The Effect of Bacillus polyfermenticus against cancer cells

Bacillus polyfermenticus & Cancer

1.The prophylactic effect of probiotic Bacillus polyfermenticus KU3 against cancer cells

Highlights

B. polyfermenticus KU3 strongly inhibited the proliferation of cancer cells.

B. polyfermenticus KU3 attenuate inflammatory activity under stimulatory conditions.

The safety was demonstrated using normal cells and enzyme production.

These results demonstrate the probiotic characteristics of B. polyfermenticus KU3.

Bacillus polyfermenticus KU3 was isolated from kimchi, a Korean dish made from fermented vegetables and its potential probiotic characteristics were investigated. The spore cell of B. polyfermenticus KU3 was highly resistant to artificial gastric juice and survived for 24 h in artificial bile acid. B. polyfermenticus KU3 did not generate the carcinogenic enzymes, β-glucosidase, N-acetyl-β-glucosaminidase, and β-glucuronidase, and adhered strongly to HT-29 human intestinal epithelial cell lines. Using the [3–4,5-dimethylthiazol-2-yl]-2,5-diphenyletrazolium bromide assay, we found that B. polyfermenticus KU3 strongly inhibited the proliferation of cancer cells such as HeLa, LoVo, HT-29, AGS, and MCF-7 cells. The supernatant of B. polyfermenticus KU3 had an anticancer effect against HeLa and LoVo cells. Conversely, the proliferation of normal MRC-5 cells was not inhibited. We also demonstrated the anti-inflammatory activity of B. polyfermenticus KU3 under inflammatory conditions, as shown by the reduction in nitric oxide and proinflammatory cytokines (TNF-α, IL-10, TGF-β2, and COX-2). These results demonstrate the probiotic characteristics of B. polyfermenticus KU3 and provide evidence for the effect of this bacterium against various cancer cells.

2. Lactic acid bacteria including the genus Lactobacillus and Bifidobacterium have been shown to exert beneficial effects in human

Probiotics are defined as live bacteria and yeasts that exert beneficial effects for health. Among their various effects, anti-cancer properties have been highlighted in recent years. Such effects include suppression of the growth of microbiota implicated in the production of mutagens and carcinogens, alteration in carcinogen metabolism and protection of DNA from oxidative damage as well as regulation of immune system. We performed a computerized search of the MEDLINE/PUBMED databases with key words: cancer, probiotics, lactobacilli, metastasis and invasion. Cell line studies as well as animal models and human studies have shown the therapeutic effects of probiotics in reduction of invasion and metastasis in cancer cells. These results support the beneficial effects of probiotics both in vitro and in vivo. However, pre-clinical or clinical studies are not enough to decide about their application.

Key Words: Probiotics, lactobacilli, cancer, metastasis, invasion

Lactic acid bacteria including the genus Lactobacillus and Bifidobacterium have been shown to exert beneficial effects in human (1). Numerous lines of evidence have shown that changed gut microbiota is associated with several common disorders including cancer. Therefore, resuming the equilibrium using the beneficial bacteria (called “probiotics”) for disease treatment and prevention has been regarded profitable (1). Probiotic bacteria have recently become the focus of research because of their anti-cancer properties. The underlying mechanisms for their anti-cancer effects are versatile including suppression of the growth of microbiota implicated in the production of mutagens and carcinogens, alteration in carcinogen metabolism, and protection of DNA from oxide damage as well as regulation of immune system (2). In addition, they have been shown to change expression of different genes participating in cell death and apoptosis (3), invasion and metastasis (4), cancer stem cell maintenance (5) as well as cell cycle control (6). Further studies have shown their modulatory effects on the cancer-related signaling pathways in a cell type specific manner (7-9). In addition, their anti- proliferative effects have been assessed in several cell line studies (10-12). Notably, a traditional fermented milk product has been shown to inhibit in vitro proliferation of MCF-7 breast cancer cells but not normal mammary epithelial cells which implies that the bioactive substances prompt responses that are specifically detected in tumor cells (13). Special attention has been given to the effects of probiotics in reduction of invasion and metastasis in cancer cells in cell line studies as well as animal models and human studies. Invasion and metastasis have been regarded as important hallmarks of malignant cells which are endowed to them through diverse and complex genetic or epigenetic aberrations as well as extrinsic signals, such as those relayed from their microenvironment (14). The schematic description of various molecules and cells involved in metastasis are summarized in Figure 1. Metastasis cascade include acquisition of the ability to interrupt the basement membrane, invasion into the stroma (local invasion), passing the blood circulation (intravasation), staying alive in the circulation before they can reach to a remote organ, and production of clinically evident metastases. During this process, cancer cells recruit numerous stromal cells to support them in each step. Consequently, cancer microenvironment not only participates in the early steps of carcinogenesis but also contributes in metastasis cascade (15). Several studies have assessed the effects of probiotics on critical steps of invasion and metastasis such as interruption of cell–cell adhesion, epithelial-mesenchymal transition, tumor microenvironment, and cancer stem cell maintenance. The results of these studies have been summarized in the following sections.

Fig 1

The schematic description of various molecules and cells involved in the metastasis. Metastasis cascade include loss of cell-cell adhesion, acquisition of the ability to interrupt the basement membrane, invasion into the stroma (local invasion), passing the blood circulation (intravasation), staying alive in the circulation before they can reach to a remote organ, extravasation and production of clinically evident metastases. Several molecules are involved in each step. TAM: tissue activated macrophage; CSC: cancer stem cell; BMDC: bone marrow derived cells

Evidence acquisition

We performed a computerized search of the MEDLINE/PUBMED, Web of Knowledge, Scopus, ProQuest and Google Scholar databases with key words: cancer, probiotics, lactobacilli, metastasis, and invasion within the maximal date range until 2017.

Cell–cell adhesion

Tight junction between epithelial and endothelial cells has a critical role in preserving cell to cell integrity. Defects in this structure underlie the invasion and thus metastasis process (16). Tight junction structure has several molecular components including zona occludens-1 (ZO-1), claudin-1, and occludin. Effective assembly and preservation of this structure is carried out through the anchorage of the transmembrane proteins by the peripheral or plaque proteins such as ZO-1. Indeed, this protein provides a scaffold to fix a number of tight junction molecules together (16). On the other hand, the matrix metalloproteinases (MMPs) are regarded as critical participants of cell invasion through their role in degradation of various extracellular matrix proteins which enables cancer cells to migrate and invade (17). Considerably, cell-free supernatants (CFS) from L. casei and L. rhamnosus GG have been shown to prevent colon cancer cell invasion suggesting that probiotic CFS has anti-metastatic bioactive substances that may participate in cell in vation decrease in vitro (18). Such decrease in cell invasion has been later found to be accompanied by a decrease in matrix metalloproteinase-9 (MMP-9) protein level in cultured metastatic human colorectal carcinoma cells and an increase in the level of the tight junction protein ZO-1 in cultured metastatic human colorectal carcinoma cells (19). In addition, perioperative probiotic treatment has been shown to maintain the liver barrier in patients undergoing colorectal liver metastases surgery (20). A more recent study has shown that L. rhamnosus and L. crispatus CFSc can decrease expression of matrix metalloproteinase-2 (MMP-2), MMP-9 in HeLa cells and increase expression of their inhibitors. L. rhamnosus showed this effect in HT-29 cells as well (4). Furthermore, L. acidophilus and L. rhamnosus GG have been shown to regulate MMP-9 expression by the up-regulation of tissue inhibitor of metalloproteinases (TIMP)-1 and down-regulation of CD147 in phorbol 12-myristate 13-acetate- differentiated human monocytes (21). CD147 is over-expressed in numerous tumor cells and enhances metastasis formation by induction of both angiogenesis and MMPs expression (22, 23). On the other hand, TIMP-1 is tissue inhibitor of MMPs and its up-regulation has resulted in the inhibition of MMP-2 and suppression of metastasis (24). Recently, it has also been reported that L. rhamnosus GG significantly down-regulates expression GLUT1 in the MDA-MB-231 cells (8). This gene encodes an important rate-limiting protein in the transport of glucose into cancer cells. Its inhibition has been shown to decrease MMP-2 expression and c-Jun NH2-terminal kinase (JNK) activation, which controls numerous targets in the metastatic cascade (25). Lipoteichoic acid (LTA) deficient L. acidophilus (NCK2025) has been shown to increase ICAM5, RUNX3, TIMP2, RASSF1Aexpression in human colon carcinoma cell line HT-29 (26). ICAM5 codes for a type I transmembrane glycoprotein that is a member of the intercellular adhesion molecule family. It has been shown to be highly methylated in a fraction of colon cancer specimens. Its methylation diminishes the cell-to-cell adhesion in the cancer cells leading to enhancement of invasive potential (27). RUNX3 inhibits cancer cell migration and invasion through up-regulation of TIMP-2, which successively prevents MMP-2 expression and function (28). RASSF1A is a genuine tumor suppressor protein that can enhance death receptor-dependent cell death through TNF-R1, TRAIL or Fas activation (29). Moreover, its methylation has been shown to be associated with colorectal cancer development (30). Considering the role of L. acidophilus (NCK2025) in restoration of expression of mentioned tumor suppressor genes, this probiotic might be efficient in suppression of metastasis. Another study has demonstrated that probiotic conditioned media treatment diminished the up-regulation of genes in the NF-κB activation pathway, and down-regulated genes participating in extracellular matrix remodeling including MMPs, tissue-type plasminogen activator urokinase (PLAU) and its receptor (PLAUR) (31). Additionally, Kefir as a probiotic-containing fermented milk product has been shown to exert cytotoxic effects on 4T1 breast tumor cells. A notable decrease in tumor size and weight, a considerable enhancement in helper T cells and cytotoxic T cells as well as significant decreases in metastasis to lung and bone marrow were detected in the kefir water-treated BALB/c mice after 4T1 cancer cells transplantation (32). Kefir has been shown to exert an anti‑proliferative effect on Caco‑2 and HT‑29 cells, and is accompanied by induction of cell cycle arrest at the G1 phase, induction of apoptosis, up-regulation in Bax:Bcl‑2 ratio and an increase in p53 independent‑p21 expression, while it does not influence either the motility and invasion of these cells in vitro or MMP expression (33). In an in vitro model of the human epithelium, L. plantarum prompted translocation of ZO-1 to the tight junction region. Besides, L. plantarum has been demonstrated to initiate Toll-like receptor 2 (TLR2) signaling, and treatment of Caco-2 monolayers with the TLR2 agonist enhanced translocation of occludin in the tight junction (34). Recently, L. rhamnosus GG has been shown to improve intestinal integrity by inhibition of miR122a leading to occludin restoration in Caco-2 colorectal cancer cells (35). Furthermore, viable L. rhamnosus GG could significantly up-regulate ZO-1, Claudin-1 and Occludin gene expression in Caco-2 cells leading to restoration of destroyed epithelial barrier (36). L. reuteri I5007 has been shown to exert similar effects in the expression of tight junction related proteins in newborn piglets (37).  Another study has assessed the ability of Caco-2 cells to degrade collagen matrix and passing from membrane following treatment with different concentrations of probiotic bacteria. Notably, L. acidophilus and L. casei supernatants and cell extracts have decreased cell invasion capacity. Invasion inhibition effect of L. acidophilus was more than that of L. casei (38). As targeting tumor cell motility within the primary tumor is capable of prevention local invasion (39), colonization of lactobacilli in the site of the primary tumor may be beneficial in the prevention of metastases.

Epithelial-mesenchymal transition (EMT)

EMT is a biological process that permits a polarized epithelial cell, which typically interacts with basement membrane through its basal surface, to undertake numerous biochemical alterations which result in acquisition of a mesenchymal cell phenotype. Such phenotype change is accompanied by increased migratory capacity and invasiveness (40). Among the different factors and pathways involved in EMT, stromal cell-derived factor 1 (SDF-1) and its receptor, CXCR4 have gained special attention. CXCR4 has been shown to enhance EMT through the Wnt/β-catenin signaling pathway. Thus, targeting of the SDF-1/CXCR4 axis has been suggested as a treatment strategy in cancer suppression (41). Anti-CXCR4 antibodies have been shown to inhibit CXCL12 mediated cancer cell adhesion, migration, and proliferation (42). Notably, L. acidophilus NCFM has been shown to exert anti-metastatic effects via down-regulation of CXCR4 expression in colon, mesenteric lymph nodes and spleen of tumor-bearing mice (43). Considering the role of MMPs in the maintenance of EMT (17), the observed role of lactobacilli in down-regulation of MMPs (4) implies a putative role for them in suppression of EMT.

Live L. casei has been demonstrated to induce apoptotic cell death in both murine (CT26) and human (HT29) colon carcinoma cell lines as well as an experimental tumor model. Tumor growth inhibition has been associated with up-regulation of the TNF-related apoptosis-inducing ligand TRAIL (10).  Previously, it has been determined that soluble TRAIL gene and actinomycin D synergistically inhibit metastasis of TRAIL-resistant colon cancer in the liver (44). Also, trail resistance has been shown to trigger EMT and increase breast cancer cell invasiveness by modulation of PTEN and miR-221 expression (45). However, there is some contradictory evidence regarding the role of TRAIL in metastasis in other cancers such as pancreatic ductal adenocarcinoma. In this cancer, TRAIL prompted the expression of the proinflammatory cytokines as well as urokinase-type plasminogen activator and increased the invasion cancer cells in vitro(46).

L. casei and L. rhamnosus GG have been

demonstrated to suppress NF-κB activation by the inhibition of IκBα destruction in intestinal epithelial cells (47-49). Besides, bacteria-free solution originating from L. plantarum has been shown to suppress various NF-κB pathways (50). As NF-κB activity is associated with EMT and metastatic potential in various cancers (51, 52), the modulation of this pathway by certain lactobacilli strains may be of practical value.

Tumor microenvironment

Tumor microenvironment is constructed via the interactions between tumoral and non-transformed cells. The latter have an active and often tumor-promoting role at all stages of tumorigenesis. The major non-malignant cell types that are detected in this microenvironment are the cells of the immune system, the tumor vasculature and lymphatics, as well as the fibroblasts, pericytes and adipocytes (53). Many animal studies have shown that the beneficial anti-metastatic effects of lactobacilli are accompanied by or exerted via modulation of microenvironment. For instance, L. casei YIT9018 has been shown to suppress pulmonary and regional lymph node metastases in mice and guinea pigs (54). Intralesional injection of L. casei YIT9018 in highly metastatic melanoma bearing C57BL/6 mice has been shown to suppress tumor growth and improve the survival of affected animals. In addition, intravenous (I.V.) injection of this strain protects the mice against pulmonary metastasis after I.V. injection of melanoma cells. Injection of these lactobacilli exerts protective effects against both the axillary lymph node metastasis and lung metastases depending on the route and timing of injections. These effects are accompanied by augmentation of natural killer (NK) cell activity as well as cytolytic activity of axillary lymph node cells (55). Another study has shown that lymph node cells activated by the subcutaneous injection of these lactobacilli participate in the suppression of the metastasis (6). Matsuzaki et al. have reported that intralesional injection of L. caseiYIT9018 into Lewis lung carcinoma-bearing mice suppresses both the growth of the primary tumors and the development of lung metastases. In the L. casei YIT9018-primed mice, intraperitoneal administration of L. casei elicits a high level of IL-2 and IFN-γ in the peritoneal cavity and enhances host immune response against tumor (56). Yazdi et al. have shown that selenium nanoparticle-enriched Lactobacillus brevis (L. brevis) elicits efficient immune responses in tumor bearing BALB/c mice, decreases the liver metastasis in metastatic form of mouse breast cancer and improves the life span of animals' life span. The immune responses include an increase in the level of IFN-γ and IL-17 as T helper 1 cytokines and enhancement in the activity of NK cells (57). Aragon et al. have demonstrated that the administration of milk fermented by L. casei CRL 431 diminishes or inhibits tumor growth with less tumor vascularity, extravasation of tumor cells, and lung metastasis. These benefits are accompanied by alterations in the immune response such as decreasing the infiltration of macrophages in both the tumor and the lungs and an increased antitumor response associated to CD8+ and CD4+ lymphocytes (58). Takagi et al. have detected anti-metastatic effects of L. casei Shirota (LcS) in transplantable tumor cells which is mediated through augmentation of NK cells cytotoxicity (41). L. rhamnosus GG has been shown to exert effective antioxidative activity via diminishing reactive oxygen species production and phagocytic capacity of the neutrophils (59). Considering the role of neutrophils in almost all steps of cancer metastasis which is exerted in response to tumor-derived incitements (60), the inhibition of their function by probiotics might be an efficient strategy which impedes metastasis. Furthermore, a constituent of polysaccharide peptidoglycan complex on LcS has been shown to exert beneficiary effects in murine model of inflammatory bowel disease and colitis-associated cancer through inhibition of IL-6/STAT3 signaling (61). Considering the constitutive activation of STAT3 in many cancers and its fundamental roles in different steps of metastasis cascade such as cell transformation and migration, angiogenesis, as well as modulation of tumor microenvironment (62), its down-regulation by lactobacilli might affect metastasis potential of cancer cells. Likewise, a recent study has shown that kefir water exerts antiangiogenic effects in breast cancer through down-regulation of the IL-1β angiogenic factor that promotes tumor invasiveness, as well as the vascular endothelial growth factor (VEGF) which is a crucial mediator for angiogenesis (32). Further, decrease of the proangiogenic factor IL-6 has been detected following treatment with probiotics in breast cancer models (58, 63-65). All data presented above support the role of probiotics in changing pro-tumoral microenvironment.

Cancer stem cells

The presence of a fraction of multipotent “cancer stem cells (CSC)” in solid tumors as well as hematological malignancies has resulted in suggestion of a new model for explanation of tumorigenesis process (42). These cells are thought to directly or indirectly participate in the induction of metastasis. Furthermore, the heterogeneity detected in CSCs has resulted to suggest a role for them in determination of complexity and organ specificity in metastases (66). Many transcription factors as well as signaling pathways are implicated in the maintenance of CSCs. Among them are the hypoxia inducible factors (HIFs) which facilitate transcriptional responses to regional hypoxia in normal tissues and in cancers. Also, they induce specific signaling pathways and transcription factors, such as Notch and Oct4, which are implicated in stem cell self-renewal and multipotency (43). Notably, L. rhamnosus has been shown to down-regulate the expression of HIF-1α in MDA-MB-231 triple negative breast cancer cell lines (8). Considering the specific activation of HIF-1α signaling in the stem cells of mouse lymphoma and human acute myeloid leukemia and the effect of their inhibitors in preferential eradication of CSCs in mouse models (38), modulation of HIF-1α signaling following treatment with lactobacilli might be of therapeutic value. Another study has revealed that a combination of eight Gram-positive bacterial strains (Streptococcus thermophilus, Bifidobac-terium longum, Bifidobacterium breve, Bifidoba-cterium infantis, L. acidophilus, L. plantarum, L. casei, and L. bulgaricus) could activate NK cells to provide enhanced differentiation of CSCs which finally has led to suppression of tumor growth, and decreased inflammatory cytokine release (40). In addition, we recently detected the over-expression of SFRP2, an antagonist of Wnt pathway in HT-29 colorectal cancer cells following L. rhamnosus treatment and in HeLa cells following L. rhamnosus and L. crispatus treatments. Takig into the account the involvement of Wnt-induced CSCs in colorectal cancer metastasis (67) as well as the role of SFRP1 in the inhibition of the transformation and invasion abilities of cervical cancer cells via modulation of Wnt signal pathway (68), lactobacilli can be considered as putative therapeutic modalities in these cancer types.

Discussion

Even with extensive work committed to the early diagnosis and prevention of cancer, micro- or macro- metastases exist in most diagnosed patients at the time of their referral to diagnostic settings. In particular, metastasis is regarded as a possible life span-dependent destiny for both the early and late stage cancer patients (55). Consequently, several studies have focused on finding substances with anti-metastatic properties. For this purpose, it is necessary to find tumor and host factors contributing in the metastasis cascade. The “seed and soil” hypothesis suggested by Paget in 1889 (69) is now extensively assented in the scientific literature (70). The progenitor cell, initiating cell, cancer stem cell, or metastatic cell are now considered as the “seed”, whereas host factors, stroma, or the organ microenvironment are regarded as the “soil” (70). The consequence of metastasis is reliant on the communication between tumor cells and receptive tissues (70). Probiotics have been shown to influence all cell types and pathways implicated in the metastasis. Previously, lactobacilli-based immunotherapy has been suggested to be used along with conventional therapeutics to overcome the failures of the traditional treatment options, especially in the treatment of cancer metastases (2). As discussed formerly, the beneficial effects of lactobacilli in the cancer therapy are not confined to their immunomodulatory effects. They have been shown to alter expression of several genes involved in cell transformation, migration and invasion. Besides, it should be emphasized that the antimetastatic properties of probiotics might be different in distinct species of these organisms. Future studies are needed to identify putative pathways or molecules that are target of strain-specific gene expression modulation. Additionally, identification of formulations with the best bioactivity and less side effects is another challenge in this regard. Probiotic lactobacilli have also been shown to protect against cyclophosphamide-caused myelo-suppression in animal models which has led to the improvement of the resistance to Candida albicans. Consequently, probiotics have been suggested as a modality to decrease immunosu-ppression in cancer patients (71). Moreover, a randomized control study in critically diseased children has shown that the intake of probiotics decreases the occurrence of acute infectious, nosocomial and antibiotic-associated diarrhea in numerous general pediatric situations (72). Nevertheless, in some immuno-compromised patients, there have been occasional cases of sepsis following probiotics intake (73). In brief, the results of recent studies on evaluation of the effects of probiotics on cancer cell invasion and metastasis have supported their beneficial effects both in vitro and in vivo. Nonetheless, pre-clinical or clinical studies are not enough to decide about their application.

Consequently, to translate the results of basic studies to clinical application and to avoid unwanted side effects, the exact component of lactobacilli which is responsible for beneficial effects should be determined in pre-clinical animal studies. Although the possibility of synergic effects of different components should be considered as well, some studies have revealed contradictory effects for different lactobacillus-derived substances (19). Additional studies for the identification of the bioactive components and their mechanism of action could lead to the application of probiotics as a nutritional modality to prevent metastasis.

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

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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

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 diarrhea^2–5. 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 DMH^7, 8. Moreover, injection of Lactobacilus casei in tumor-bearing mice exerted anti-tumor activity^9, 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 ErbB2^18, 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 receptor^16, 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 cancer^22, 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 (C_T) at which the exponential growth in fluorescence from the probe passes a certain threshold value (C_T). Relative gene expression was determined by the difference in the C_T values of the target genes after normalization to RNA input level, using C_T value of GAPDH. Relative quantification was represented by standard 2^−ΔCT calculations. ΔC_T = (C_{T-target gene} − C_T-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 studies^6–8. 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

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

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

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 methods^17–19. 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

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

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 tumorigenesis^34, 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

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

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 rate^6–8, 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 fibriosolvensMDT-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 TLR4^43, 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 dependent^46, 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

4.Anticancer effects of probiotics in cancer cells/ cell lines

Substantial research using human cancer cells/cell lines has demonstrated that probiotics possess antiproliferative or proapoptotic activities in these cells, among which colonic cancer cells and gastric cancer cells were most commonly studied. According to the report by Lee et al., the cytoplasmic fractions of Lactobacillus acidophilus, Lactobacillus casei, and Bifidobacterium longum showed significant antitumor activities in some cancer cell lines (25). Studies by Russo et al. and Orlando et al. indicated the antiproliferative role of the cytoplasmic extracts from Lactobacillus rhamnosus strain GG (LGG) in both human gastric cancer cells and colonic cancer cells (30– 32), while another probiotic product named Bifidobacterium adolescentis SPM0212 inhibited the proliferation of three human colon cancer cell lines including HT-29, SW 480, and Caco-2 (33). Other probiotic products or strains that exerted antitumor activities against human colon cancer cells included Bacillus polyfermenticus (34), Lactobacillus acidophilus 606 (35), LGG/Bb12 (36), and LGG/Bifidobacterium animalis subsp. lactis (37). In addition, Cousin et al. reported that fermented milk containing Propionibacterium freudenreichii enhanced the cytotoxicity of camptothecin that was used as a chemotherapeutic agent for gastric cancer (38). An in vitro study using human colorectal carcinoma cells demonstrated the inhibitory activity of probiotics against cell invasion (39). Other studied cell types included cervical cancer cells (40), breast cancer cells (41), and myeloid leukemia cells (42). In Table 1 we summarized the antiproliferative role of probiotic strains and their products toward various cancer cells. Since in vitro studies using cell lines indicated that probiotics had proapoptotic effects on carcinoma cells (43–47), probiotics-based regimens might be used as an adjuvant treatment during anticancer chemotherapy.

Anticancer effects of probiotics in experimental models

 To further investigate the anticancer effects of probiotics, researchers have conducted animal model experiments using rats and mice. The outcomes of most studies turned out to be encouraging and showed potential clinical applications. As indicated in Table 2, treatment with Lactobacillus acidophilus, Butyrivibrio fibrisolvens, Bacillus polyfermenticus, Lactobacillus plantarum, Lactobacillus fermentum, or combination of L. acidophilus and Bifidobacterium bifidum significantly inhibited the colonic cancer development in rats or mice injected with a carcinogen 1,2-dimethylhydrazine (DMH) (9,48–54). Oral administration of probiotics (Lactobacillus casei, Clostridium butyricum, combination of Lactobacillus rhamnosus and Bifidobacterium lactis, combination of Lactobacillus acidophilus, Lactobacillus helveticus, and Bifidobacterium spp., or combination of Bifidobacterium lactis and resistant starch) in rats decreased the incidence of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) and colon cancer (15,21,55–57), while administration of beetroot juice fermented by Lactobacillus brevis and Lactobacillus paracasei provided protection against ACF formation in N-nitroso-N-methylurea

(MNU)-treated rats (58). According to the reports by Urbanska et al. and Chen et al., either Lactobacillus acidophilus or Saccharomyces boulardii exhibited inhibitory role against colorectal tumorigenesis in a mouse model carrying a germline APC mutation (59,60). Administration of probiotics dramatically mitigated enteric dysbacteriosis, ameliorated intestinal inflammation, and decreased liver tumor growth, suggesting an optional avenue for therapeutic prevention of hepatocellular carcinoma development (61). Long-term consumption of Lactobacillus casei in combination with soymilk achieved a beneficial effect for breast cancer prevention in chemically treated rats (62), and Lactobacillus salivarius was proven to inhibit the incidence of 4-nitroquinoline 1-oxide (4NQO)-induced oral cancer in rats (63). In addition, probiotics provided adequate protection of animals against radiation, chemical, or UV-induced damages (64–66). Nevertheless, these results should be interpreted with caution because most of the tumors were induced by various chemical agents, which was quite different from the natural process of carcinogenesis.

Anticancer effects of probiotics in clinical trials

Clinical studies have shown that certain probiotics are useful in the control of various intestinal disorders, including viral diarrhea, chemotherapy/radiotherapy or antibiotic-associated diarrhea, and postoperative inflammatory diseases. In a study that included 206 patients receiving radiotherapeutic treatment, L. rhamnosus (Antibiophilus) relieved the gastrointestinal toxicity related to radiation (67), while another study demonstrated the effectiveness of L. casei DN-114 001 on stool consistency of patients submitted to pelvic radiotherapy (68). According to the report by Delia et al., administration of VSL#3 (a mixture of 8 probiotics) to patients who were undergoing pelvic radiotherapy prevented the occurrence and severity of diarrhea (69,70). Combination of L. acidophilus and B. bifidum (Infloran) also had significant benefits on the stool consistency and the reduction of radiation-induced diarrhea (71). Of the conventional therapies for cancers, chemotherapy might change the human gut microbiota, resulting in favor of the colonization with Clostridium difficile and Enterococcus faecium (72). In patients who were diagnosed with colorectal cancer and submitted to chemotherapy, LGG effectively reduced the frequency of severe diarrhea and abdominal discomfort (73), and enteral administration of Bifidobacterium breve strain Yakult improved the intestinal environments of patients who received chemotherapy for pediatric malignancies (74). In addition, substantial evidence demonstrated that perioperative administration of probiotics effectively reduced the postoperative infectious complications (75–81). As for the preventative role of probiotics in tumor formation, El-Nezami et al. demonstrated that 5-wk supplementation of probiotics reduced the urinary excretion of aflatoxin B(1)-N(7)-guanine (AFB-N(7)-guanine), a marker for hepatocyte carcinogenesis (82), and synbiotic consumption for 12 wk significantly reduced colorectal cancer risk (83). It is generally considered that probiotic supplementation can reduce the risk of breast cancer development in perimenopausal women. However, Bonorden et al. and Nettleton et al. reported that shortterm soy and probiotic supplementation did not markedly affect the concentrations of reproductive hormones in these women (84,85). It seems that long-term consumption of probiotics is necessary to achieve chemopreventive effect on the neoplastic development. For example, Ishikawa et al. demonstrated that probiotics prevented atypia of colorectal tumors in patients who were administered L. casei for 4 yr (86). Three months of yogurt consumption did not enhance cell-mediated immune function in young women (87), while regular consumption of L. casei strain Shirota (LcS) and soy isoflavone since adolescence was inversely associated with the incidence of breast cancer in Japanese women (88).

Mechanisms through which probiotics exert their functions

 Generally speaking, the probiotics mentioned above exert their antitumor roles through improvement of intestinal microbiota, degradation of potential carcinogens, modulation of gut-associated and systemic immune, and enhancement of local and systemic antioxidant activity (for a review, see Ref. 89). As discussed in more details in the following contents, the anticarcinogenic effect may not be attributable to a single mechanism but rather to a combination of events.

Effects on intestinal microbiota homeostasis and bacterial translocation

Increased proportion of colonic bacteria with proinflammatory characteristics has been implicated in neoplastic formation. Accumulating evidence supports the hypothesis that probiotics have preventative effects on colorectal carcinogenesis by improving the intestinal environment. Probiotic lactobacilli significantly reduced the prevalence of colon cancer by modification of enteric flora in mice (90), and administration of probiotics reduced the bacterial overgrowth and the bacterial translocation in adult Wistar rats after 80% gut resection (91). A study administering probiotics to goats indicated that the supplement was able to modify microflora balance by increasing the LAB and Bifidobacterium and reducing Enterobacteriaceae like Salmonella/Shigella, resulting in the decrease of fecal mutagen concentration and fecal putrescine (92). In human beings, administration of Lactobacillus rhamnosus LC705 together with Propionibacterium freudenreichii ssp. shermanii JS increased the fecal counts of lactobacilli and propionibacteria and decreased the activity of b-glucosidase (93), an essential part of the bacterial glycolytic enzymes that might contribute to the development of colon cancer by generating carcinogens (94). Enterotoxigenic Bacteroides fragilis (ETBF) is a bacterium that is associated with diarrheal disease, inflammatory bowel disease, and colorectal cancer. In a study containing 32 adults who were found to be carriers of ETBF, probiotic yogurt was demonstrated to be effective for decreasing the cell number of ETBF (95).

The protective effects on intestinal barrier or DNA damage of intestinal epithelium

One feature in the promotion stage of colorectal carcinogenesis is the disruption of tight junctions, leading to a loss of integrity across the intestinal barrier. Commane et al. indicated that the fermentation products of proand prebiotics prevented disruption of the intestinal epithelial barrier (96), while Ko et al. demonstrated that L. plantarum inhibited the decrease in transepithelial electrical resistance of Caco-2 cells (97). Administration of probiotic products to patients undergoing biliary drainage improved the intestinal permeability and attenuated the inflammatory response (98,99). In addition, probiotics were proven to decrease the mutagen-induced DNA damage or DNA adduct formation in the colonic epithelium (100–103). An in vitro study using rat intestinal epithelial cells showed the preventative role of probiotics against enterocyte apoptosis and loss of intestinal barrier function caused by 5-fluorouracil (5-FU) (104), while an in vivo study with rats demonstrated that combination of resistant starch and B. lactis facilitated the apoptotic response to carcinogen-induced DNA damage of the rat colorectal cells (105). With this point of view, probiotics exert their functions similar to the tumor suppressor protein p53, which triggers cell apoptosis when the DNA damage is at high levels (106).

Modulation of gut-associated and/or systemic immune functions

Up to now, studies on the immune-regulatory role of probiotics in human beings were very limited and the number of study subjects was very small. Takeda and Okumura reported that daily intake of L. casei for 3 wk provided a positive effect on natural killer (NK) cell activity of health volunteers (107), while supplementation of synbiotics containing LGG, B. lactis, and oligofructose for 12 wk showed little effects on the systemic immune system of colon cancer patients (108). On the contrary, considerable reports demonstrated that administration of probiotics (or synbiotics) significantly decreased the occurrence of colon cancers in animal models through immunomodulatory effects. As

summarized in Table 3, the NK cell number or cell cytotoxicity were increased in rats or mice treated with probiotic products (9,23,25,109,110). In addition, the probiotic products enhanced the host immune functions by increasing the number of CD4/CD8-positive lymphocytes (25,55,57,66,111–114) or the phagocytic activity of macrophages (115). Long-term colonic inflammation promotes carcinogenesis and histological abnormalities such as dysplasia, a precursor of colorectal adenomas. In a study using the immortalized polyclonal human colon carcinoma cell line Caco-2, B. lactis sp. 420 showed the potential antiinflammatory and anticarcinogenic properties by modulating the host expression profiles of cyclooxygenases (116), while another study using the mouse model demonstrated that probiotics increased the production of conjugated linoleic acids possessing anti-inflammatory and anticarcinogenic properties (117). More importantly, probiotics showed anti-inflammatory activities through regulating the production of inflammatory mediators such as interleukins, interferons, and cytokines (9,11,14,23,24,66,109–112,118,119), resulting in

Figure 1. Illustration for the suppressive effects of probiotics on tumor formation and growth. Probiotics can exert their functions locally and systemically. Oral administration of probiotics can provide protection of intestinal epitheliums, modulate the homeostasis of the intestinal microflora, and inhibit the potential pathogens and carcinogenesis in the gut (┤). Together with the enhancement of antioxidant activities (!), probiotics can increase the number/activity of immune cells (!) and control the inflammatory reaction, resulting in the prevention of tumor formation. In addition, probiotics can act on cancer cells by promoting cell apoptosis (I ) and inhibiting cell proliferation or invasion (┤), resulting in the suppression of tumor growth.

the effective control of inflammation and carcinogenesis. From Table 3 we can conclude that the probiotics regulate more than one indicator of the host immunity/ inflammation in many cases. In addition, there are isolated reports citing that administration of LAB results in increased activity of antioxidative enzymes (49,52,120), which provided beneficial effects on gut-associated and/or systemic antioxidant defense against carcinogen-induced damage. Conclusion and perspectives Probiotics have obtained increasing medical importance because of their beneficial effects upon the host health. As illustrated in Figure 1, oral administration of probiotics has multiple effects such as normalization of the intestinal microflora, improvement of the gastrointestinal barrier, and inhibition of potential pathogens or carcinogenesis in the gut. Together with the enhancement of systemic immune or/and anti-inflammatory activities, probiotics may play a part in the suppression of tumor formation and growth. While laboratory-based studies have demonstrated encouraging outcomes that probiotics or synbiotics possess antitumor effects, the benefits should not be exaggerated before we get more results from human subjects. Randomized double-blind, placebo-controlled clinical trials should be done to gain the acceptance of the broader medical community and to explore the potential of probiotics as an alternative therapy for cancer control.

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