|Year : 2012 | Volume
| Issue : 3 | Page : 417-423
Combination of Ononis hirta and Bifidobacterium longum decreases syngeneic mouse mammary tumor burden and enhances immune response
Wamidh H Talib1, Adel M Mahasneh2
1 Department of Clinical Pharmacy and Therapeutics, Applied Science University, Amman, Jordan
2 Department of Biological Sciences, University of Jordan, Amman, Jordan
|Date of Web Publication||17-Nov-2012|
Wamidh H Talib
Department of Clinical Pharmacy and Therapeutics, Applied Science University, Amman
Source of Support: None, Conflict of Interest: None
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.
Keywords: Anticancer activity, bacteriolytic therapy, Bifidobacterium longum, Ononis hirta methanol extract
|How to cite this article:|
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
|How to cite this URL:|
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 [serial online] 2012 [cited 2020 Jan 22];8:417-23. Available from: http://www.cancerjournal.net/text.asp?2012/8/3/417/103523
| > 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. 
Cancer development is a multistep process including induction of genetic instability, abnormal expression of genes, abnormal signal transduction, angiogenesis, metastasis, and immune evasion.  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.  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. 
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. ,,
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.  Many researches focused on Clostridium or Clostridium spores as anticancer agents alone or in combination with conventional therapies. 
Compared with Clostridium, Bifidobacterium is nontoxic, nonspore forming, and naturally found in the alimentary canal of human digestive system.  Many phytochemicals were proved to target cancer development.  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. 
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|| |
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.
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 μg/ml L-glutamine, 40 μg/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 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.  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.  Exponentially growing 66CL-4-GFP cells were washed and seeded at 17000 cells/well (in 200 μl 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 μl 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 μl of the medium were aspirated and 15 μl of the MTT solution were added to the remaining medium (100 μl) in each well. After 4 h contact with the MTT solution, blue crystals were formed. A volume of 100 μl 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 LD 50 (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 LD 50 doses. 
For LD 50 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 LD 50 . The LD 50 value was derived using the arithmetical method of Karber. 
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 × 10 6 /100 μl. 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 × 10 6 cells suspended in 100 μl 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 μl) of the vehicle (5% tween 20 in PBS). Group 2 was exposed to daily intraperitoneal injection (100 μl) of Ononis hirta methanol extract for 14 days at concentration =28.5 mg/kg (10% of the determined LD 50 value). Group 3 received an intratumoral injection (100 μl, 1.5 × 10 7 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 μl, 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 × width 2 × 0.5.  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 μm 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.
At the end of the therapy, mice with complete tumor regression were subcutaneously inoculated with 1.5 × 10 7 cells (in 100 μl 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 μl) 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.  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 μm nylon syringe filter . The HPLC (Shimadzu Nexera HPLC system; UV detector) analysis was performed under the following conditions: XR-ODS C18 column (2.2 μm, 3 mm ×50 mm) was used with 80:20 (v/c) acetonitrile phosphate buffer (pH =2.5) as a mobile phase. A sample (20 μl) 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].
|Figure 1: (a) HPLC fi ngerprint of Ononis hirta methanol extract. (b) HPLC chromatogram of authentic samples of quercetin, brucine, quinine, strychnine, and reserpine|
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The results are presented as mean ± SEM of three independent 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.
| > Results and Discussion|| |
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 IC 50 values of 16.66, 21.03, 40.53, and 48.20 μg/ml, respectively. Aqueous and butanol extracts showed limited activity against both cell lines with IC 50 values exceeding 200 μg/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. ,,,
According to the American National Cancer Institute (NCI), the criteria of cytotoxic activity for the crude extracts is an IC 50 <sub> 30 μg/ml.  The methanol and chloroform extracts showed high antiproliferative potential against 66 cl-4-GFP cell line with IC 50 values of 16.66 and 21.03 μg/ml, respectively. On the other hand, both extracts were less toxic against Vero cell line with IC 50 values above 40 μg/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 in the extract or to tissue specific response.  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.
|Table 1: Percentage yield and IC50 determination of different Ononis hirta solvent extracts|
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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. , 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.  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. 
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 (IC 50 = 16.88 μg/ml) against the mouse cell line (66 cl-4-GFP), which was used to inoculate mice in the in vivo study.
The pilot LD 50 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 LD 50 value was calculated according to the method of Akhila and alwar, 2007  [Table 2]. The LD 50 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 Hypericum hookerianum stem inhibited the increase in tumor size and body weight in Ehrilch ascites carcinoma in Swiss albino mice.  The same tumor was treated with the ethanolic extract of Butea monosperma and the results showed significant reduction in tumor size. 
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.  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. 
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.  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. 
|Figure 2: Distribution of Bifi dobacterium longum within tumors. Bifi dobacterium 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 Bifi dobacterium longum (stained blue) clustered within colonies. (b) Arrows showed Bifi dobacterium longum cells in higher magnifi cation|
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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.
|Figure 3: Hematoxylin/eosin staining of tumors treated with (a) vehicle, (b) Ononis hirta methanol extract, (c) Bifi dobacterium longum, and (d) a combination of Ononis hirta methanol extract and Bifi dobacterium longum. N: Necrotic area|
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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. 
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.  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.  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.  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. 
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 × 10 6 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 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.  Previous studies showed that tumors are immunogenic since tumor-specific antibodies and reactive T cells were detected in untreated patients.  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. 
Our results showed that the use of viable Bifidobacterium longum enhances anticancer immune response and caused lysis of cancer cells in tumor core. Previous studies reported the bacteriolytic activity of viable bacteria by producing hydrolytic enzymes.  Such dual activity cannot be achieved by using killed bacteria, which can generate anticancer immune response,  but cannot cause any lysis of tumor cells.
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.
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.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3]