Salmonella have been demonstrated to inhibit tumor growth. However, the mechanism of Salmonella-induced tumor cell death is less defined. Autophagy is a cellular process that mediates the degradation of long-lived proteins and unwanted organelles in the cytosol. Tumor cells frequently display lower levels of basal autophagic activity than their normal counterparts and fail to increase autophagic activity in response to stresses. Autophagy is involved in the cell defense elimination of bacteria. The signaling pathways leading to activation of Salmonella-induced autophagy in tumor cells remain to be elucidated. We used autophagy inhibitor (3-Methyladenine) and apoptosis inhibitor (Z-VAD-FMK) to demonstrate that Salmonella may induce cell death via apoptosis and autophagic pathway. Meanwhile, we suggested that Salmonella induce autophagy in a dose- and time-dependent manner. The autophagic markers were increased after tumor cell infected with Salmonella. In addition, the protein express levels of phosph-protein kinase B (P-AKT), phosph-mammalian targets of rapamycin (P-mTOR), phosph-p70 ribosomal s6 kinase (P-p70s6K) in tumor cells were decreased by western analysis after Salmonella infection. In conclusion, our results point out that Salmonella induce the autophagic signaling pathway via downregulation of AKT/mTOR pathway. Herein, our findings that Salmonella in controlling tumor growth may induce autophagic signal pathway.
The use of preferentially replicating bacteria as oncolytic agents is one of the innovative approaches for the treatment of tumor. This is based on the observation that some obligate or facultative anaerobic bacteria are capable of multiplying selectively in tumors and inhibiting their growth. Salmonella have been employed as an antitumor agent that is capable of preferentially amplifying within tumors and inhibiting their growth.1 Previous studies demonstrated that the induction of tumor apoptosis was correlated with Salmonella accumulation n the tumor sites.1, 2 Bacteria in tumors induced apoptosis by multiple mechanisms including competition for nutrients, stimulation of immune response.3 Moreover, toxin from bacteria may induce the apoptosis of tumor. As bacterial replication in tumors and subsequent lysis of tumor cells may induce cell-mediated immune responses to tumor cells, higher oncolysis could account, in part, for an increased infiltrate of immune cells in tumors. The cells undergoing bacteria-induced cell death exhibit heterogeneous morphological features.4, 5 It is clear that more than one mechanism is involved in the bacteria-induced killing of cells.6
Autophagy is a cellular process that mediates the degradation of long-lived proteins and unwanted organelles in the cytosol. Autophagy pathway interacts with intracellular bacteria in a variety of ways.7 Autophagy is involved in the cell defense elimination of bacteria. The signaling pathways leading to the activation of bacteria-induced autophagy in tumor cells remain to be elucidated. Autophagy is regulated by a multitude of factors, including nutritional status, hormones and intracellular signaling pathways.8 Malignant cells frequently display lower levels of basal autophagic activity than their normal counterparts and fail to increase autophagic activity in response to stresses.9 To date, a possible interaction of Salmonella with tumor cells has not been examined. Herein, we propose a role for Salmonella in controlling tumor growth by inducing apoptosis and autophagy.
Tumor-targeting potential of Salmonella in tumor-bearing mice and inhibition of subcutaneous tumor growth at distance by Salmonella
We monitored the kinetics of bacterial distribution in murine melanoma K1735 (Figure 1a) and B16F10 (Figure 1b) mice models after injection with 2 × 106 colony-forming units (c.f.u.) of Salmonella. The bacterial amount was much higher in tumors than that in livers and spleens in both tumor models of mice at each examined time. They were approximately three to four orders of magnitude higher than those found in livers or spleens. Antitumor effects of Salmonella were evaluated in terms of tumor growth of the mice bearing K1735 or B16F10 tumors. As shown in Figures 1c and d, tumor growth was significantly retarded in mice treated with Salmonella in comparison with that in PBS-treated control mice. Supplementary Figures S1a and b demonstrated that the survival of the mice injected with Salmonella was significantly prolonged compared with that of the mice injected with PBS. As indicated in Supplementary Figures S1c and d, mice treated with S.C. had a 9% lower average body weight compared with mice treated with PBS. The body weight of mice recovered after 1 week. Two tumor models had similar results. To examine the effect of Salmonella on cell death in melanoma cells, cells were incubated with different multiplicities of infection of Salmonella and then analyzed by cell viability assay. We also observed that Salmonella induced melanoma cell death in a dose-dependent manner in K1735 (Figure 1e) and B16F10 (Figure 1f) cells in vitro. Taken together, these results indicate that systemic delivery of Salmonella can target tumor sites and delay tumor growth.
Salmonella induced apoptosis-related and -independent cell death
The induction of tumor death was correlated with Salmonella accumulation in the tumor sites. When the amount of Salmonella accumulated in tumor sites, Salmonella significantly induced tumor cell death. We next investigated whether autophagy has a role in Salmonella-induced cell death in melanoma cells. Either the pan-caspase inhibitor Z-VAD-FMK or 3-MA, an autophagy inhibitor, could partially protect the cells from Salmonella-induced death, as assessed by cell viability assay. Determination of cell viability by trypan blue exclusion assay shows that the treatment of melanoma cells with Z-VAD-FMK resulted in the inhibition of Salmonella-induced apoptosis (Figure 2a). Treatment of Z-VAD-FMK decreased the expression of cleaved-caspase 3 after Salmonella infection in two tumor models (Figures 2a and b). In marked contrast, treatment of 3-MA also decreased the cell death after Salmonella infection. 3-MA could partially protect melanoma cell from Salmonella-induced cell death (Figures 2b and c), suggesting that 3-MA protected cells from autophagy-related cell death. Immunoblot analysis revealed that treatment with Salmonella in melanoma cells enhanced the conversion of microtubule-associated protein 1 light chain 3 (LC3)-I to LC3-II (Figures 2b and c). Z-VAD-FMK and 3-MA partially protected cells from Salmonella-induced death (Figures 2e and f). The results point out that Salmonella induced apoptotic and autophagic cell death in melanoma cells.
Salmonella induced autophagy in melanoma
As our results revealed that Salmonella induced nonapoptotic cell death as well as caspase-dependent apoptotic cell death in melanoma cells, we sought to examine whether Salmonella induced autophagic cell death in melanoma cells. During the autophagic process, LC3 is concentrated in autophagosome membranes, and the punctate fluorescence produced by green fluorescent protein (GFP)-fused LC3 (GFP-LC3) can be used as a good indicator of autophagy.10 We transfected the GFP-LC3 expression plasmid into melanoma to observe autophagy. As shown in Figure 3a, control cells showed diffuse cytoplasmic distribution of green fluorescence, whereas punctate fluorescence of GFP-LC3 was significantly observed in Salmonella-treated cells. The percentage of GFP-LC3 punctuated dots in cells were significantly increased in the cells infected with Salmonella compared with PBS group (Figure 3b). We also used transmission electron microscopy to observe the ultrastructure of autophagy in Salmonella-treated cells. Whereas PBS-treated cells exhibited few autophagic features, numerous autophagic vacuoles were observed in melanoma cells treated with Salmonella. The presence of double-membrane containing cellular organelles was observed in Salmonella-treated cell at higher magnification (Figure 3c). Autophagy formation is associated with various signaling pathway. The above findings prompted us to further explore the detailed mechanism underlying the autophagic effects of Salmonella in melanoma. The AKT/mTOR/p70S6K signaling pathway negatively regulates autophagy.11 We next examined the AKT/mTOR/p70S6K signaling pathway in Salmonella-induced autophagy. In a dose- (Figure 4) and time-dependent (Supplementary Figure S2) manner, treatment of Salmonella decreased the phosphorylation of AKT, mTOR and p70S6K, indicating downregulation of the AKT/mTOR/p70S6K pathway by Salmonella in K1735 cells (Figure 4a). Furthermore, very similar results were observed when Salmonella was treated with B16F10 cells (Figure 4b). Modulation of beclin 1 expression can affect the induction of autophagy.9 Figure 4 also showed that treatment of Salmonella in melanoma cells dramatically increased the expression of beclin1 and enhanced of the conversion of LC3-1 to LC3-II, which is indicative of autophagic induction.11 p62 binds directly to LC3 proteins via a specific sequence motif. The protein is itself degraded by autophagy.12 As shown in Figure 4 and Supplementary Figure S2, treatment of Salmonella in melanoma cells dramatically decreased the expression of p62. These results suggested that Salmonella induced melanoma autophagy. Taken together, these results indicated that induction of autopagy by Salmonella in melanoma cells was associated with the downregulation of AKT/mTOR/p70S6K pathway.
Salmonella induced autophagy via the downregulation of AKT signaling pathway
We found that Salmonella induced autophagy by reducing AKT phosphorylation. The AKT/mTOR/p70S6K signaling pathway was reversed by transfecting constitutively active AKT plasmid. Suppressive effect of Salmonella on the AKT/mTOR/p70S6K signaling pathway was relieved by transfecting constitutively active AKT in K1735 (Figure 5a) and B16F10 (Figure 5b) cells. Transfection of constitutively active AKT plasmid reduced the expression of becline 1 and the conversion of LC3-I to LC3-II by Salmonella treatment in comparison with vector only control transfection. The Salmonella-induced cell death was also reduced after Salmonella treatment by transfecting constitutively active AKT plasmid (Figures 5c and d). Our results suggest that downregulation of AKT is required for Salmonella-induced autophagy in melanoma cells.
Salmonella induced apoptosis and autophagy in vivo
Although Salmonella was effective in inducing autophagy in vitro, autophagic marker was not observed in vivo. To investigate the apoptosis and autophagy in vivo after Salmonella treatment, mice bearing melanoma were injected with Salmonella, and the levels of apoptotic cells and autophagic marker in the tumors were determined by terminal dUTP nick-end labeling (TUNEL) and immunohistochemistry (Figure 6). TUNEL assay shows an increase in the amount of cells undergoing apoptosis in the Salmonella-treated tumors compared with PBS-treated tumors (Figure 6a). There was 3–10-fold increase in the number of apoptotic cells induced by Salmonella compared with that induced by PBS (Figure 6b). Meanwhile, the expression of becline 1and LC3 were significantly upregulated after Salmonella treatment compared with the groups treated with PBS (Figure 6c). Induction of autophagy was confirmed by western blotting for LC3 in B16F10 model (Figure 6d). LC3-II was increased by Salmonella treatment. The effects of Z-VAD-FMK and 3-MA were further confirmed in Salmonella-treated B16F10 tumor model. Tumor growth was not suppressed more significantly with combination treatment than with inhibitor treatment only (Figure 6d). Taken together, these results indicate that Salmonella therapy resulted in retarding tumor growth and increasing apoptosis and autophagy in the tumors.
Some anaerobic and facultative anaerobic bacteria represent novel therapeutic agents that have been recently applied in cancer therapy. Systemic administration of Salmonella in tumor-bearing mice leads to its preferential accumulation in tumor sites and thus retards tumor growth. Salmonella can effectively eradicate primary and metastatic tumors including bone, prostate, breast, pancreas and sarcoma.13, 14, 15 Many studies suggest the clinical potential of bacterial treatment for critical metastatic tumor targets.16, 17, 18 In this regard, we investigated the antitumor activity of Salmonella in the murine melanoma model. Autophagy is an evolutionarily conserved process. Autophagy is a mutifaceted process and alterations in autophagic signaling pathways are frequently found in tumor cells. The AKT/mTOR/p70S6K pathway is known to be a negative regulator of autophagy. We observed that the levels of phosphorylated AKT, mTOR and p70S6K were significantly decreased in Salmonella-infected melanoma cells compared with control groups. These results indicated that Salmonella can induce autophagic activities in addition to caspase-dependent cell death in melanoma cells in vitro and in vivo.
Autophagy has a very important role in keeping cellular homeostasis. The removal of invading bacteria is crucial for bacterial infection. The ability of autophagy eliminates invasive bacteria or provides a niche for bacterial replication. Previous study demonstrated that autophagy inhibited Salmonella replication in cells.19 Autophagy defends the mammalian cytosol against bacterial invasion. Recent studies discovered that the autophagy receptor CALCOCO2/NDP52, which detects cytosol-invading Salmonella, preferentially binds LC3.20 Meantime, autophagy is activated following bacterial invasion of epithelial cells through a process requiring epithelial cell-intrinsic signaling via the innate immune adaptor protein. Thus, autophagy is an important epithelial cell-autonomous mechanism of antibacterial defense that protects against dissemination of Salmonella.21 When the amount of Salmonella accumulates in tumor sites, tumor cells want to clean Salmonella and induce strong autophagic response, resulting in cell death. Autophagy is considered to have oppositive roles, promotion and suppression, in tumor cells.22 Autophagy can provide energy for tumor cells under such stress conditions and thereby have a tumor-promoting role.23 On the other hand, autophagy has a tumor-suppressing role. Beclin 1 has antitumor activity. Although beclin 1 has multiple functions, these observations suggest a tumor-suppressing role of autophagy. Herein, Salmonella dramatically increased the expression of beclin 1 in melanoma cells.
Salmonella-induced cell death of melanoma cells was associated with the induction and processing of the autophagy marker LC3 (Figure 2). Salmonella-induced LC3-II conversion was blocked by 3-MA. However, when autophagy was inhibited by 3-MA, Salmonella still induced tumor cell death through the activation of caspase 3. Although autophagy and apoptosis constitute distinct processes, their signaling pathways are interconnected through various mechanisms of crosstalk. Thus, either 3-MA or the caspase inhibitor Z-VAD-FMK alone influenced the effect of Salmonella in melanoma cells, and in combination they did not significantly rescue Salmonella-induced cell death. Autophagy may co-occur with apoptosis in tumor cells exposed to Salmonella. Furthermore, at later stages of infection, autophagy may partially participate in the execution of tumor cell death by enhancing apoptosis. When apoptosis is blocked infected tumor cells undergo increased autophagy. These data suggest that Salmonella treatment efficiently induces both autophagy and apoptosis, which partner to induce cell death cooperatively by modifying beclin-1 and caspase expression. Apoptosis and autophagy exist crosstalk between two pathways.23 In this study we found that Salmonella induced both apoptosis and autophagy. Both apoptosis and autophagy cooperate to lead to tumor cell death after Salmonella infection. In fact, the simultaneous activation of both pathways has been found in preclinical and clinical studies. The treatment of adenovirus carrying XIAP-associated factor 1 induced apoptosis and autophagy in gastric cancer cells.24 Imatinib activated apoptosis and autophagy pathway in Kaposi’s sarcoma.25 The role of Salmonella in apoptosis and autophagy may be important for oncolytic Salmonella.
Previously, we showed that Salmonella significantly upregulated IFN-γ, which may be responsible for recruiting peripheral immune cells to the tumor in wild-type mice, but not in T-cell-deficient mice. We suggested the T cell is involved in the regulation of Salmonella-induced host antitumor immunity in tumor-bearing mice. Thus, our studies may provide a cellular basis for understanding the recruitment of effector immune cells and the synergism between the oncolytic effect of Salmonella and adaptive antitumor immune mechanisms.26
This study may not only evaluate therapeutic efficacy of Salmonella for the treatment of cancer but also elucidate the mechanisms underlying antitumor activities mediated by Salmonella, which involve cellular mechanisms.
Materials and methods
Bacteria, cell lines, reagents, plasmid and mice
The Salmonella enterica serovar choleraesuis (S. Choleraesuis; S.C.) (ATCC 15480) vaccine strain was obtained from the Bioresources Collection and Research Center (Hsinchu, Taiwan). This rough variant of S.C., which is designated vaccine 51, was obtained by spreading an 18-hour broth culture of the virulent strain 188 of the S. Choleraesuis serovar Dublin over the surface of a dried nutrient agar plate, adding a drop of a suspension of salmonella anti-o phage No. 1 and selecting for a phage-resistant colony after incubation at 37 °C for 24 h.27, 28 Murine K1735,29 B16F1030, 31 melanoma cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 50 μg ml−1 gentamicin, 2 mM L-glutamine and 10% heat-inactivated fetal bovine serum at 37 °C in 5% CO2. Murine k1735 cells were kindly provided by Dr MC Hung (The University of Texas MD Anderson Cancer Center). Caspase family inhibitor (Z-Val-Ala-DL-Asp-FMK; Z-VAD-FMK) was purchased from Enzo Life Sciences Inc. (Farmingdale, NY, USA). Autophagy inhibitor (3-methladenine; 3-MA) were purchased from Merk (Darmstadt, Germany). Constitutively active AKT plasmid was kindly provided by Dr Chiau-Yuang Tsai (Department of Molecular Immunology, Osaka University).32 Six-to-eight-week-old female C3H/HeN and C57BL/6 mice were obtained from the National Laboratory Animal Center of Taiwan. The animals were maintained in a specialized pathogen-free animal care facility in 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 number: 99-20-N).
Groups of mice were subcutaneously (s.c.) inoculated with 106 tumor cells. When the tumors had grown to diameters between 50 and 100 mm3, the mice were intravenously (i.v.) injected with 2 × 106 c.f.u. of S.C. These groups of mice were killed at various time points post infection and the numbers of Salmonella in the tumors, livers and spleens were determined on LB agar plates; these data were expressed as c.f.u. per gram of tissue. In a separate experiment, palpable tumors were measured every 3 days in two perpendicular axes using a tissue caliper and the tumor volumes were calculated as follows: (length of tumor) × (width of tumor)2 × 0.45.
Infection of tumor cells with Salmonella. The body weight and survival of mice were monitored daily. To inhibit autophagy and/or apoptosis, the mice were injected with 3-MA (24 mg kg−1) and/or Z-VAD-FMK (10 mg kg−1) intraperitoneally (i.p.) every 3 days for a total of 5 times (days 3, 6, 9, 12 and 15). Groups of mice were s.c. inoculated with 106 tumor cells. When the tumors had grown to diameters between 50 and 100 mm3, the mice were i.v. injected with 2 × 106 c.f.u. of S.C. at day 7. The palpable tumors were measured every 3 days in two perpendicular axes using a tissue caliper.
Cell viability assay
Cells were pretreated with various inhibitors for 4 h, then Salmonella (multiplicity of infection=0.1, 1 10) was added to cells for 24 h. In a parallel experiment, the adherent cells were measured for cell survival. Cell survival was assessed using the trypan blue exclusion assay.
The protein content in each sample was determined by bicinchoninic acid protein assay (Pierce Biotechnology, Rockford, IL, USA). Proteins were fractionated on SDS-PAGE, transferred onto Hybond-enhanced chemiluminescence nitrocellulose membranes (Amersham, Little Chalfont, UK) and probed with antibodies against LC3 (Novus Biologicals, Littleton, CO, USA), becline 1(Novus Biologicals), p62 (Novus Biologicals), the mammalian target of rapamycin (mTOR) (Cell Signaling, Danvers, MA, USA), phosphor-mTOR (Cell Signaling), AKT (Santa Cruz Biotechnology, Inc. Santa Cruz, CA, USA), phosphor-AKT (Santa Cruz Biotechnology, Inc.), p70 S6 kinase (p70S6K) (Cell Signaling), phosphor-p70S6K (Cell Signaling) or monoclonal antibodies against β-actin (AC-15, Sigma Aldrich). Horseradish peroxidase-conjugated goat anti-mouse IgG or anti-rabbit IgG (Jackson, West Grove, PA, USA) was used as the secondary antibody and protein–antibody complexes were visualized by enhanced chemiluminescence system (Amersham). The signals were quantified with ImageJ software (rsbweb.nih.gov/ij).33
Transmission electron microscopy
The melanoma cells infected with Salmonella for 90 min were fixed for 10 min in 50% Karnovsky fixative. Cells were collected and centrifuged at 1500 g for 5 min. The pellet was washed and stored in 70% Karnovsky fixative at 4 °C until embedding and then analyzed by transmission electron microscopy. The sections were observed with a JEOL JEM-1400 electron microscope (Tokyo, Japan).
Analysis of intracellular autophagic vacuoles
The GFP-LC3 was used to detect autophagy as described previously.10 The melanoma cells were transfected with 5 μg of the GFP-LC3 expression plasmid using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). The tranfected cells were infected with Salmonella for 90 min and the fluorescence of GFP-LC3 was visualized by fluorescence microscopy. Cell number was counted to normalize the measurement and the percentage in cells was calculated.
To analyze autophagic marker in the tumors, groups of mice that had been inoculated s.c. with 106 melanoma cells at day 0 were injected i.v. with 2 × 106 c.f.u. of Salmonella at day 10, and the control mice received PBS. The tumors were excised and snap-frozen on day 20. Cryostat sections (5 μm) were prepared, fixed and incubated with rabbit LC3 (Abgent, San Diego, CA, USA) or rabbit anti-beclin-1 (Novus Biologicals) antibodies. After sequential incubation with the appropriate peroxidase-labeled secondary antibody and aminoethyl carbazole as the substrate chromogen, the slides were counterstained with hematoxylin. TUNEL assay was used to detect cell apoptosis in the tumor area and was performed according to the manufacturer’s protocol (Promega, Madison, WI, USA). TUNEL-positive cells were counted under the microscope. The apoptosis index was defined by the percentage of TUNEL-positive among the total cells of each sample.34
The unpaired, two-tailed Student’s t-test was used to determine differences between groups for the comparison of control group. A survival analysis was performed using the Kaplan–Meier survival curve and log-rank test. A P-value <0.05 was considered to be statistically significant.
Lee CH, Wu CL, Tai YS, Shiau AL . Systemic administration of attenuated Salmonella choleraesuis in combination with cisplatin for cancer therapy. Mol Ther 2005; 11: 707–716.
Ganai S, Arenas RB, Sauer JP, Bentley B, Forbes NS . In tumors Salmonella migrate away from vasculature toward the transition zone and induce apoptosis. Cancer Gene Ther 2011; 18: 457–466.
Lee CH . Engineering bacteria toward tumor targeting for cancer treatment: current state and perspectives. Appl Microbiol Biotechnol 2012; 93: 517–523.
Chen LM, Kaniga K, Galán JE . Salmonella spp. are cytotoxic for cultured macrophages. Mol Microbiol 1996; 21: 1101–1115.
Boise LH, Collins CM . Salmonella-induced cell death: apoptosis, necrosis or programmed cell death? Trends Microbiol 2001; 9: 64–67.
Hernandez LD, Pypaert M, Flavell RA, Galán JE . A Salmonella protein causes macrophage cell death by inducing autophagy. J Cell Biol 2003; 163: 1123–1131.
Kirkegaard K, Taylor MP, Jackson WT . Cellular autophagy: surrender, avoidance and subversion by microorganisms. Nat Rev Microbiol 2004; 2: 301–314.
Klionsky DJ, Emr SD . Autophagy as a regulated pathway of cellular degradation. Science 2000; 290: 1717–1721.
Pattingre S, Levine B . Bcl-2 inhibition of autophagy: a new route to cancer? Cancer Res 2006; 66: 2885–2888.
Kabeya Y, Mizushima U, Ueno T, Yamamoto A, Kirisako T, Noda T et al. LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J 2000; 19: 5720–5728.
Yo YT, Shieh GS, Hsu KF, Wu CL, Shiau AL . Licorice and licochalcone-A induce autophagy in LNCaP prostate cancer cells by suppression of Bcl-2 expression and the mTOR pathway. J Agri Food Chem 2009; 57: 8266–8273.
Lee YR, Hu HY, Kuo SH, Lei HY, Lin YS, Yeh TM et al. Dengue virus infection induces autophagy: an in vivo study. J Biomed Sci 2013; 20: 65.
Zhao M, Yang M, Li XM, Jiang P, Baranov E, Li S et al. Tumor-targeting bacterial therapy with amino acid auxotrophs of GFP-expressing Salmonella typhimurium. Proc Natl Acad Sci USA 2005; 102: 755–760.
Hoffman RM . Bugging tumors. Cancer Discov 2012; 2: 588–590.
Liu F, Zhang L, Hoffman RM, Zhao M . Vessel destruction by tumor-targeting Salmonella typhimurium A1-R is enhanced by high tumor vascularity. Cell Cycle 2010; 9: 4518–4524.
Nagakura C, Hayashi K, Zhao M, Yamauchi K, Yamamoto N, Tsuchiya H et al. Efficacy of a genetically-modified Salmonella typhimurium in an orthotopic human pancreatic cancer in nude mice. Anticancer Res 2009; 29: 1873–1878.
Yam C, Zhao M, Hayashi K, Ma H, Kishimoto H, McElroy M et al. Monotherapy with a tumor-targeting mutant of S. typhimurium inhibits liver metastasis in a mouse model of pancreatic cancer. J Surg Res 2010; 164: 248–255.
Zhao M, Yang M, Ma H, Li X, Tan X, Li S et al. Targeted therapy with a Salmonella typhimurium leucine-arginine auxotroph cures orthotopic human breast tumors in nude mice. Cancer Res 2006; 66: 7647–7652.
Birmingham CL, Brumell JH . Autophagy recognizes intracellular Salmonella enterica serovar Typhimurium in damaged vacuoles. Autophagy 2006; 2: 156–158.
von Muhlinen N, Akutsu M, Ravenhill BJ, Foeglein Á, Bloor S, Rutherford TJ et al. An essential role for the ATG8 ortholog LC3C in antibacterial autophagy. Autophagy 2013; 9: 784–786.
Tattoli I, Sorbara MT, Philpott DJ, Girardin SE . Bacterial autophagy: the trigger, the target and the timing. Autophagy 2012; 8: 1848–1850.
Hsu KF, Wu CL, Huang SC, Wu CM, Hsiao JR, Yo YT et al. Cathepsin L mediates resveratrol-induced autophagy and apoptotic cell death in cervical cancer cells. Autophagy 2009; 5: 451–460.
Eisenberg-Lerner A, Bialik S, Simon HU, Kimchi A . Life and death partners: apoptosis, autophagy and the cross-talk between them. Cell Death Differ 2009; 16: 966–975.
Sun PH, Zhu LM, Qiao MM, Zhang YP, Jiang SH, Wu YL et al. The XAF1 tumor suppressor induces autophagic cell death via upregulation of Beclin-1 and inhibition of Akt pathway. Cancer Lett 2011; 310: 170–180.
Basciani S, Vona R, Matarrese P, Ascione B, Mariani S, Cauda R et al. Imatinib interferes with survival of multi drug resistant Kaposi's sarcoma cells. FEBS Lett 2007; 581: 5897–5903.
Lee CH, Hsieh JL, Wu CL, Hsu PY, Shiau AL . T cell augments the antitumor activity of tumor-targeting Salmonella.. Appl Microbiol Biotechnol 2011; 90: 1381–1388.
Lee CH, Hsieh JL, Wu CL, Hsu HC, Shiau AL . B cells are required for tumor-targeting Salmonella in host. Appl Microbiol Biotechnol 2011; 92: 1251–1260.
Chang WW, Kuan YD, Chen MC, Lin ST, Lee CH . Tracking of mouse breast cancer stem-like cells with Salmonella. Exp Biol Med 2012; 237: 1189–1196.
Chen MC, Chang WW, Kuan YD, Lin ST, Hsu HC, Lee CH . Resveratrol inhibits LPS-induced epithelial-mesenchymal transition in mouse melanoma model. Innate Immun 2012; 18: 685–693.
Lee CH, Wu CL, Chen SH, Shiau AL . Humoral immune responses inhibit the antitumor activities mediated by Salmonella enterica serovar Choleraesuis. J Immunother 2009; 32: 376–388.
Lee CH, Wu CL, Shiau AL . Endostatin gene therapy delivered by Salmonella choleraesuis in murine tumor models. J Gene Med 2004; 6: 1382–1393.
Shiau AL, Shen YT, Hsieh JL, Wu CL, Lee CH . Scutellaria barbata inhibits angiogenesis through downregulation of HIF-1 α in lung tumor. Environ Toxicol 2012, e-pub ahead of print 13 February 2012 doi:10.1002/tox.21763.
Hsu SC, Lin JH, Weng SW, Chueh FS, Yu CC, Lu KW et al. Crude extract of Rheum palmatum inhibits migration and invasion of U-2 OS human osteosarcoma cells by suppression of matrix metalloproteinase-2 and -9. Biomedicine 2013; 3: 120–129.
Chang WW, Lai CH, Chen MC, Liu CF, Kuan YD, Lin ST et al. 2013. Salmonella enhance chemosensitivity in tumor through connexin 43 upregulation. Int J Cancer 2013; 133: 1926–1935.
This work was supported by National Science Council from Taiwan (NSC101-2320-B039-012-MY3) and China Medical University (CMU102-S-29). We are grateful for the technical assistance of Electron Microscope Laboratory of Tzong Jwo Tang, School of Medicine, Fu Jen Catholic University.
The authors declare no conflict of interest.
Supplementary Information accompanies this paper on Gene Therapy website
About this article
Cancer Letters (2019)
Salmonella-Based Targeted Cancer Therapy: Updates on A Promising and Innovative Tumor Immunotherapeutic Strategy
TheSalmonellaeffectors SseF and SseG inhibit Rab1A-mediated autophagy to facilitate intracellular bacterial survival and replication
Journal of Biological Chemistry (2018)
Enhanced Immunotherapy Based on Photodynamic Therapy for Both Primary and Lung Metastasis Tumor Eradication
ACS Nano (2018)
Journal of Immunology Research (2018)