjected to xylene (20 min),
followed by decreasing ethanol concentrations 100%, 95%,
and 70% (20 min each), and then distilled water. Sections
were dried and exposed to bacterial gram stain using
standard protocol. Briefly, sections were stained using crystal
violet (2 min), followed by Gram’s iodine (1 min) and then
decolorized using acetone (few seconds). Safranin was applied
as a counterstain (2 min) before washing with water. Finally,
slides were dried and examined under the light microscope.
Rechallenge study
At the end of the therapy, mice with complete tumor regression
were subcutaneously inoculated with 1.5 × 107 cells (in 100 l
PBS) of 66 cl-4-GFP mouse cell line. Mice were monitored for
5 months for any tumor development.
Qualitative thin layer chromatography
Qualitative thin layer chromatography (TLC) was conducted
for Ononis hirta ethanol extract. Aliquots (50–75 l) of the
extracts were applied 1 cm from the base of the TLC plates
(0.25 mm, Nacherey-Nagel, Germany). Serial mixtures of
chloroform and methanol (from 0% to 100%) were used as
eluents. Development of the chromatograms was done in a
closed tank in which the atmosphere had been saturated with
the eluent vapor by lining the tank with filter paper wetted
with the eluent. For flavonoids and terpenoids identification,
plates were sprayed with p-anisaldehyde/sulfuric acid
reagent and were carefully heated at 105°C for optimal color
development.[16] For alkaloids detection, plates were sprayed
with iodoplatinic acid and were dried in the fume hood.
High-performance liquid chromatography (HPLC) analysis
The dried extract was dissolved in ethanol and subsequently
filtered using 0.45 m nylon syringe filter. The HPLC (Shimadzu
Nexera HPLC system; UV detector) analysis was performed under
the following conditions: XR-ODS C18 column (2.2 m, 3 mm
×50 mm) was used with 80:20 (v/c) acetonitrile phosphate
buffer (pH =2.5) as a mobile phase. A sample (20 l) was
injected into the column and eluted at room temperature with
a constant flow rate of 0.4 ml/min. Wavelength of detection
was set at 265 nm. The following pure compound, quercetin,
brucine, quinine, strychnine, and reserpine were analyzed under
the same conditions and the retention time was used to confirm
the chromatographic peaks of the plant extract [Figure 1].
Statistical analysis
The results are presented as mean ± SEM of three independent
a
b
Figure 1: (a) HPLC fingerprint of Ononis hirta methanol extract.
(b) HPLC chromatogram of authentic samples of quercetin, brucine,
quinine, strychnine, and reserpine
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Talib and Mahasneh: Combined bacteriolytic therapy of solid tumors
experiments. Statistical differences among fractions in the
in vitro study and groups in the in vivo study were determined
by one-way analysis of variance (ANOVA) followed by student
t test. Differences were considered significant at P < 0.05.
in the extract or to tissue specific response.[22] Methanol and
chloroform extracts fall within the NCI criteria in their activity
against 66 cl-4-GFP and are thus considered as of promising
anticancer potential.
RESULTS AND DISCUSSION
Phytochemical screening revealed the presence of flavonoids,
terpenoids, and alkaloids in Ononis hirta extracts. The results of
TLC analysis were confirmed in HPLC which revealed the presence
of quercetin, brucine, quinine, strychnine, and reserprine
[Figure 1]. The antiproliferative activity of total flavonoids
and alkaloids isolated from different plants were reported.[23,24]
Out of 27 flavonoids tested against several tumor and normal
cell lines, seven showed high antiproliferative activity toward
cancer cell lines, while the toxicity was very limited toward
normal cell lines.[25] On the other hand, new alkaloids isolated
from Zanthoxylum leprieurii showed high activity and selectivity
against lung carcinoma cells and colorectal adenocarcinoma cells
while it was less toxic against normal cell line tested.[26]
Antiproliferative activity of Ononis hirta extracts
Six solvent extracts from Ononis hirta were evaluated for their
antiproliferative activity against 66 cl-4-GFP and Vero cell lines.
Among all tested extracts, the most potent activity was that
of the methanol extract, followed by chloroform, n-hexane,
and ethanol extracts with IC50 values of 16.66, 21.03, 40.53,
and 48.20 g/ml, respectively. Aqueous and butanol extracts
showed limited activity against both cell lines with IC50 values
exceeding 200 g/ml [Table 1]. The results of this part showed
that the extracts derived from chloroform were more active
against the two cell lines than aqueous and butanol extracts
[Table 1]. This may indicate that the nonpolar active principles
are responsible for most of the antiproliferative activity in
these plants. This result agrees with many previous researches
that reported the bioactivity of nonpolar principles in plants
like Achillea santolina, Typhonium flagelliforme, Schisandra
sphenanthera, and Scutellaria barbata.[17-20]
According to the American National Cancer Institute (NCI),
the criteria of cytotoxic activity for the crude extracts is an
IC50 < 30 g/ml.[21] The methanol and chloroform extracts
showed high antiproliferative potential against 66 cl-4-GFP cell
line with IC50 values of 16.66 and 21.03 g/ml, respectively. On
the other hand, both extracts were less toxic against Vero cell
line with IC50 values above 40 g/ml for both. Such selectivity
was absent among other extracts. This selective toxicity could
be due to the sensitivity of the cell line to the active compounds
Table 1: Percentage yield and IC50 determination of different
Ononis hirta solvent extracts
Extract number
1
2
3
4
5
6
Vincristin sulfate
Yield (w:w %)
4.56
7.50
4.57
5.95
3.20
13.72
–
IC50 value (
g/ml)±SEM
66 cl-4-GFP
48.20±2.51
21.03±0.97
16.66±1.77
>200
40.53±2.13
>200
45 nM
Vero
50.35±1.20
42.74±2.18
41.87±2.72
>200
86.60±0.59
>200
>100 nM
In vivo study
The methanol extract of Ononis hirta (aerial parts) was selected
for the in vivo study because it showed the highest activity
in vitro (IC50 = 16.88 g/ml) against the mouse cell line (66
cl-4-GFP), which was used to inoculate mice in the in vivo study.
The pilot LD50 study revealed that the highest nonlethal
concentration was 310 mg/kg and the lowest lethal
concentration was 450 mg/kg.
Five concentrations were prepared within the determined
range (310–450 mg/kg) and used to inject five groups of mice
(N = 6). The LD50 value was calculated according to the method
of Akhila and alwar, 2007[14] [Table 2]. The LD50 value of the
Ononis hirta (aerial parts) methanol extract was 285 mg/kg.
Treatment of tumor-bearing mice with Ononis hirta (aerial
parts) methanol extract showed significant (P < 0.05)
inhibition of tumors where a reduction in body weight was
recorded (−1.61%) in the treated mice compared with the
untreated control group (+14.57%). Measuring tumor size
showed that Ononis hirta (aerial parts) methanol extract had
the ability to significantly (P < 0.05) reduce the increase in
tumor size (+302.81%) compared with the size of the control
group (+592.31%) [Table 3]. The ability of plant extracts
to reduce body weight and tumor size was reported in
some studies. Previous work using the methanol extract of
Table 2: LD50 determination of Ononis hirta methanol extract
Group
1
2
3
4
5
6
Total number of
mice (N)
6
6
6
6
6
6
Dose (mg/kg)
vehicle
310
330
350
390
450
Number of dead
animals
0
0
1
1
2
4
Dose difference
(mg/kg) (a)
–
0
20
20
40
60
Mean mortality (b)
Probit (a×b)
–
0
0.5
1
1.5
3
–
0
10
20
60
180
a: Dose difference = higher dose – lower dose. b: Mean mortality = (mortality in the second concentration + mortality in the first concentration) × 0.5. LD50 = least
lethal dose ∑ (a X b)/N = 330 (270/6) = 330 45 mg/kg = 285 mg/kg, where N = total number of mice in each group, a = dose difference and b = mean mortality
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Talib and Mahasneh: Combined bacteriolytic therapy of solid tumors
Table 3: Effect of different treatments on mice weight and tumors size
Treatment
Negative control
Ononis hirta extract (a)
Bifidobacterium longum (b)
(a)+(b)
Initial body
weight (g)
23.00
23.60
23.20
23.90
Final body
weight (g)
26.35
23.22
24.38
23.19
% change in
body weight
+14.57
-1.61
+5.09
-2.97
Initial tumor size
(mm3) ±SEM
3.30±0.14
3.90±0.45
3.50±0.20
4.00±0.31
Final tumor size
(mm3) ±SEM
22.50±1.08
15.60±2.32
6.50±0.93
2.66±0.25
% change in
tumor size
+592.30
+302.80
+85.50
-33.5
% tumor
regression
0
0
10
30
% change in weight = (final weight – initial weight)/initial weight × 100. % change in tumor size = (final size – initial size)/initial size × 100
Hypericum hookerianum stem inhibited the increase in tumor
size and body weight in Ehrilch ascites carcinoma in Swiss
albino mice.[27] The same tumor was treated with the ethanolic
extract of Butea monosperma and the results showed significant
reduction in tumor size.[28]
An increase in the average body weight (+5.09%) of mice
treated with Bifidobacterium longum was recorded. This
increase is very limited compared with the increase in the body
weight of the untreated control group (+14.57%). Also the
tumor size was significantly (P < 0.05) inhibited (+85.51%) by
Bifidobacterium longum compared with that of the untreated
control group (+592.31%) [Table 3].
This indicates the ability of the anaerobic bacteria to retard
tumor increase in size, which is documented in the literature
for Clostridium novyi-NT which was used to target two murine
cell lines CT26 colorectal cancer and RENCA renal cell carcinoma
inoculated in Balb/C mice.[15] The combination of Ononis hirta
(aerial parts) methanol extract and Bifidobacterium longum
in this study showed the highest reduction in body weight
(−2.97%) and tumor size (−33.5%) [Table 3]. This is expected
since using both agents will apply higher stress on tumor
growth. In a similar study, a combination of Clostridium
novyi-NT spores with chemotherapeutic drug resulted in a
significant antitumor effect where 50% of treated mice were
completely cured, while using single agent alone showed
reduction in tumor size but no complete cure.[9]
In order to study the distribution of the Bifidobacterium
longum inside the tumor, tumor sections from mice that
received injections of Bifidobacterium longum were stained
using gram stain. Figure 2 showed the concentration of the
bacteria in clusters within the hypoxic region in the tumor
core. Such distribution of Bifidobacterium longum in cancerous
tissues was observed in other studies.[9] It is believed that
the abnormal vasculature and poor oxygen supply enhanced
the accumulation of the anaerobic bacteria Bifidobacterium
longum in the central part of the tumor and this was observed
in earlier studies.[5]
Hematoxylin/eosin staining of tumor sections treated with
Ononis hirta methanol extract showed large necrotic areas
with infiltrating inflammatory cells in the area around the
necrotic regions compared with the control group where
limited necrosis was observed [Figure 3]. This indicates the
ability of compounds in this extract to attack tumor by direct
toxicity and through induction of immune response.
Previous studies showed that the ethanol extract of Piper
longum reduced tumor size, increased the total white
blood cells count, and enhanced antibody production in
tumor-bearing mice.[29]
In our study, large necrotic areas were also detected in tumor
sections treated with Bifidobacterium longum with more
inflammatory cells infiltrating the tissue [Figure 3]. Bacterial
cells are highly immunogenic since they are foreign to the
host immune system. Such immunogenecity stimulates
an inflammatory reaction that causes a potent immune
response against the bacteria together with cancer cells.[15]
An example of the ability of bacterial cells to induce immune
response is the Freund’s adjuvant which is used to enhance
the immune response. Freund’s adjuvant is composed of
inactivated bacteria and oil.[30] After injection of the adjuvant
together with other components, the immune response will be
directed against the adjuvant in addition to the components
in the injected mixture.[31] Thus, it is reasonable to say that
Bifidobacterium longum is expected to augment the immune
response against implanted tumors. Sekine et al. (1995) used
cell wall extract prepared from Bifidobacterium infantis as
immunomodulator to target implanted tumors.[32]
In our attempts to enhance the antitumor potential, a
combination of the two treatments was used to target solid
tumors in mice. In this part, tumor-bearing mice received
Ononis hirta methanol extract treatment in addition to
Bifidobacterium longum treatment. Extensive areas of necrosis
were observed in addition to inflammatory cells infiltration
[Figure 3]. The necrotic regions were larger than those
observed in other treatments. Such response could be a result
of the cumulative effects of the cytotoxic activity of Ononis
hirta methanol extract and the immunomodulatory effect
of Bifidobacterium longum. As a result of this combination
therapy, 3 mice out of 10 (30%) were completely cured, while
only 1 mouse (10%) was cured in the group treated with
Bifidobacterium longum alone. Although there is a reduction
in tumor size in the group treated with plant extract,
this treatment was unable to completely cure any of the
tumor-bearing mice [Table 3].
To determine whether the cured mice had developed a
protective immune response against cancer cells, mice that
have been cured were injected subcutaneously with 1 × 106
tumor cells. All mice were resistant to tumor growth. The
cured mice were observed for 3 months and they showed no
symptoms of tumor growth or systemic infection. We believe
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421
Talib and Mahasneh: Combined bacteriolytic therapy of solid tumors
a
b
Figure 2: Distribution of Bifidobacterium longum within tumors. Bifidobacterium longum was concentrated within few colonies in the central part
of the tumor. The peripheral parts of the tumors showed limited distribution of the bacteria. (a) Arrows showed Bifidobacterium longum (stained
blue) clustered within colonies. (b) Arrows showed Bifidobacterium longum cells in higher magnification
lysis of cancer cells in tumor core. Previous studies reported
the bacteriolytic activity of viable bacteria by producing
hydrolytic enzymes.[9] Such dual activity cannot be achieved by
using killed bacteria, which can generate anticancer immune
response,[35] but cannot cause any lysis of tumor cells.
a
c
The combination used in this study showed promising ability
to activate the immune system to produce memory cells able
to protect mice from developing new tumors.
b
d
Figure 3: Hematoxylin/eosin staining of tumors treated with (a) vehicle,
(b) Ononis hirta methanol extract, (c) Bifidobacterium longum, and
(d) a combination of Ononis hirta methanol extract and Bifidobacterium
longum. N: Necrotic area
that the spread of Bifidobacterium longum was controlled by
the strong immune response against bacterial cells and the
availability of oxygen (which is toxic for anaerobic bacteria) in
tissues outside the tumor core. The potent immune response
against anaerobic bacteria was reported in another study that
showed an increase in some cytokines like IL6, MIP-2, G-CSF,
TIMP-1, and KC and overstimulation of neutrophils, monocytes,
and lymphocytes.[15] Previous studies showed that tumors are
immunogenic since tumor-specific antibodies and reactive T
cells were detected in untreated patients.[33] On the other hand,
tumor cells can protect themselves from the immune system by
different mechanisms including shedding of tumor antigens,
lack of MHC class I molecules, and masking tumor antigens.[34]
Our results showed that the use of viable Bifidobacterium
longum enhances anticancer immune response and caused
422
Activation of the immune system by anaerobic bacteria in
addition to the multiple intervening points of the crude plant
extract may represent a promising strategy to target solid
tumors. However, further purification and testing of crude
extract is needed in order to identify the active ingredients
and further studies are required to fully understand the
immune response induced by the Bifidobacterium longum
(DSMZ 20090).
ACKNOWLEDGEMENTS
The authors would like to thank the University of Jordan for the
financial support. We also would like to thank Prof. Bob Sanders
(Department of Genetics and Microbiology, University of Texas, Austin,
USA) for providing the mouse mammary cancer cell line (66CL-4-GFP)
and Dr. Maha Shumaf (Faculty of Medicine, University of Jordan) for
her assistance in evaluating tumor sections.
REFERENCES
1.
2.
3.
4.
Talib WH, Mahasneh AM. Antimicrobial, cytotoxicity and
phytochemical screening of jordanian plants used in traditional
medicine. Molecules 2010;15:1811-24.
Boik J. Natural compounds in cancer therapy. 1st ed. USA: Oregon
Medical Press; 2001
Brown JM, Wilson WR. Exploiting tumor hypoxia in cancer
treatment. Nat Rev Cancer 2004;4:437-47.
Tannock IF. Conventional cancer therapy: Promise broken or promise
delayed? Lancet 1998;35 Suppl 2:S9-16.
Journal of Cancer Research and Therapeutics - July-September 2012 - Volume 8 - Issue 3
Talib and Mahasneh: Combined bacteriolytic therapy of solid tumors
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
Xu J, Liu X, Zhou S, Wei M. Combination of immunotherapy with
anaerobic bacteria for immunogene therapy of solid tumors. Gene
Ther Mol Biol 2009;13:36-52.
Neergheen VS, Bahorun T, Taylor EW, Jen LS, Aruoma OI. Targeting
specific cell signaling transduction pathways by dietary and
medicinal phytochemicals in cancer chemoprevention. Toxicology
2009;278:229-41.
Ryan RM, Green J, Lewis CE. Use of bacteria in anti-cancer therapies.
Bioessays 2005;28:84-94.
Chakraborty A, Chowdhury BK, Bhattacharyya P. Clausenol
and clausenine-two carbazole alkaloids from Clausena anisata.
Phytochemistry 1995:40:295-8.
Dang LH, Bettegowda C, Huso DL, Kinzler KW, Vogelstein B.
Combination bacteriolytic therapy for the treatment of experimental
tumors. Proc Natl Acad Sci U S A 2001;98:15155-60.
Pawelek JM, John KB, Bermudes D. Bacteria as tumor-targeting
vectors. Lancet Oncol 2003;4:548-56.
Talib WH, Mahasneh AM. Antiproliferative activity of plant extracts
used against cancer in traditional medicine. Sci Pharm 2010;78:33-45.
Mahasneh AM, El-Oqlah AA. Antimicrobial activity of extracts
of herbal plants used in the traditional medicine in Jordan.
J Ethnopharmacol 1999;64:271-6.
Lau CB, Ho CY, Kim CF, Leung KN, Fung KP, Tse TF, et al. Cytotoxic
activities of Coriolus versicolor (Yunzhi) extract on human
leukemia and lymphoma cells by induction of apoptosis. Life Sci
2004;75:797-808.
Akhila J, Alwar M. Acute toxicity studies and determination of median
lethal dose. Curr Sci 2007;93:917-20.
Agrawal N, Bettegowda C, Cheong I, Geschwind JF, Drake CG,
Hipkiss EL, et al. Bactriolytic therapy can generate a potent immune
response against experimental tumors. Proc Natl Acad Sci U S A
2004;101:15172-7.
Masoko P, Mmushi T, Mogashoa M, Mokgotho M, Mampuru L,
Howard R. In vitro evaluation of the antifungal activity of Sclerocarya
birrea extracts against pathogenic yeast. Afr J Biotechnol 2008;7:3521-6.
Abu-Dahab R, Afifi F. Antiproliferative activity of selected medicinal
plants of Jordan against a breast adenocarcinoma cell line (MCF7).
Sci Pharm 2007;75:121-36.
Huyke C, Engel K, Simon-Haarhaus B, Quirin KW, Schempp CM.
Composition and biological activity of different extracts from
Schisandra sphenanthera and Schisandra chinensis. Planta Med
2007;73:1116-26.
Yu J, Liu H, Lei J, Tan W, Hu X, Zan G. Antitumor activity of chloroform
fraction of Scutellaria barbata and its active constituents. Phytother
Res 2007;21:817-22.
Lai CS, Mas RH, Nair NK, Majid MI, Mansor SM, Navaratnam V.
Typhonium flagelliforme inhibits cancer cell growth in vitro and
induces apoptosis: An evaluation by the bioactivity guided approach.
J Ethnopharmacol 2008;118:14-20.
Itharat A, Houghton PJ, Eno-Amooquaye E, Burke PJ, Sampson JH,
Raman A. In vitro cytotoxic activity of Thai medicinal plants used
traditionally to treat cancer. J Ethnopharmacol 2004;90:33-8.
22. Kirana C, Record I, McIntosh G, Jones G. Screening for antitumor
activity of 11 Species of Indonesian Zingiberaceae using human
MCF-7 and HT-29 cancer cells. Pharm Biol 2003;41:271-6.
23. Vijayan P, Vijayaraj P, Setty PH, Hariharpura RC, Godavarthi A,
Badami S, et al. The cytotoxic activity of the total alkaloids isolated
from different parts of Solanum pseudocapsicum. Biol Pharm Bull
2004;24:528-30.
24. Park HJ, Kim MJ, Ha E, Chung JH. Apoptotic effect of hesperidin
through caspase 3 activation in human colon cancer cells, SNU-C4.
Phytomedicine 2008;15:147-51.
25. Kawaii S, Tomono Y, Katase E, Ogawa K, Yano M. Antiproliferative
activity of flavonoids on several cancer cell lines. Biosci Biotechnol
Biochem 1999;63:896-9.
26. Ngoumfo RM, Jouda JB, Mouafo FT, Komguem J, Mbazoa CD, Shiao TC,
et al. In vitro cytotoxic activity of isolated acridones alkaloids
from Zanthoxylum leprieurii Guill. et Perr. Bioorg Med Chem
2010;18:3601-5.
27. Dongre SH, Badami S, Natesan S, Raghu HC. Antitumor activity
of the methanol extract of hypericum hookerianum stem against
Ehrlich ascites carcinoma in Swiss albino mice. J Pharmacol Sci
2007;103:354-9.
28. Rekha J, Jayakar B. Anti cancer activity of ethanolic extract of Leaves
of Butea monosperma (Lam) Taub. Curr Pharm Res 2011;1:106-10.
29. Sunila ES, Kuttan G. Immunomodulatory and antitumor activity of
Piper longum Linn. and piperine. J Ethnopharmacol 2004;90:339-46.
30. Means TK, Wang S, Lien E, Yoshimura A, Golenbock DT, Fenton MJ.
Human toll-like receptors mediate cellular activation by Mycobacterium
tuberculosis. J Immunol 1999;163:3920-7.
31. Cheadle EJ, Jackson AM. Bugs as drugs for cancer. Immunology
2002;107:10-9.
32. Sekine K, Ohta J, Onishi M, Tatsuki T, Shimokawa Y, Toida T, et al.
Analysis of antitumor properties of effector cells stimulated with a
cell wall preparation (WPG) of Bifidobacterium infantis. Biol Pharm
Bull 1995;18:148-53.
33. Old LJ. Cancer vaccine collaborative 2002: Opening address. Cancer
Immun 2003;3 Suppl 1:1-8.
34. Coico R, Sunshine G, Benjamini E. Immunology a Short Course. 5th ed.
USA: Wiley; 2003.
35. Wang LS, Zhu HM, Zhou DY, Wang YL, Zhang WD. Influence of
whole peptidoglycan o f bifidobacterium on cytotoxic effectors
produced bymouse peritoneal macrophages. World J Gastroenterol
2001;7:440–3.
Cite this article as: Talib WH, Mahasneh AM. Combination of Ononis hirta
and Bifidobacterium longum decreases syngeneic mouse mammary tumor
burden and enhances immune response. J Can Res Ther 2012;8:417-23.
Source of Support: The financial support for this work was provided by the
University of Jordan, Amman-Jordan, Conflict of Interest: No.
Journal of Cancer Research and Therapeutics - July-September 2012 - Volume 8 - Issue 3
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