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. Author manuscript; available in PMC: 2015 May 5.
Published in final edited form as: Int J Cancer. 2010 Aug 15;127(4):780–790. doi: 10.1002/ijc.25011

The anti-cancer effect of probiotic Bacillus polyfermenticus on human colon cancer cells is mediated through ErbB2 and ErbB3 inhibition

Elise L Ma 1, Yoon Jeong Choi 1, Jinyoung Choi 1, Charalabos Pothoulakis 1, Sang Hoon Rhee 1, Eunok Im 1
PMCID: PMC4420487  NIHMSID: NIHMS266254  PMID: 19876926

Abstract

A wealth of data implicates that ErbB receptors have essential roles in tumor development. Probiotic bacteria are known to exert an anti-cancer activity in animal studies. Bacillus polyfermenticus (B.P.), a probiotic bacterium, has been clinically used for a variety of gastrointestinal disorders in East Asia. Here we investigated the effect of B.P. on the growth of tumors and its putative mechanism of actions. Conditioned medium of B.P. cultures (B.P. CM) inhibited the growth of human colon cancer cells including HT-29, DLD-1 and Caco-2 cells. Moreover, B.P. CM suppressed colony formation of HT-29 cells cultured on soft agar and reduced carcinogen-induced colony formation of normal colonocytes. Furthermore, data from the mouse xenograft model of human colon cancer cells showed reduced tumor size in B.P. CM-injected mice when compared to E.coli conditioned medium-injected mice. Exposure of B.P. CM to HT-29 cells for 24 h, 48 h and 2 weeks reduced ErbB2 and ErbB3 protein expression as well as mRNA levels. Moreover, cyclin D1 expression which is required for ErbB-dependent cell transformation was decreased by B.P. CM. Furthermore, transcription factor E2F-1 which regulates cyclin D1 expression was also decreased by B.P. CM. These results show that B.P. inhibits tumor growth and its anti-cancer activity occurs, at least in part, through suppressing ErbB2 and ErbB3. Taken together, our study suggests that this probiotic may be clinically used as a prophylactic treatment to prevent colon cancer development.

Keywords: colon cancer, probiotics, Bacillus polyfermenticus, ErbB2, ErbB3

INTRODUCTION

Probiotic is a living microorganism which exerts health benefits when ingested in adequate amounts1. Use of probiotic therapy has progressively increased for prevention and treatment of gastrointestinal disorders including irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), pathogenic bacterial or viral infection, and antibiotic-associated diarrhea2–5. A wealth of evidence emerging from laboratory studies indicates anti-cancer activity of probiotics. Certain strains of lactic acid bacterium have been shown to be antigenotoxic toward carcinogens including 1,2-dimethylhydrazine (DMH) and N’-nitro-N-nitrosoguanidine6. These bacteria have also been found to inhibit the formation of aberrant crypt foci as early neoplastic lesions induced by azoxymethane (AOM) or DMH7, 8. Moreover, injection of Lactobacilus casei in tumor-bearing mice exerted anti-tumor activity9, 10. However, the mechanisms by which probiotics may inhibit colon cancer are still poorly understood.

A commercially available probiotic bacterium, Bacillus polyfermenticus (B.P.), first found in the air by Dr. Terakado in 1933, is clinically used to treat a variety of intestinal disorders11. Due to its endospore-forming feature, B.P. is relatively resistant to digestive enzymes, gastric acid, and bile salts and presents longer in the gastrointestinal track11. B.P. also produces the antimicrobial agent bacteriocin11. Oral administration of B.P. to humans stimulates IgG production and modulates the number of CD4+, CD8+, or NK cells12. Moreover, antiproliferative effects of B.P. have been reported in Caco-2 colon cancer cells when the live bacterium was added in cell culture media13. The formation of aberrant crypt foci by DMH was also suppressed in rats supplemented with live B.P.14. However, the molecular mechanism of B.P. on anti-cancer activity has not been investigated yet.

The ErbB receptor family consists of four members including ErbB1/epidermal growth factor receptor (EGFR)/HER1, ErbB2/HER2/Neu, ErbB3/HER-3, and ErbB4/HER-4 which are often activated as homo- and/or heterodimer complexes15. ErbB2 is a preferred partner of the other ErbBs, and formation of heterodimers with ErbB3 and ErbB4 is important for cancer development16. The overexpression of ErbB2 is observed in many human cancers including bladder, breast, colon, and lung cancers17. Moreover, high levels of ErbB2 in solid tumors are strongly correlated to poor prognosis17. Furthermore, the expression of ErbB3 is observed in many tumors that express ErbB218, 19. Due to substitutions in critical residues in its kinase domain, ErbB3 is an impaired kinase and can only become phosphorylated when dimerized with another ErbB receptor16, 20. This occurs most often with ErbB2, which is the most oncogenic member of the family21. Anti-cancer therapies targeting ErbB family receptors have gained their strength due to vast clinical data over years. Anti-ErbB2 antibodies (Trastuzumab/Herceptin and Pertuzumab) have been used for breast cancer22, 23. Small molecule tyrosine kinase inhibitors (gefitinib and erlotinib) have been evaluated in clinical trials for patients with lung cancer15.

In the present study, we investigated the effects of B.P. on the growth of colon cancer cells in vitro and the development of colon cancer in vivo. Our results show that B.P. inhibited the growth of various cancer cell lines including colon, breast, cervical and lung cancers and suppressed colony formation of HT-29 colon cancer cells and AOM-treated NCM460 colonocytes. Moreover, peritumoral injection of B.P. to tumors implanted in the skin of nude mice suppressed tumor growth. The molecular mechanism by which B.P. inhibited tumor growth involved reduced expression levels of ErbB2 and ErbB3 protein and mRNA. Moreover, B.P. inhibited E2F-1-dependent transcriptional regulation of cyclin D1 which may play a role in the anti-tumorigenic effect of B.P. Together, these results suggest that B.P. exerts an anti-cancer activity by inhibiting the ErbB receptor-dependent pathway.

Materials and methods

Reagents

Anti-mouse and anti-rabbit antibodies conjugated to horseradish peroxidase were obtained from Amersham Biosciences (Piscataway, NJ). Rabbit monoclonal ErbB2, ErbB3, and cyclin D1 as well as rabbit polyclonal Akt, cleaved caspase-3, histone H2A and HSP90 antibodies were obtained from Cell Signaling Technology (Beverly, MA). Mouse monoclonal E2F-1 antibody was purchased from Active Motif (Carlsbad, CA). Mouse monoclonal β-actin antibody was purchased from Sigma (St. Louis, MO). The RasGAP antibody was a crude polyclonal rabbit antisera extracted as previously described24. Purified rat polyclonal CD31 antibody and its isotype control rat IgG were from BD Pharmingen (San Diego, CA). Rabbit monoclonal Ki67 antibody was from Vector Laboratories (Burlingme, CA) and its isotype control rabbit IgG was from BD Pharmingen. Biotinylated anti-rat and anti rabbit antibodies were purchased from Vector Laboratories. Nuclear extraction kit was purchased from Active Motif. Azoxymethane (AOM) and MG132 were purchased from Sigma. Human recombinant Fas ligand (Fas L) was purchased from Calbiochem (San Diego, CA). All other reagents were purchased from Sigma.

Cell cultures

Human cancer cell lines were purchased from ATCC (Manassas, VA). Human colon cancer cell lines HT-29, DLD-1, and Caco-2 were maintained in McCoy’s 5A Medium Modified (ATCC), DMEM (Invitrogen, Carlsbad, CA) and MEM (ATCC), respectively. Human skin cancer cell line A375, breast cancer cell line MCF-7, cervical cancer cell line HeLa, and lung cancer cell line A549 were maintained in DMEM. The culture medium was supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Invitrogen) and 10 units/ml penicillin and 100 µg/ml streptomycin (Invitrogen) at 37 °C in air supplemented with 5% CO2.

Human colonic epithelial cells (NCM460) were cultivated in M3D medium (INCELL Corp., San Antonio, TX) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Invitrogen) and 10 units/ml penicillin and 100 µg/ml streptomycin (Invitrogen) at 37 °C in air supplemented with 5% CO2 as previously described25.

Preparation of B.P. conditioned medium and E.coli conditioned medium

Freeze-dried B.P. (4×109 CFU/g) was provided by BINEX Co. (Busan, South Korea). Frozen Escherichia coli (E.coli) stock (DH10B) was purchased from Invitrogen (Camarillo, CA). For conditioned medium of B.P. (B.P. CM) and E.coli (E.C. CM), we followed the previously described protocol by Grabig et al26. Briefly, B.P. (2×109 CFU) and E.coli (2.4×109 CFU) were incubated for 16 hours at 37°C in 100 mL Luria-Bertani broth (Invitrogen). Cultures were then collected by centrifugation (1,000 g for 15 min), pellets were washed twice in phosphate-buffered saline and then resuspended in M3D medium containing 10% fetal calf serum without antibiotics. After 2 hours of incubation at 37°C in 5% CO2, culture medium was collected and filtered through a 0.22 µM-pore-size filter. B.P CM or E.C. CM was then mixed with complete culture medium (1:2 ratio) before treatments.

Cell proliferation assay (XTT assay and trypan blue exclusion assay)

HT-29, DLD-1, Caco-2, A375, MCF-7, HeLa, and A549 (1 × 104) cells were plated for each well of 24-well plates and stabilized overnight. B.P. CM mixed with complete medium (1:2) was added to cells for 7 or 14 days. Medium was changed every 4 days. At the end of experiments, XTT labeling mixture (Roche Applied Science, Mannheim, Germany) was prepared according to the manufacturer’s protocol and added to the cells. After 4 hours of incubation, colorimetic intensity was measured on a spectrophotometer (Spectra Max M5, Molecular Devices, Sunnyvale, CA) with absorbance at 470 nm and a reference wavelength at 650 nm. For the trypan blue exclusion assay, cells were harvested and suspended in culture medium. An equal volume of trypan blue solution (0.08%, Invitrogen) was added to the cell suspension and total cell number was counted under the microscope.

Soft Agar Colony Forming Assay

HT-29 cells (5 × 104) or NCM460 cells (5 × 104) were cultured either in complete culture medium or B.P. CM mixed with the culture medium in a ratio of 1 to 2 for 2 weeks. Medium was changed every 3 days. After 2 weeks of incubation, cells were suspended in 0.45% agar (Difco, Lawrence, KS) and plated on 6-well plates (1 × 105 cells per well) coated with a layer of 0.5% agar (Difco) as previously described27. After solidification, medium was added. AOM (1 mg/ml) was added to NCM460 cells. After two weeks of incubation in 37 °C, three random fields were photographed per well in a 5× bright field with a Zeiss Observer D1 inverted microscope with an AxioCam digital camera (Carl ZEISS, Germany) and processed with Adobe Photoshop. In 2.5” × 3.3” photos, colonies of 4 mm diameter and larger were counted as significant.

Xenograft model of human colon cancer

DLD-1 colon cancer cells (6×105 cells in 200 µl of culture medium/per mouse) were injected subcutaneously into the flank of 8-week-old female CD-1 nude mice. Mice were monitored every day and tumor size was measured every other day with calipers. At the end of the experiment (20 days after the injection of cancer cells), tumors were excised from euthanized mice and tumor size was measured. Tumor volume was calculated as (length × width2) × 0.528. For peritumoral B.P. CM or E.C. CM treatment, B.P. CM or E.C. CM (200 µl volume per mouse) was injected around the tumor site every other day beginning 4 days after the initial injection of cancer cells into the mice. The tumors were fixed in 10% buffered formalin, paraffin-embedded, cut into 6 µm sections and stained with H&E.

Immunohistochemistry

For Ki67 and CD31 staining, excised tumors were embedded in OCT and frozen immediately. Five-micron sections were cut and then processed for peroxidase immunohistochemistry using Ki67 (1:200 dilution) or CD31 antibody (1:100 dilution) as previously described28. Hematoxylin solution (Vector Laboratories) was used for counterstaining. For the quantification of the Ki67, we counted 500 tumor cells with distinct positive nuclei staining in consecutive high power fields in the most positively stained area of the section on the slide29. We then calculated the percentage of positively stained cells. For the quantification of the CD31 staining, we counted the number of vessels with distinct positive staining from 8 different slides per group.

Quantitative real-time PCR

Total RNA from mouse colon tissues was isolated using ‘RNeasy Plus Mini Kit’ (QIAGEN) and an equal amount of RNA (2 µg) was transcribed into cDNA using ‘High Capacity Reverse Transcription Kit’ (Applied Biosystems). Subsequently, quantitative real-time PCR was performed on ‘Applied Biosystems 7500 Fast Real-Time PCR System’ with TaqMan Universal Master Mix, using the standard conditions from Applied Biosystems. Annealing/extension temperature was 60 °C (1 min). The primer pairs and FAM™ dye-labeled TaqMan® MGB (minor groove binding) probes or GAPDH gene for the internal control were purchased from Applied Biosystems. The level of expression was calculated based upon the PCR cycle number (CT) at which the exponential growth in fluorescence from the probe passes a certain threshold value (CT). Relative gene expression was determined by the difference in the CT values of the target genes after normalization to RNA input level, using CT value of GAPDH. Relative quantification was represented by standard 2−ΔCT calculations. ΔCT = (CT-target gene − CT-GAPDH)30. Each reaction was performed in triplicate.

Quantitative real time PCR with human colon cancer cDNA panel

We used a commercially available (Origene Technologies, Inc.) cDNA panel made from tissues (n=5) of normal and colon cancer patients, which were selected from mixed ages, gender and ethnic groups at various clinical stages (S1–S4). This cDNA panel contains dried and first strand cDNA and the amount of cDNA is normalized using a house-keeping gene, β-actin. Quantitative real-time PCR was performed as described above using ErbB2 and ErbB3 primers. Any detail information of the patients and pathological evaluation is available at www.origene.com/qPCR/getTissueScan.aspx.

Immunoblot analysis

Equal amounts of protein from cell lysates were subjected to SDS-PAGE analysis and immunoblotting using the appropriate antibodies was performed as we previously described31. For the nuclear extraction, a nuclear extraction kit was used according to the manufacturer’s protocol (Active Motif, Carlsbad, CA). The incubation conditions for each antibody is as follows: anti-Akt antibody (1:2000, overnight in 4 °C), anti-β-actin antibody (1:5000, 1 h in room temperature), anti-cleaved caspase-3 (1:1000, overnight in 4 °C), anti-Cyclin D1 antibody (1:1000, overnight in 4 °C), anti-ErbB2 antibody (1:1000, overnight in 4 °C), anti-ErbB3 antibody (1:1000, overnight in room temperature), anti-E2F-1 antibody (1:500, overnight in 4 °C), anti-H2A antibody (1:500, overnight in room temperature) or anti-RasGap antibody (1:4000, 1h in room temperature).

Characterization of B.P. CM

B.P. CM was incubated with proteinase K (1, 10, and 100 µg/ml) for 1 h at 37 °C and then subjected to IL-8 ELISA (Invitrogen). B.P. CM was also sorted based on the molecular weight cut-offs by centrifugation (1,000g, 10 min) with Amicon ultra centrifugal filters (Millipore, Temecula, CA) then subjected to IL-8 ELISA. In addition, B.P. CM was heat inactivated for 45 min at 100 °C and then subjected to IL-8 ELISA.

ELISA of human IL-8

For human IL-8, NCM460 cells were supplemented with B.P. CM after proteinase K treatment or centrifugation with molecular weight cut-off filter devices or heat-inactivation. The culture supernatant was collected and then the concentration of human IL-8 was determined by ELISA (Invitrogen). Experiments were carried out in triplicate, and results are shown as mean pg/ml.

Statistical analysis

Results are represented as the mean ± SD. Paired and 2-tailed Student’s t tests were used to compare results from the experiments. A p value of less than 0.05 was considered statistically significant.

Results

Probiotic B.P. inhibits the growth of cancer cells

Probiotic bacteria are known to exert anti-cancer activity in animal studies6–8. Additionally, co-culture of live bacteria with Caco-2 colon cancer cells for 72 h exerted an anti-proliferative effect13. Moreover, probiotic bacteria in fermented milk exerted anti-proliferative effect in MCF-7 breast cancer cells32. In this study, however, the presence of live bacteria was not required for this effect suggesting the presence of soluble, biologically active compounds. To this end, we made conditioned medium of B.P. cultures (B.P. CM) which may contain various active compounds. We first investigated the anti-cancer effect of B.P. on various cancer cells using trypan blue exclusion and XTT assays. B.P. CM significantly inhibited cell proliferation of human colon cancer cells [HT-29 (35% or 56%), DLD-1 (69% or 33%), and Caco-2 (99% or 95%)] when treated for 7 days or 14 days, respectively (Figure 1A–C). We next asked whether B.P. CM-induced growth inhibition was due to increased cell death. To test this, we performed immunoblotting for the cleaved caspase-3 as a marker for apoptosis. B.P. CM did not induce apoptosis in HT-29 colon cancer cells while a known apoptosis inducer Fas L (100 ng/ml) increased cleaved caspase-3 (Figure 1D). In addition, B.P. CM suppressed the growth of other adenocarcinoma cells including A375 (skin), MCF-7 (breast), HeLa (cervical) and A549 (lung) (Figure 1E). These data suggest that B.P. exerts its anti-cancer activity by suppressing tumor cell growth.

Fig 1.

Fig 1

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Conditioned medium of B.P. (B.P. CM) inhibits the growth of human cancer cells. HT-29 (A), DLD-1 (B), or Caco-2 (C) human colon cancer cell lines were cultured in the presence of B.P. CM or culture medium (control) for 7 or 14 days. At the end of the experiment, representative photographs were taken in HT-29 cells indicating reduced cell numbers in the B.P. CM-treated group. The cells were harvested, mixed with a trypan blue dye, and the cell number was counted. Error bars represent SD of triplicate samples. (A) * p<0.001 versus control; (B) * p<0.05 versus control; (C) * p<0.001 versus control. (D) HT-29 cells were cultured in the presence of B.P. CM or culture medium (Control) for 7 or 14 days or Fas L (100 ng/ml) for 24 h. At the end of the experiment, the cells were lysed and subjected to the immunoblot analysis for cleaved caspase-3 or β-actin. (E) A375 (skin), MCF-7 (breast), HeLa (cervical), and A549 (lung) human cancer cells were cultured in the presence of B.P. CM or culture medium (control) for 7 or 14 days and the growth of cells were measured by XTT assay or trypan blue exclusion assay.

To further investigate the anti-cancer activity of B.P., we next performed clonogenicity assays by measuring colony formation of the cells when cultured in soft agar. HT-29 colon cancer cells were cultured in the presence of B.P. CM for 2 weeks before being plated into soft agar. After additional 2 weeks of culture on soft agar, the number of colonies was counted. In the cells pre-treated with B.P. CM, the number of colonies was significantly reduced (by 90%) than untreated control cells (Figure 2A). Moreover, we further tested whether B.P. prevents normal colonocytes from carcinogen-triggered tumorigenesis. Non-transformed NCM460 cells were pre-treated with B.P. CM for 2 weeks before being plated into soft agar. When the cells were cultured in soft agar for 2 additional weeks, the carcinogenic agent AOM (1 mg/ml) was added throughout the experimental period. Treatment of AOM significantly increased number of colonies formed in normal colonocytes (Figure 2B). However, B.P. CM pretreatment prevented AOM-induced colony formation by 58% (Figure 2B). It is also noted that B.P. CM alone did not affect colony formation of normal colonocytes, but inhibited colony formation in cancer cells (Figure 2A and B). These results indicate that B.P. CM inhibits proliferation of colon cancer cells and prevents carcinogen-induced tumorigenesis of normal colonocytes.

Fig 2.

Fig 2

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B.P. CM reduces colony formation of colon cancer cells or AOM-treated non-transformed colonocytes. (A) HT-29 colon cancer cells pretreated with B.P. CM for 2 weeks were plated on a soft agar and incubated for additional 2 weeks. At the end of the experiment, photographs were taken and the number of colonies was counted as described in materials and methods section. The data are shown as mean ± SD; * p<0.009 versus control. Bar, 200 µm. (B) Non-transformed colonic epithelial cells (NCM 460) pretreated with B.P. CM for 2 weeks, were plated on a soft agar with or without AOM (1 mg/ml) and incubated for 2 additional weeks. At the end of the experiment, photographs were taken and the number of colonies was counted. The data are shown as mean ± SD; * p<0.009 versus control.

B.P. CM reduces tumor growth in a mouse xenograft model

To test an anti-cancer effect of B.P. in vivo, we used the mouse xenograft model of human colon cancer28. DLD-1 human colon cancer cells were subcutaneously injected into the flank of nude mice. Four days after cell injection, B.P. CM or conditioned medium of E. coli cultures (E.C. CM) was injected into the peritumoral region every other day until the end of the experiment. Tumor size was measured every other day and calculated into tumor volume. We observed that tumors from B.P. CM-injected mice grew smaller and slower than tumors from E.C. CM-injected mice (Figure 3A). At day 20 after implanting tumor cells, the tumor xenograft was excised to examine the tumor size and weight. Tumor weight and size were greatly reduced in B.P. CM-injected mice than E.C. CM-injected mice (Figure 3B). Tumor necrosis is a critical factor for modulating tumor development and growth, and therefore necrotic regions inside tumors often reveal inflammatory infiltrates. To test whether the reduced tumor growth in B.P. CM-injected mice is associated with the altered tumor necrosis, we evaluated H&E stained tumor sections of both E.C. CM and B.P. CM-injected mice. We found that both B.P. CM and E.C. CM tumors have similar amount (size) of necrotic areas and leukocytes infiltration (Figure 3C). Since B.P. CM inhibited cancer cell growth in vitro (Figure 1), we next tested whether B.P. CM can also modulate the growth of xenograft tumors. To test this, we performed immunohistochemical staining of the proliferation marker, Ki67 in the B.P. CM and E.C. CM-injected tumors. B.P. CM-injected tumors showed reduced intensity of Ki67 staining compared to E.C. CM-injected tumors suggesting that B.P. CM inhibited the tumor cell growth (Figure 3D with quantification in E). Moreover, tumor angiogenesis is essential for the growth of solid tumors due to their requirement of nutrient and oxygen33. Therefore, we evaluated angiogenesis by staining B.P. CM or E.C. CM-injected tumors with an angiogenesis marker CD31. The staining of CD31 was greatly reduced in B.P. CM-injected tumors compared to E.C. CM-injected tumors suggesting B.P. CM-inhibited tumor angiogenesis might be alternative cause for the growth inhibition effect by B.P. CM (Figure 3D with quantification in F). Collectively, these data suggest that B.P. CM reduces tumor growth not by inducing tumor necrosis and leukocyte infiltration, but by suppressing cell proliferation and angiogenesis.

Fig 3.

Fig 3

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B.P. CM inhibits tumor growth in a mouse xenograft model of human colon cancer. The DLD-1 colon cancer cells were subcutaneously injected into the flank of nude mice. Six days after cell injection, B.P. CM or conditioned medium of E.coli cultures (E.C. CM) was injected into the peritumoral region every other day. (A) Tumor volume of B.P. CM or E.C. CM was measured. The data are shown as mean ± SD; * p<0.01 versus E.C. CM, n=22 per group. (B) Gross appearance of xenografts of B.P. CM or E.C. CM injected mice is shown at day 20 in the upper panel. The photo of excised tumors at day 20 is shown in the lower panel. Each index in the ruler represents 1 mm. (C) Excised tumors were fixed, sectioned, and stained with H&E for histological evaluation. Bar, 100 µm. (D) Immunohistochemical staining of excised tumors for Ki67 and CD31. Bar, 50 µm. (E) The quantification of Ki67 staining intensity was shown. * p<0.01 versus E.C. CM. (F) The quantification of CD31 staining intensity was shown. * p<0.0001 versus E.C. CM.

The mRNA levels of ErbB2 and ErbB3 were increased in colon cancer

The overexpression of ErbB2 and ErbB3 was observed in many human cancers as shown by immunohistochemical staining methods17–19. We further tested whether the mRNA levels of ErbB2 and ErbB3 are altered at the different stages of human colon cancer. We used a commercially available cDNA panel made from tissues (n=5) of normal and colon cancer patients, which were selected from mixed ages, gender and ethnic groups at various clinical stages (S1–S4). This cDNA panel contains dried and first strand cDNA and the amount of cDNA is normalized using a house-keeping gene, β-actin. Quantitative real time PCR results indicated that the mRNA levels of both ErbB2 and ErbB3 were increased in samples from colon cancers than normal tissues (Figure 4A and B). This result provides new information that not only the protein levels of ErbBs (as shown by other groups) but also mRNA levels of ErbB2 and ErbB3 are also increased in human colon cancer at various clinical stages supporting their important role in colon cancer development.

Fig 4.

Fig 4

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The mRNA levels of ErbB2 and ErbB3 are increased in human colon cancer. Quantitative real time PCR of ErbB2 (A) or ErbB3 (B) was performed using a cDNA panel made from tissues of normal and colon cancer patients, which were selected from mixed ages, gender and ethic groups at various clinical stages (S1–S4). * p<0.05 versus normal.

B.P. CM inhibits ErbB2 and ErbB3 expression

Since ErbB2 and ErbB3 are critical players in colon cancer development, we next tested whether B.P. CM modulates ErbB2 and ErbB3 expression. Incubation of HT-29 colon cancer cells with B.P. CM for 24 h, 48 h or 2 weeks, greatly reduced ErbB2 and ErbB3 expression (Figure 5A and B). Next, to test whether the effect of B.P. on the expression level of ErbBs was due to its serum-binding/depletion, we treated B.P. CM in the absence of serum. We found that the reduced expression level of ErbB3 by B.P. CM was not altered by serum deficiency suggesting that inhibitory effect of B.P. CM on ErbB expression was not due to its serum-binding or depletion effect (Figure 5C). We next asked the mechanism by which B.P. reduces ErbB2 and ErbB3 protein levels. Among several negative regulators of ErbBs, E3 ubiquitin ligases and an inhibitor of HSP90, such as geldanamycin, target ErbBs for degradation. Therefore, we tested whether a molecular chaperone HSP90 which binds to ErbBs, is involved in B.P. CM-induced ErbBs degradation. However, our result indicates that the level of HSP90 was not changed by B.P. CM treatment (Figure 5D). We next tested whether the proteosome inhibitor MG132 can block B.P. CM-induced ErbB2 degradation. As shown in Figure 5E, MG132 (1 µM) did not block degradation of ErbB2. To test whether B.P. CM regulates mRNA levels of ErbB2 and ErbB3, the HT-29 cells were treated with B.P. CM for 24 h, 48 h or 2 weeks. Quantitative PCR results showed that B.P. CM decreased mRNA levels of ErbB2 and ErbB3 (Figure 5F and G). These results suggest that B.P. CM regulates ErbB2 and ErbB3 expression by reducing mRNA at the transcriptional level.

Fig 5.

Fig 5

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B.P. CM reduces the protein expression and mRNA levels of ErbB2 and ErbB3. The HT-29 colon cancer cells were incubated with B.P. CM for 24 h, 48 h, or 2 weeks. The cells were lysed and subjected to the immunoblot analysis for ErbB2 (A) or ErbB3 (B) or β-actin. (C) The HT-29 cells were incubated with B.P. CM or culture medium (Control) for 24 h in the presence or absence of serum. The cells were lysed and subjected to the immunoblot analysis for ErbB3 or Akt. (D) The HT-29 colon cancer cells were incubated with B.P. CM for 24 h. The cells were lysed and subjected to the immunoblot analysis for Hsp90 or β-actin. (E) The HT-29 cells were pre-treated with MG132 (1 µM) for 30 min and then treated with B.P. CM for additional 24 h. The cell lysates were made and subjected to the immunoblot analysis for ErbB2 or β-actin. (F and G) The HT-29 colon cancer cells were incubated with B.P. CM for 24 h or 2 weeks. The mRNA was isolated and subjected to a quantitative PCR for ErbB2 (F) or ErbB3 (G).

B.P. CM regulates cyclin D1 and E2F-1

Induction of cyclin D1 by growth factors and oncogenes contributes to tumorigenesis34, 35. Moreover, a recent report indicated that overexpression of ErbB2 in transgenic mice and breast cancer cells increased cyclin D1 proteins levels, and this ErbB-dependent cyclin D1 expression is regulated by the transcription factor E2F-136. Since our results in Figure 5 indicated that B.P. CM inhibits ErbBs, we investigated whether B.P. CM regulates the transcriptional activator E2F-1, leading to the reduced cyclin D1 protein levels. We found that E2F-1 level was also reduced by B.P. CM when the cells were treated with B.P. CM for as early as 6 h to 2 weeks suggesting B.P. CM regulates cyclin D1 through E2F-1 (Figure 6A). Moreover, the expression of cyclin D1 was reduced by B.P. CM treatment for 24 h, 48 h or 2 weeks (Figure 6B). These results indicate that B.P. CM inhibits ErbBs and their downstream molecules including the cell cycle regulator cyclin D1 and its transcriptional regulator E2F-1 to block ErbB-dependent tumorigenesis.

Fig 6.

Fig 6

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B.P. CM decreased E2F-1 and Cyclin D1 expression. (A) The HT-29 colon cancer cells were incubated with B.P. CM for 6 h, 18 h, 24 h, or 2 weeks. Nuclear extracts were made and subjected to the immumoblot analysis for E2F-1, β-actin or H2A. (B) The HT-29 colon cancer cells were incubated with B.P. CM for 24 h, 48 h, or 2 weeks. Total cell lysates were prepared and subjected to the immumoblot analysis for Cyclin D1, β-actin or RasGAP.

Characterization of B.P. CM

To identify the active components in the conditioned medium of B.P., we performed some preliminary experiments. First, B.P. CM was treated with various concentrations (1, 10, and 100 µg/ml) of proteinase K for 1 h at 37 °C and then treated to colonic epithelial cells. As shown in Figure 7A, increased IL-8 cytokine production by B.P. CM was gradually reduced by proteinase K treatment concentration-dependently. Second, B.P. CM was divided into 2 fractions based on the molecular weight after centrifugation (1,000g, 10 min) with the filter device. We found that the fraction of B.P. CM whose molecular weight is more than 30 KDa exerted the same activity like B.P. CM as shown in Figure 7B. Third, heat-inactivated (100°C, 45 min) BPCM was still able to induce the responses as shown in Figure 7C. Based on these preliminary results, we speculate that heat-stable bacterial proteins whose molecular weight is more than 30 KDa may be the active components of B.P. CM.

Fig 7.

Fig 7

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Active components in B.P. CM may be heat-stable bacterial proteins whose molecular weight is more than 30 KDa. (A) B.P. CM was treated with various concentrations (1, 10, and 100 µg/ml) of proteinase K for 1 h at 37 °C and then treated to colonic epithelial cells. (B) B.P. CM was divided into 2 fractions based on the molecular weight after centrifugation (1,000g, 10 min) with the filter device. Two fractions of B.P. CM were treated to colonic epithelial cells. (C) Heat-inactivated (100°C, 45 min) BPCM was treated to colonic epithelial cells. The supernatant (A–C) was collected and subjected to IL-8 ELISA. * p<0.05 versus untreated B.P. CM.

DISCUSSION

There are two major findings in this study: First, the observation that the new probiotic bacterium B.P. suppresses tumor growth as shown by inhibition of cancer cell growth, failure of colony formation, and reduced tumor volume of xenograft tumors in nude mice. Second, that B.P. inhibits ErbB2 and ErbB3 expression and reduced E2F-1 and cyclin D1 expression. These findings suggest a possibility that probiotic bacterium can be used as a chemopreventive therapy. Additionally, these data also suggest a putative mechanism by which probiotic bacterium exerts an anti-cancer activity.

There is a large body of studies suggesting a preventive effect of probiotics on colon cancer development. Evidence in human studies has shown that the consumption of probiotics, fermented milk, yogurt or other dairy products containing Lactobacillus or Bifidobacterium is causally related to prevention of colon cancer development37. Moreover, ingestion of probiotics in the diet and/or direct injection to animals prevented carcinogens including AOM and DMH-induced aberrant crypt foci formation, reduced tumor incidence, volume, multiplicity and increased animal survival rate6–8, 38. Several mechanisms by which probiotic bacteria may suppress colon cancer development have been suggested. Those mechanisms involve increasing a production of inflammatory cytokines (IL-6, TNF-α) in the host, altering enzyme activities (NADPH-cytochrome P-450 reductase, glutathione S-transferase, COX-2) in the colon, reducing the mutagenicity by inhibiting the uptake of potential carcinogens, or producing anti-proliferative and anti-tumorigenic compounds [6, 32, 38–41, reviewed in37].

In this study, we showed that the conditioned medium of probiotic B.P. suppressed the growth of cancer cells both in vitro and in vivo. It seems that the mechanism of B.P.’s anti-cancer effect is by producing anti-proliferative, anti-tumorigenic and anti-angiogenic compounds because conditioned medium of B.P. exerted these effects. Butyrate known to be produced from the bacterial strain Butyrivibrio fibriosolvens MDT-1 decreased ACF formation and inhibited tumor growth in animals41. Additionally, propionate and acetate, produced from Propionibacterium acidipropionici, also induced apoptosis of colorectal cancer cells42. Our preliminary experiments suggest heat stable bacterial proteins whose molecular weight is more than 30 KDa may be the active compounds in B.P. CM. Based on above references and our preliminary data, our ongoing studies are to further identify anti-proliferative compounds which B.P. produces to exert its anti-cancer activity.

It is also possible that B.P. releases microbial products including lipopolysaccharide (LPS), flagellin, and/or bacterial CPG DNA among others and trigger activation of pattern recognition receptors such as Toll-like receptors (TLRs). Recent reports indicated that LPS inhibited epithelial tumor growth when the cells express TLR443, 44. Indeed, our unpublished data indicate that B.P. alleviated mouse colitis, but B.P. failed to protect mice from colitis in the mice genetically lacking TLR2 or TLR4. These results indicate that B.P. may produce TLR2 and TLR4 ligands. It is also possible that B.P. produces TLR5 ligand flagellin. We reported that activation of TLR5 inhibited tumor growth in the xenograft model of human colon cancer indicating that bacterial flagellin can be used for the anti-tumor therapy28. In addition, a recent study showed that flagellin protected normal cells from cell death induced by radiation without affecting tumor radiosensitivity45. However, we found that B.P. CM did not induce apoptosis of cancer cells suggesting the anti-tumor effect of B.P. CM was not through modulating apoptosis even though B.P. CM might contain bacterial flagellin. Since our data suggest that B.P. CM reduced ErbB2 and ErbB3 mRNA levels, it is likely that microbial products of B.P. activate downstream signaling which may activate a transcriptional repressor for ErbBs or increase production of cytokines or other molecules which in turn regulate ErbBs transcription. Sequentially, reduced ErbBs mRNA levels contribute to anti-proliferative and/or anti-tumorigenic effect of B.P.

The activating transcription factor E2F-1 is essential for regulating genes which are implicated in cell proliferation and DNA replication46. Therefore, deregulation of E2F activity is a hallmark of many human cancers34. Several different mechanisms are known to regulate E2F. E2Fs are downstream targets of the retinoblastoma protein (pRB) family members which are major regulators of the cell proliferation machinery47. E2Fs can act as a transcriptional activator or suppressor of genes depending on their association with pRB family members48.

Additionally, for high-affinity binding to pRB and to other E2F consensus site, E2Fs are required to bind to a DP (DRTF1 polypeptide) family member46. In contrast, a cyclin A-dependent kinase phosphorylates DP-1 that binds directly to E2Fs inhibits E2F activity48. Moreover, the ubiquitin-proteasome pathway also regulates the activity of the E2Fs49. E2F target genes include cyclins, pRB and Myc among many which their expression is cell-cycle dependent46, 47. A recent report indicated that ErbBs regulate E2F-1 which subsequently controls cell cycle protein cyclin D136. In this case, cyclin D1 is the critical component for ErbB to exert its oncogenic activity. In our study, B.P. reduced ErbBs expression and this led to decreased E2F-1 and cyclin D1 expressions later on. Reduced expressions of these two critical proteins for cell cycle and proliferation provides an explanation for the anti-proliferative effect of B.P.

In summary, our study shows that B.P. suppresses cancer cell growth in vitro and tumor growth in vivo. B.P. exerts its anti-cancer effect through reduction of ErbB2 and ErbB3 and their downstream signaling molecules E2F-1 and cyclin D1. Our results suggest a novel mechanism by which probiotics prevent colonic tumorigenesis through regulating ErbBs. Future studies will evaluate a preventive effect of B.P. against colon cancer development.

This manuscript presents a novel finding that probiotic bacterium exerts the anti-cancer effect by inhibiting ErbBs. These results will advance our understanding of the molecular mechanism by which probiotics suppress colon cancer, and suggest a possibility that probiotic bacteria can be used as a prophylactic treatment to prevent cancer development.

Acknowledgement

This work was supported by research grant from BINEX Co. (SHR), a Young Clinical Scientist Award from “FAMRI, Inc.” (EI and SHR) and by NIH/NIDDK PO1 DK33506 and RO1 DK072471 (CP), 1KO1 DK079015 (SHR), and 1KO1 DK083336 (EI).

Abbreviation

B.P.

Bacillus polyfermenticus

B.P. CM

conditioned medium of B.P. cultures

IBS

irritable bowel syndrome

IBD

inflammatory bowel disease

DMH

1,2-dimethylhydrazine

AOM

azoxyemthane

EGFR

epidermal growth factor receptor

E.C. CM

conditioned medium of Escherichia coli

TLR

Toll-like receptors

Footnotes

Competing interests: None.

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