Abstract
We have developed a bacterial cancer therapy strategy using the genetically-engineered strain Salmonella typhimurium A1-R (A1-R). A1-R is auxotrophic for leu and arg which attenuates bacterial growth in normal tissue but allows high tumor virulence. A1-R is effective against metastatic human and murine cancer cell lines in clinically-relevant nude-mouse models. However, A1-R treatment of tumors in immunocompetent mouse models with high doses is limited by toxicity. The current study evaluated a traditional Chinese medicine (TCM) herbal mixture in combination with A1-R therapy in a syngeneic metastatic immunocompetent mouse model of highly aggressive lung cancer. In a model of Lewis lung carcinoma, the combination of a TCM herbal mixture and S. typhimurium A1-R enabled bacteria to be safely administered at the large dose of 2×107 colony forming units once a week i.v. with increased treatment efficacy and reduced toxicity compared to monotherapy with A1-R. The herbal mixture prevented body weight loss, spleen weight gain and liver infection by A1-R, as well as hemorrhagic lesions on the skin, liver, and spleen, all observed with A1-R monotherapy. The results of the present study suggest that the combination of A1-R and TCM has important potential for therapy of highly aggressive types of cancer, including those which are resistant to standard therapy.
Salmonella typhimurium (S. typhimurium), which is a facultative anaerobe, was previously attenuated with purine and other auxotrophic mutations and used for cancer therapy (1). In a Phase I clinical trial on patients with metastatic melanoma, the S. typhimurium strain VNP20009, attenuated by msbB and purI mutations, as well as amino acid auxotrophic mutations, was safely administered to patients. However, VNP20009 poorly colonized the patients' tumors, perhaps because it was overattenuated (2).
Subsequently, the S. typhimurium strain A1-R (A1-R) was developed for tumor targeting. S. typhimurium A1-R is auxotrophic for leu and arg, which prevents it from mounting a continuous infection in normal tissues. A1-R has no other attenuating mutations as VNP20009 does. S. typhimurium A1-R was able to eradicate primary and metastatic tumors in monotherapy in nude mouse models of prostate, breast, and pancreatic cancer, as well as sarcoma and glioma (3-10). Tumors with a high degree of vascularity were more sensitive to A1-R, and vascular destruction appears to play a role in antitumor efficacy of A1-R (11).
Use of green fluorescent protein (GFP) for imaging A1-R offers advantages of real-time visualization of single bacteria in vivo (12) that demonstrate the extent of tumor targeting. For example, dual-color labeling of cancer cells with GFP in the nucleus and red fluorescent protein (RFP) in the cytoplasm allows simultaneous imaging of intracellularly infecting GFP-expressing bacteria and of the apoptotic behavior of the infected cancer cells (3).
In contrast to nude mice, immunocompetent mice cannot be treated with high doses of S. typhimurium A1-R due to toxicity (13). To enhance efficacy and reduce side effects caused by S. typhimurium A1-R therapy in immunocompetent mice, we evaluated a traditional Chinese medicine (TCM) mixture in combination with S. typhimurium A1-R for targeting lung cancer in a syngeneic mouse model.
Materials and Methods
Preparation of herbs. Chinese herbs, including Huangqi, Dihuang, Maimendong, and Mudanpi (Pacific College of Oriental Medicine, San Diego, CA, USA), were mixed with phosphate-buffered saline (PBS) (14). A 30 mg herb mixture in 300 μl (PBS) per mouse was given to mice by gavage.
Cancer cell line. RFP-labeled Lewis lung carcinoma cells (LLC-RFP) (AntiCancer Inc., San Diego, CA, USA) were maintained in RPMI-1640 medium (Irvine Scientific, Santa Ana, CA, USA) supplemented with 10% fetal bovine serum, 2 mM glutamine (GIBCO/BRL, Life Technologies, Inc., Grand Island, NY, USA) at 37°C in a CO2 incubator (13).
Mice. Nude mice (female, 6-7 weeks) (AntiCancer Inc., San Diego, CA, USA) were used for evaluation of toxicity, and C57/BL6 (C57) immunocompetent mice (female, 6-7 weeks) were used for efficacy evaluation of the combination of S. typhimurium A1-R and the herbal mixture. Mice were fed with an autoclaved laboratory rodent diet. All animal studies were conducted in accordance with the principles and procedures outlined in the NIH Guide for the Care and Use of Laboratory Animals under Assurance number A3873-1.
Lewis lung carcinoma-RFP cell injection. LLC-RFP cells (1×106) were injected into the tail vein in order to obtain experimental metastasis in the C57 mice. By day 5, tumor colonies in the lung were visible.
Preparation of S. typhimurium A1-R. GFP-expressing Salmonella typhimurium A1-R bacteria were grown overnight in Luria Bertani (LB) medium (Fisher Sci., Hanover Park, IL, USA) and then diluted 1:10 in LB medium. Bacteria were harvested at late-log phase, washed with phosphate-buffered saline (PBS), and then diluted in PBS (Omega Sci., San Diego, CA, USA) (3).
Bacterial dosing. Nude mice (n=30) were used for toxicity testing. Ten mice in group 1 served as untreated controls. Ten mice in group 2 were treated with intravenous S. typhimurium A1-R monotherapy [5×107 CFU/100 μl] once a week via tail-vein injection. Ten mice in group 3 were treated with S. typhimurium A1-R [5×107 CFU/100 μl] once a week via tail vein in combination with the herbal mixture (30 mg in 300 μl PBS) orally by gavage twice a day. On days 3 and 6, five mice from each group were sacrificed. The livers were removed and imaged with an IV100 scanning laser microscope (Olympus Corp., Tokyo, Japan).
C57/BL6 mice (n=30) were injected via the tail vein with LLC-RFP cells (2×106). Ten mice in group 1 served as untreated controls. Ten mice in group 2 were treated with intravenous S. typhimurium A1-R [2×107 CFU/100 μl] once a week via tail-vein injection beginning on day 3 after LLC-RFP injection. Ten mice in group 3 were treated with S. typhimurium A1-R [2×107/CFU/100 μl] once a week via tail-vein injection and the herbal mixture (30 mg in 300 μl PBS) twice a day, orally by gavage, beginning on day 3 after LLC-RFP injection. Five mice from each group were sacrificed on day 34 after treatment. Lung and spleen weights were measured. The remaining mice were used for survival determination at 70 days post-initial treatment.
Monitoring and evaluation. The mice were monitored weekly for signs of toxicity, including inactivity, inability to groom, ruffling of fur and body weight. At sacrifice, lungs were excised from lung cancer-bearing mice and were weighed and imaged.
Small-animal imaging systems. The Olympus OV100 Small Animal Imaging System (Olympus Corp.), comprising an MT-20 light source (Olympus Biosystems, Planegg, Germany) and DP70 CCD camera (Olympus Corp.), was used (15).
An Olympus IV100 scanning laser microscope (Olympus Corp.) was also used. Novel stick objectives (as small as 1.3 mm) were designed specifically for high-resolution imaging (16).
Statistical analysis. All statistical analyses were performed using SYSTAT 12.0 (SYSTAT, Inc., Chicago, IL, USA). The experimental data are expressed as the mean ± SD. The two-tailed Student's t-test was used for statistical analysis. The Kaplan-Meier test was used to analyze survival.
Results
Herbal mixture reduced side effects of high-dose S. typhimurium A1-R infection in nude mice without tumors. Initial experiments were performed in nude mice to determine dosing parameters of a herbal mixture to reduce side-effects of high dose S. typhimurium A1-R. In contrast to bacterial treatment alone, there were no bleeding foci on the skin, liver, or spleen after high-dose S. typhimurium A1-R infection [5×107 CFU/100 μl] in combination with the herbal mixture (data not shown).
Lower distribution of S. typhimurium A1-R bacteria in the liver of nude mice when combined with the herbal mixture. S. typhimurium A1-R [5×107 CFU/100 μl PBS] was injected once into the tail vein of the nude mice without tumors as monotherapy or in combination with the herbal mixture twice a day by gavage until the end of study. On day 3 and day 6 after S. typhimurium A1-R injection, livers were removed and imaged with an IV100 scanning laser microscope. On day 3, the average number of GFP-labeled S. typhimurium A1-R clones in the liver was higher in the monotherapy-treated animals (48 ± 9.8) than in the animals treated with S. typhimurium A1-R combined with the herbal mixture (18.6 ± 12.3) (p=0.003). On day 6, the average number of GFP-labeled S. typhimurium A1-R clones in the liver was also higher in the A1-R monotherapy group (1.6±0.89) than in the combination group (0) (p=0.04). These results indicate that the herbal mixture appeared to protect the liver from bacterial infection (Figure 1A,B).
Lower spleen weight in immunocompetent mice with Lewis lung carcinoma (LLC-RFP) tumors treated with S. typhimurium A1-R in combination with the herbal mixture. LLC-RFP cells (2×106) were injected in the tail vein of C57 mice. Beginning on day 3 after LLC injection, S. typhimurium A1-R [2×107 CFU/100 μl PBS] was injected in the tail vein of the mice once a week for five weeks, and the herbal mixture was given in addition by gavage twice a day for five weeks. On day 34, all animals were sacrificed. Gross necropsy demonstrated that mice treated with S. typhimurium A1-R combined with the herbal mixture had a significantly lower mean spleen weight (0.29 ± 0.001g) compared to the mice treated with S. typhimurium A1-R monotherapy (0.58 ± 0.26g) (p=0.036). Body weight loss was prevented by the combination of the herbal mixture with S. typhimurium A1-R compared to use of A1-R monotherapy (p=0.027) at day 21 after treatment (Figure 2A,B).
Efficacy of the herbal mixture in combination with S. typhimurium A1-R on LLC experimental metastasis in immunocompetent mice. LLC-RFP cells (2×106) were injected in the tail vein of C57 mice. Beginning on day 3, experimental metastases in the lung were detected. At this time, S. typhimurium A1-R (2×107 CFU/100 μl PBS) was injected into the tail vein of the mice once a week. The herbal mixture was administered twice daily by gavage. On day 34, all animals were sacrificed. The excised lungs were weighed and imaged with the OV100. Fewer LLC-RFP metastases were found in the mice treated with monotherapy (11.6 ± 6.07) and the combination therapy (4.2 ± 3.03) than in the untreated mice (19.8 ± 4.21) (S. typhimurium A1-R vs. control: p=0.038; S. typhimurium A1-R plus herbal mixture vs. control: p=0.0001); S. typhimurium A1-R in combination with the herbal mixture vs. S. typhimurium A1-R monotherapy: p=0.04 (Figures 3 and 4). The mean lung weight was lower in S. typhimurium A1-R monotherapy-treated mice (0.44 ± 0.13g) and combination therapy-treated mice (0.26 ± 0.11g) compared to untreated control mice (1.58 ± 0.35g) (S. typhimurium A1-R vs. control: p<0.001; S. typhimurium A1-R plus herbal mixture vs. control: p<0.001); S. typhimurium A1-R plus herbal mixture vs. S. typhimurium A1-R monotherapy (p=0.04) (Figure 5).
Survival of immunocompeted mice with LLC is prolonged by S. typhimurium A1-R and S. typhimurium A1-R and herbal mixture. LLC-RFP cells (2×106) were injected in the tail vein of C57 mice. Beginning on day 3, S. typhimurium A1-R (2×107 CFU) was injected in the tail vein once a week for 10 weeks. The herbal mixture was given twice a day for 10 weeks. The average survival time of the S. typhimurium A1-R monotherapy-treated mice was 59 days and the mice treated with S. typhimurium A1-R combined with the herbal mixture was 66 days compared with 36 days for the untreated mice (S. typhimurium A1-R vs. control: p=0.022; S. typhimurium A1-R plus herbal mixture vs. control: p=0.002); S. typhimurium A1-R + herbal mixture vs. S. typhimurium A1-R monotherapy: (p=0.459) (Figure 6).
Discussion
Herbal mixtures have been used in the treatment of cancer in TCM for thousands of years in China, Japan, and other Asian countries (17, 18). The herbal mixture used in the present study contained four herbs, Huangqi, Maimendong, Mudanpi, and Shendihuang. As this study shows, the herbal mixture combined with S. typhimurium A1-R has advantages in terms of increasing anti-metastatic efficacy of S. typhimurium A1-R and reducing side-effects associated with its monotherapy, including body weight loss in immunocompetent mice.
The results of the present study with a highly metastatic lung tumor suggest that a combination of S. typhimurium A1-R and the herbal mixture has clinical potential for very aggressive cancer, including those types which respond poorly to conventional therapy.
Acknowledgements
This work was supported in part by NCI grant CA126023.
Footnotes
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Conflicts of Interest
None of the Authors have a conflict of interest regarding this study.
- Received February 11, 2013.
- Revision received March 19, 2013.
- Accepted March 20, 2013.
- Copyright© 2013 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved