Salmonella -Mediated Cytolethal Distending Toxin Transfer Inhibits Tumor Growth

Abstract

The Salmonella enterica serovar Choleraesuis (S.C.) has potential as an antitumor agent because of its tumor-targeting characteristics. S.C. can also be used for specific delivery of therapeutic agents and continuous release during replication. Previously, we successfully used S.C. as a vector to transfer a therapeutic gene and oncolytic virus, which suggested that modified S.C. is suitable for incorporating other antitumor agents into a single system. Cytolethal distending toxin B (CdtB) produced by Campylobacter jejuni can induce tumor cell apoptosis. Here we coated CdtB with poly(allylamine hydrochloride) (PAH) to yield PAH-CdtB. Treatment of cells with PAH-coated CdtB induced apoptosis, demonstrating that the compound retained antitumor activity. Furthermore, S.C. coated with PAH-CdtB (CdtB-S.C.) maintained tumor-targeting activity and had an enhanced antitumor effect. Measurement of the cytotoxic effect of CdtB-S.C. in vitro in a tumor cell line showed increased apoptosis whereas treatment of tumor-bearing mice with CdtB-S.C. reduced tumor growth and prolonged survival. Taken together, our results provide evidence that Salmonella carrying CdtB could have application for the treatment of tumors.

Introduction

Specific tumor-targeting therapeutic agents are urgently needed for effective cancer treatment. The facultative anaerobe Salmonella can exert antitumor effects on multiple types of tumors through a variety of mechanisms.^1

–6 Because of its ability to target small metastatic and primary tumors, Salmonella has been used as a tumor-targeting therapeutic agent that can slow the growth of a broad range of tumors in mice and humans.^7

–11 Systemic Salmonella treatment allows tumor targeting and specific delivery of therapeutic agents to distant tumor sites, and the high concentrations of antitumor factors can be used to reach the interior of solid tumors.^12,13

Cytolethal distending toxin (Cdt) is a Campylobacter jejuni genotoxin that has three subunits: CdtA, CdtB, and CdtC.¹⁴ CdtA and CdtC facilitate translocation of CdtB across the target cell membrane. On reaching the cytoplasm, CdtB promotes cell apoptosis by inducing DNA damage.¹⁵ Several studies demonstrated that Cdt has potential as an antitumor agent.^16
–18

In this study, we sought to encapsulate CdtB into poly(allylamine hydrochloride) (PAH) to modify the CdtB surface charge (PAH-CdtB). The polymer-modified CdtB could translocate across the cell membrane in the absence of CdtA and CdtC. The negatively charged surface of the Salmonella enterica serovar Choleraesuis (S.C.) also attracted the positively charged PAH-CdtB. Together the results demonstrated that bacterial genotoxin delivery into tumor sites, using tumor-targeting Salmonella, could be a promising candidate for the treatment of tumors.

Materials and Methods

Bacteria, CdtB, cells, reagents, and mice

A vaccine strain of Salmonella choleraesuis (Salmonella subsp. choleraesuis [Smith] Weldin serovar Dublin [ATCC 15480]) (S.C.) was obtained from the Bioresource Collection and Research Center (BCRC, Hsinchu, Taiwan).² Mouse 4T1 (breast tumor) and B16F10 (melanoma) cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 1% glutamine, and gentamicin (50 μg/mL) at 37°C in 5% CO₂. Recombinant histidine (His)-tagged CdtB was purified by chromatography (Clontech, Palo Alto, CA) and assessed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Poly(allylamine hydrochloride) (PAH, MW 15,000), fluorescent PAH, 4′,6-diamidino-2-phenylindole (DAPI), and anti-His-tagged antibody were purchased from Sigma-Aldrich (St. Louis, MO). For subcutaneous tumor models, mice were injected with B16F10 cells (10⁶) or 4T1 cells (10⁶) via the left flank. C57BL/6 and BALB/c mice were purchased from the National Laboratory Animal Center of Taiwan. The animals were maintained in a pathogen-free animal care facility under isothermal conditions with regular photoperiods. The experimental protocol adhered to the rules of the Animal Protection Act of Taiwan and was approved by the Laboratory Animal Care and Use Committee of the China Medical University (permit No. 101-20-N). Groups of C57BL/6 or BALB/c mice were inoculated with B16F10 or 4T1 cells (10⁶). After 5 or 7 days, when the tumors were approximately 50–100 mm³, Salmonella (10⁶ colony-forming units [cfu]), PAH-CtdB (20 μg), CdtB-S.C. (10⁶ cfu), or phosphate-buffered saline (PBS) was injected intraperitoneally. Tumor size was measured every 3 days in two perpendicular axes, using a tissue caliper, and tumor volumes were calculated as (length of tumor) × (width of tumor)² × 0.45. All mice were monitored for survival and tumor growth.¹⁹

Preparation of PAH-modified CdtB (PAH-CdtB) and CdtB-S.C.

CdtB (50 μg) was dissolved in 1 mL of PAH solution (1–8 μg/mL), incubated with shaking for 30 min, and then centrifuged (15 min at 13,200 g). The excess PAH solution was discarded, and PAH-CdtB was dispersed and washed three times in deionized water. S.C. (10⁶ cfu) was washed with deionized water three times. The washed S.C. was added to 1 mL of PAH-CdtB solution (PAH, 4 μg/mL), incubated with shaking for 30 min at 37°C, and then centrifuged (10 min at 12,000 g). The excess PAH-CdtB solution was removed and the CdtB-S.C. was washed three times in water.

Cell viability assay

The supernatants from excess PAH (1–8 μg/mL), PAH-CdtB (20 μg), Salmonella (multiplicity of infection [MOI], 10), or CdtB-S.C. (MOI, 10) were added to cells for 24 h. Cell survival was assessed using the WST-1 assay (Dojindo Laboratories, Tokyo, Japan).²⁰

Western blot analysis

The bicinchoninic acid protein assay (Pierce Biotechnology, Rockford, IL) was used to determine the protein content in each sample. Proteins were fractionated by SDS–PAGE and stained with Coomassie blue. In the caspase-3 detection assay, proteins were transferred onto Hybond enhanced chemiluminescence nitrocellulose membranes (Pall, Port Washington, NY) and detected with antibodies against caspase-3 (Cell Signaling, Danvers, MA) and β-actin (Sigma-Aldrich). Suitable secondary antibodies were used for binding to primary antibodies, and protein–antibody complexes were visualized with an enhanced chemiluminescence system (GE Healthcare, Little Chalfont, UK). The signals were quantified with ImageJ software (https://rsbweb.nih.gov/ij).²¹

Characterization of PAH-CdtB and CdtB-S.C.

The ζ potential values of CdtB, S.C., PAH-CdtB, and CdtB-S.C. were measured in deionized water with a dynamic light-scattering system (Zetasizer ZS90; Malvern Instruments, Malvern, UK).²² PAH-CdtB and fluorescent PAH-CdtB were prepared as previously described. CdtB, PAH-CdtB, and fluorescent PAH-CdtB were treated with B16F10 cells for 2 h. The samples were then washed with PBS, fixed in 3.7% paraformaldehyde, incubated with 6 × His-tagged antibody at room temperature for 40 min, and subsequently incubated with Texas Red-conjugated anti-mouse antibody (diluted 1:100; KPL, Guildford, UK) at room temperature for 1 h. Nuclei were stained with DAPI (50 μg/mL), and the stained samples were observed by fluorescence microscopy at a magnification of × 200. 4T1 and B16F10 cells were infected with CdtB-S.C. (MOI, 10) for 2 h before fixation in 3.7% paraformaldehyde, incubation with rabbit anti-Salmonella serum (1:100)²³ and 6 × His-tagged antibody at room temperature for 30 min, followed by incubation with fluorescence-conjugated anti-rabbit antibody (KPL) and Texas Red-conjugated anti-mouse antibody (KPL) at room temperature for 1 h. Nuclei were stained with DAPI.

TUNEL assay

Apoptotic cells in tumor sections were detected by a TUNEL (terminal deoxynucleotidyltransferase dUTP nick-end labeling) assay performed according to the manufacturer's protocol (Promega, Madison, WI). TUNEL-positive cells were observed by fluorescence microscopy. The TUNEL-positive percentage was defined as the percentage of TUNEL-positive cells among the total cells of each field.² Nuclei were stained with DAPI.

Statistical analysis

An unpaired, two-tailed Student's t-test was used to determine differences between groups. A survival analysis was performed using a Kaplan-Meier survival curve and log-rank test. A p value less than 0.05 was considered to be statistically significant.

Results

Encapsulation of CdtB into PAH

In this study, we determined whether CdtB can be encapsulated in PAH and delivered using tumor-targeting Salmonella. Using dynamic light scattering, the net surface charge of the CdtB particles was determined to be −33.53 ± 4.16 mV (Fig. 1A). Because of the negative surface charge of CdtB, positively charged PAH can adsorb onto the negatively charged particle surface (CdtB). After coincubating PAH (4 μg/mL) and CdtB, the negative surface charge of the particles was −18.8 ± 2.21 mV (Fig. 1A). To determine the optimal concentration of PAH for modifying CdtB, increasing amounts of PAH (1–8 μg/mL) were incubated with CdtB (50 μg). Because CdtB can induce tumor cell apoptosis by degrading DNA and PAH can substitute for CdtA and CdtC to facilitate translocation of CdtB into cells, we assessed the antitumor activity of PAH-CdtB in a cell viability assay (Fig. 1B and D). PAH could also influence cell survival, so we measured the effect of excess PAH in the cell supernatant that did not adsorb onto CdtB to examine whether PAH alone could induce apoptosis (Fig. 1B and D).

Figure 1.

Encapsulation of cytolethal distending toxin subunit B (CdtB) into poly(allylamine hydrochloride) (PAH). CdtB (50 μg) incubated with PAH at 4 μg/mL is designated PAH-CdtB. Salmonella incubated with PAH-CdtB (20 μg) is designated CdtB-S.C. (A) ζ potential values for CdtB, PAH-CdtB, Salmonella, and CdtB-S.C. (B) CdtB (50 μg) incubated with various PAH concentrations (1–8 μg/mL). After centrifugation, the PAH-CdtB pellets were dissolved in 1 mL of water and B16F10 cells were added at 10⁴ per well. Excess PAH in the supernatant was also incubated with B16F10 cells (10⁴ per well). Cell viability was measured after 24 h by WST-1 assay. The data are reported as means ± SD (n = 6) (**p < 0.01). (C) After treatment for 24 h, B16F10 proteins were collected and levels of cleaved caspase-3 were detected by Western blotting. The values show protein expression relative to β-actin. (D) The same procedure as described in (B) was used except that 4T1 cells were added and (E) 4T1 proteins were collected for analysis as described for (C).

When two mouse tumor cell lines, 4T1 (breast tumor) and B16F10 (melanoma), were treated with supernatant of CdtB coated with PAH at 8 μg/mL, cell viability decreased compared with untreated control cells (Fig. 1B and D).

Treatment of the two tumor cell lines with PAH-CdtB also reduced cell viability, and this reduction was correlated with upregulated amounts of the apoptosis marker cleaved caspase-3 (Fig. 1C and E). PAH-CdtB prepared with PAH at 4 μg/mL retained the antitumor activity of CdtB and supernatants from PAH-CdtB prepared with PAH at 4 μg/mL had no effect on cell viability. As such, PAH-CdtB prepared with PAH at 4 μg/mL was used for subsequent analyses.

Characterization of PAH-coated CdtB

PAH was fluorescently labeled (FA-PAH) and CdtB was stained with anti-6 × His antibody (Texas Red) to observe the dynamics of PAH-modified CdtB. PHA adsorbed on the CdtB surface (Fig. 2A). Furthermore, an immunofluorescence assay showed that PAH-CdtB (red) absorbed onto the surface of Salmonella (green; Fig. 2B). To determine the numbers of PAH-CdtB adhered to the surface of Salmonella cells, various concentrations (10–50 μg/mL) of PAH-CdtB were incubated with Salmonella (10⁶ cfu) before centrifuging to pellet the Salmonella cells. CdtB (20 μg) was detectable in the Salmonella pellet, but not in the supernatant as shown by Coomassie blue assay (Fig. 2C). Whereas PAH-CdtB at 20 μg/mL completely adhered to the Salmonella, when excess amounts of PAH-CdtB (30–50 μg) were added, PAH-CdtB was detectable in the supernatant. The surface charges of Salmonella increased by 25% after PAH-CdtB adherence relative to untreated cells (Fig. 1A). Taken together, these results showed that PAH-CdtB could absorb onto Salmonella to form CdtB-S.C.

Figure 2.

Characteristics of PAH-CdtB and CdtB-S.C. (A) B16F10 cells treated with PAH-CdtB (20 μg) were observed by fluorescence microscopy. PAH was fluorescently labeled and CdtB was stained with a 6 × histidine (His)-tag antibody (Texas Red). 4′,6-Diamidino-2-phenylindole (DAPI) was used to stain cell nuclei. (B) B16F10 and 4T1 cells (10⁴) treated with CdtB-S.C. (10⁵ colony-forming units [cfu]) were observed by fluorescence microscopy. Salmonella was stained with anti-Salmonella antibody (fluorescein isothiocyanate [FITC]) and CdtB was stained with a 6 × His-tagged antibody (Texas Red). DAPI was used to stain cell nuclei. (C) The amounts of PAH-CdtB on the surface of Salmonella. CdtB-S.C. cells and supernatants were collected. CdtB from the solutions was measured in a Coomassie blue assay. Color images available online at www.liebertpub.com/hum

CdtB-Salmonella inhibits tumor growth in vitro and in vivo

After successfully establishing optimal CdtB-S.C., we evaluated the antitumor activity of CdtB-S.C. in vitro and in vivo. A cell viability assay revealed that among the CdtB preparations, CdtB-S.C. had the strongest antitumor activity in vitro (Fig. 3A). Although both PAH-CdtB and Salmonella could retard tumor cell growth, CdtB-S.C. also dramatically inhibited cell proliferation. This antitumor effect was associated with the upregulation in levels of cleaved caspase-3 observed by Western blotting of lysates from B16F10 or 4T1 cells treated with CdtB-S.C. (Fig. 3B).

Figure 3.

CdtB-Salmonella inhibited tumor cell proliferation. (A) B16F10 or 4T1 cells (10³) were treated with PAH-CdtB (0.2 μg), Salmonella (10⁴), or CdtB-S.C. (10⁴) for 24 h and cell numbers were measured in the WST-1 assay (*p < 0.05; **p < 0.01). (B) After treatment, B16F10 or 4T1 proteins were collected and expression of cleaved caspase-3 was detected by Western blot. Values show relative expression with respect to β-actin.

Using B16F10 and 4T1 tumor models in mice, the antitumor activity of CdtB-S.C. was evaluated. B16F10 tumor growth was significantly reduced after CdtB-S.C. treatment. Compared with the CdtB-S.C. group and the Salmonella group, systemic treatment with CdtB-Salmonella promoted stronger tumor growth inhibition (Fig. 4A). The size of CdtB-S.C.-treated melanoma tumors was significantly smaller than that seen for the PBS, PAH-CdtB, and Salmonella groups (Fig. 4C). These results suggested that CdtB-S.C. treatment significantly increased survival of mice with established melanoma tumors (Fig. 4E). Although PAH-CdtB had no antitumor activity after systemic injection, antitumor activity of CdtB-S.C. in 4T1 tumor models was observed (Fig. 4B and D). As expected, Salmonella treatment enhanced the survival of 4T1 tumor-bearing mice. The combination of Salmonella and CdtB produced the longest survival times of tumor-bearing mice (Fig. 4F). These results suggest that the antitumor activity of CdtB-S.C. is a general phenomenon that is preserved across various tumor models.

Figure 4.

Antitumor activity of CdtB-S.C. C57BL/6 and BALB/c mice were inoculated subcutaneously with (A) 10⁶ B16F10 cells or (B) 10⁶ 4T1 cells on day 0 and were treated intraperitoneally with phosphate-buffered saline (PBS), PAH-CdtB (20 μg), Salmonella (10⁶ cfu), or CdtB-S.C. (10⁶ cfu) on day 4 (B16F10) or day 7 (4T1). After systemic injection, (A) B16F10 and (B) 4T1 tumor volumes were observed every 3 days (n = 10, mean ± SEM; **p < 0.01). The effect of PBS, PAH-CdtB, Salmonella, or CdtB-S.C. on tumor growth in (C) B16F10 and (D) 4T1 tumor models was also assessed. After systemic injection, Kaplan-Meier survival curves of mice bearing (E) B16F10 and (F) 4T1 tumors were generated. (Salmonella, S.C.) (p < 0.01 for CdtB-S.C. vs. PBS and CdtB; p < 0.05 for S.C. vs. PBS and Cdt-S.C. vs. S.C.). Color images available online at www.liebertpub.com/hum

Additive antitumor effects of CdtB-S.C.

Although Salmonella monotherapy could inhibit the growth of established melanoma and breast tumors, Salmonella carrying CdtB had better antitumor efficacy. Whereas PAH-CdtB treatment exerted no antitumor effect, CdtB-S.C. reduced tumor growth and prolonged the survival of mice compared with Salmonella treatment alone. The combination of CdtB and Salmonella could induce cell death by promoting apoptosis. To explore this possibility, the number of apoptotic cells in mice bearing B16F10 or 4T1 tumors treated with PBS, PAH-CdtB, Salmonella, or CdtB-Salmonella was analyzed by TUNEL assay. Notable increases in TUNEL signals were observed in mice treated with Salmonella and, in particular, in those treated with CdtB-Salmonella (Fig. 5A and B). However, systemic PAH-CdtB treatment failed to induce tumor apoptosis. The results also indicated an increase in the numbers of cells undergoing apoptosis in the Salmonella-treated tumors compared with PBS- or PHS-CdtB-treated tumors (Fig. 5C and D). There was a 1.47- to 1.80-fold increase in the intensity of apoptotic signals induced by CdtB-S.C. compared with that induced by Salmonella in two tumor models (Fig. 5C and D).

Figure 5.

Increased numbers of apoptotic cells in tumor-bearing mice treated with Cdt-S.C. Groups of four C57BL/6 and BALB/c mice inoculated subcutaneously with B16F10 (10⁶) and 4T1 (10⁶) on day 0 were treated intraperitoneally with PBS, PAH-CdtB (20 μg), Salmonella (10⁶ cfu), or CdtB-S.C. (10⁶ cfu) on day 4 (B16F10) or day 7 (4T1). On day 10, (A) B16F10 and (B) 4T1 tumors were collected and apoptotic cells in tumor sections were detected by TUNEL assay to determine the percentages of apoptotic cells in (C) B16F10 and (D) 4T1 tumor sections. (Salmonella, S.C.) (n = 4, mean ± SD; *p < 0.05; **p < 0.01). Color images available online at www.liebertpub.com/hum

Discussion

Many bacterial toxins, such as anthrax toxin, diphtheria toxin, and Shiga toxin, have been used in tumor therapy.^24
–26 C. jejuni secretes Cdt into target cells and damages cellular DNA to induce cell apoptosis.¹⁸ Some studies found that Cdt acts as an antitumor agent in various cancers including lung cancer, oral cancer, gastric cancer, and prostate cancer.^14

–17 Although Cdt has strong antitumor activity after intratumoral injection and can also significantly enhance the efficacy of radiation therapy, appropriate vectors or routes are required for Cdt delivery in tumor therapy.^17,27 The use of chitosan/heparin nanoparticles for encapsulation promoted CdtB-mediated effects on gastric cancer in vitro and in vivo.²⁸ Salmonella can target and replicate in primary and metastatic tumors after systemic administration. Salmonella that accumulates at tumor sites could compete for nutrients in the tumor microenvironment and induce tumor cell death via apoptosis and autophagy.²⁹ Several types of tumors, such as melanoma, hepatoma, prostate cancer, lung cancer, and bladder cancer, can be targeted by Salmonella to reduce tumor volume to a greater degree compared with conventional therapy.² Thus, Salmonella that can specifically deliver therapeutic agents to tumor sites has significant potential in the development of tumor therapy. Previously, Salmonella was used as a gene transfer vector for cancer therapy or vaccines.³⁰ Salmonella successfully delivered therapeutic genes and oncolytic viruses to tumor sites, whereas PAH modification reduced host side effects to improve treatment outcomes.¹³ Tumor immune escape can also be overcome by Salmonella, which targets tumor sites by stimulating host cytokine production; reducing expression of tumor immune checkpoints, such as indoleamine 2,3-dioxygenase; and enhancing T-cell immune responses through pathogen-associated molecular pattern receptors.^31,32 Host CD4 T cells also play important roles in Salmonella-associated antitumor activity.³³ Thus, CdtB combined with immune modulators such as Salmonella could have multiple advantages for tumor therapy.

Here we have developed a new strategy to modify cationic bacterial genotoxin-coated Salmonella that can efficiently deliver CdtB for tumor therapy. To facilitate delivery of CdtB into cells, PAH was substituted for CdtA and CdtC and also provided positive charges that promote adsorption of CdtB onto the surface of tumor-targeting Salmonella. One Salmonella cell can carry 0.02 ng of CdtB to tumor sites, where it inhibits tumor growth. These results demonstrated that Salmonella in combination with bacterial toxins could represent a new strategy for tumor therapy by coupling apoptotic inducers such as CdtB with the pleiotropic activities of Salmonella.

Footnotes

Acknowledgments

This work was supported by grants from the Ministry of Science and Technology, Taiwan (MOST 104-2320-B-039-042-MY3) and the NSYSU-KMU joint research project (#NSYSUKMU 107-P004).

Author Disclosure

The authors declare no conflicts of interest.

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