Attenuated Salmonella Typhimurium with truncated LPS and outer membrane-displayed RGD peptide for cancer therapy.

Gram-negative, facultatively anaerobic bacteria Salmonella Typhimurium is a candidate agent or delivery vector for cancer therapy. Effective targeted therapies in addition to radiotherapy, chemotherapy and surgery have been urgently needed as an alternative or supplement. This study expected to further improve the tumor-targeting ability of Salmonella bacteria through genetic modifications. Based on an auxotrophic Salmonella bacterial strain (D2), we constructed Salmonella mutants with altered LPS length to facilitate displaying the RGD4C targeting peptide on the outer membrane surface of Salmonella. The expression of RGD4C peptide in fusion with OmpA was identified by outer membrane protein extraction and WB detection in different mutant strains. However, flow cytometry analysis following immunofluorescence staining demonstrated that the extracellular length of Salmonella LPS did affect the surface display of RGD4C peptide. The strain D2-RGD4C that synthesized intact LPS including lipid A, core oligosaccharides and O antigen polysaccharides could hardly display RGD4C peptide, showing the same fluorescence signal intensity as the strains not expressing RGD4C peptide. Among different strains, D2 ∆rfaJ-RGD4C that synthesized truncated LPS including lipid A and partial core oligosaccharides was capable of displaying RGD4C peptide most efficiently and showed the highest ability to target HUVECs expressing αV integrin and tumor tissue with abundant neovascularization. Animal experiments also demonstrated that this tumor-targeting attenuated Salmonella strain to simultaneously deliver endostatin and TRAIL, two agents with different anti-tumor activities, could significantly inhibit tumor growth and prolong mouse survival. Thus, our studies revealed that Salmonella could be genetically engineered to improve its tumor targeting via the truncation of LPS and surface display of targeting peptides, thereby eliciting superior anti-tumor effects through targeted delivery of drug molecules.

Optimized Attenuated Salmonella Typhimurium Suppressed Tumor Growth and Improved Survival in Mice.

The gram-negative facultative anaerobic bacteria Salmonella enterica serovar Typhimurium (hereafter S. Typhimurium) has always been considered as one candidate of anti-tumor agents or vectors for delivering drug molecules. In this study, we compared several widely studied S. Typhimurium strains in their anti-tumor properties aiming to screen out the best one for further optimization and use in cancer therapy. In terms of the motility, virulence and anti-tumor efficacy, the three strains 14028, SL1344, and UK-1 were similar and obviously better than LT-2, and UK-1 showed the best phenotypes among them. Therefore, the strain UK-1 (D) was selected for the following studies. Its auxotrophic mutant strain (D1) harboring ∆aroA and ∆purM mutations was further optimized through the modification of lipid A structure, generating a new strain named D2 with stronger immunostimulatory activity. Finally, the ∆asd derivative of D2 was utilized as one live vector to deliver anti-tumor molecules including the angiogenesis inhibitor endostatin and apoptosis inducer TRAIL and the therapeutic and toxic-side effects were evaluated in mouse models of colon carcinoma and melanoma. After intraperitoneal infection, engineered Salmonella bacteria equipped with endostatin and/or TRAIL significantly suppressed the tumor growth and prolonged survival of tumor-bearing mice compared to PBS or bacteria carrying the empty plasmid. Consistently, immunohistochemical studies confirmed the colonization of Salmonella bacteria and the expression of anti-tumor molecules inside tumor tissue, which were accompanied by the increase of cell apoptosis and suppression of tumor angiogenesis. These results demonstrated that the beneficial anti-tumor efficacy of attenuated S. Typhimurium bacteria could be improved through delivery of drug molecules with powerful anti-tumor activities.

The Protective Effects of 5-Methoxytryptamine-α-lipoic Acid on Ionizing Radiation-Induced Hematopoietic Injury.

Antioxidants are prospective radioprotectors because of their ability to scavenge radiation-induced reactive oxygen species (ROS). The hematopoietic system is widely studied in radiation research because of its high radiosensitivity. In the present study, we describe the beneficial effects of 5-methoxytryptamine-α-lipoic acid (MLA), which was synthesized from melatonin and α-lipoic acid, against radiation-induced hematopoietic injury. MLA administration significantly enhanced the survival rate of mice after 7.2 Gy total body irradiation. The results showed that MLA not only markedly increased the numbers and clonogenic potential of hematopoietic cells but also decreased DNA damage, as determined by flow cytometric analysis of histone H2AX phosphorylation. In addition, MLA decreased the levels of ROS in hematopoietic cells by inhibiting NOX4 expression. These data demonstrate that MLA prevents radiation-induced hematopoietic syndrome by increasing the number and function of and by inhibiting DNA damage and ROS production in hematopoietic cells. These data suggest MLA is beneficial for the protection of radiation injuries.