The role of endotoxin/lipopolysaccharide in surgically induced tumour growth in a murine model of metastatic disease

Surgical removal of a primary tumour is often followed by rapid growth of previously dormant metastases. Endotoxin or lipopolysaccharide, a cell wall constituent of Gram-negative bacteria, is ubiquitously present in air and may be introduced during surgery. BALB/c mice received a tail vein injection of 105 4T1 mouse mammary carcinoma cells. Two weeks later, animals were subjected to surgical trauma or an intraperitoneal injection of endotoxin (10 μg per animal). Five days later, animals which underwent open surgery, laparoscopy with air sufflation or received an endotoxin injection displayed increased lung metastasis compared to anaesthetic controls. These increases in metastatic tumour growth were reflected in increased tumour cell proliferation and decreased apoptosis within lung metastases. Circulating levels of the angiogenic cytokine, vascular endothelial growth factor (VEGF), were also elevated in these groups and correlated with increased plasma levels of endotoxin. Endotoxin treatment for 18 h (>10 ng ml–1) directly up-regulated VEGF production by the 4T1 tumour cells in vitro. Metastatic tumour growth in mice undergoing carbon dioxide laparoscopy, where air is excluded, was similar to anaesthetic controls. These data indicate that endotoxin introduced during surgery is associated with the enhanced growth of metastases following surgical trauma, by altering the critical balances governing cellular growth and angiogenesis. © 1999 Cancer Research Campaig

Requirement of a Membrane Potential for the Posttranslational Transfer of Proteins into Mitochondsria

Posttranslational transfer of most precursor proteins into mitochondria is dependent on energization of the mitochondria. Experiments were carried out to determine whether the membrane potential or the intramitochondrial ATP is the immediate energy source. Transfer in vitro of precursors to the ADP/ATP carrier and to ATPase subunit 9 into isolated Neurospora mitochondria was investigated. Under conditions where the level of intramitochondrial ATP was high and the membrane potential was dissipated, import and processing of these precursor proteins did not take place. On the other hand, precursors were taken up and processed when the intramitochondrial ATP level was low, but the membrane potential was not dissipated. We conclude that a membrane potential is involved in the import of those mitochondrial precursor proteins which require energy for intracellular translocatio

Cell-Free Synthesis of the Mitochondrial ADP/ATP Carrier Protein of Neurospora crassa

ADP/ATP carrier protein was synthesized in heterologous cell-free systems programmed with Neurospora poly(A)-containing RNA and homologous cell-free systems from Neurospora. The apparent molecular weight of the product obtained in vitro was the same as that of the authentic mitochondrial protein. The primary translation product obtained in reticulocyte lysates starts with formylmethionine when formylated initiator methionyl-tRNA (fMet-tRNAfMet) was present. The product synthesized in vitro was released from the ribosomes into the postribosomal supernatant. The evidence presented indicates that the ADP/ATP carrier is synthesized as a polypeptide with the same molecular weight as the mature monomeric protein and does not carry an additional sequence

Transport of Proteins into Mitochondria

The mitochondrial ADP/ATP carrier is an integral transmembrane protein of the inner membrane. It is synthesized on cytoplasmic ribosomes. Kinetic data suggested that this protein is transferred into mitochondria in a posttranslational manner. The following results provide further evidence for such a mechanism and provide information on its details. 1. In homologous and heterologous translation systems the newly synthesized ADP/ATP carrier protein is present in the postribosomal supernatant. 2. Analysis by density gradient centrifugation and gel filtration shows, that the ADP/ATP carrier molecules in the postribosomal fraction are present as soluble complexes with apparent molecular weights of about 120000 and 500000 or larger. The carrier binds detergents such as Triton X-100 and deoxycholate forming mixed micelles with molecular weights of about 200000–400000. 3. Incubation of a postribosomal supernatant of a reticulocyte lysate containing newly synthesized ADP/ATP carrier with mitochondria isolated from Neurospora spheroplasts results in efficient transfer of the carrier into mitochondria. About 20–30% of the transferred carrier are resistant to proteinase in whole mitochondria. The authentic mature protein is also largely resistant to proteinase in whole mitochondria and sensitive after lysis of mitochondria with detergent. Integrity of mitochondria is a prerequisite for translocation into proteinase resistant position. 4. The transfer in vitro into a proteinase-resistant form is inhibited by the uncoupler carbonyl-cyanide m-chlorophenylhydrazone but not the proteinase-sensitive binding. These observations suggest that the posttranslational transfer of ADP/ATP carrier occurs via the cytosolic space through a soluble oligomeric precursor form. This precursor is taken up by intact mitochondria into an integral position in the membrane. These findings are considered to be of general importance for the intracellular transfer of insoluble membrane proteins. They support the view that such proteins can exist in a water-soluble form its precursors and upon integration into the membrane undergo a conformational change. Uptake into the membrane may involve the cleavage of an additional sequence in some proteins, but this appears not to be a prerequisite as demonstrated by the ADP/ATP carrier protein

Cyclo-oxygenase inhibition reduces tumour growth and metastasis in an orthotopic model of breast cancer

The effect of selective and non-selective cyclo-oxygenase inhibition on tumour growth and metastasis in an orthotopic model of breast cancer was investigated. 4T1 mammary adenocarcinoma cells were injected into the mammary fat pad of female BALB/c mice. When tumours reached a mean tumour diameter of 8.4±0.4 mm, mice were randomised into three groups (n=6 per group) and received daily intraperitoneal injections of the selective cyclo-oxygenase-2 inhibitor, SC-236, the non selective cyclo-oxygenase inhibitor, Indomethacin, or drug vehicle. Tumour diameter was recorded on alternate days. From 8 days after initiation of treatment, tumour diameter in animals treated with either SC-236 or indomethacin was significantly reduced relative to controls. Both primary tumour weight and the number of lung metastases were significantly reduced in the SC-236 and indomethacin treated mice. Microvessel density was reduced and tumor cell apoptosis increased in the primary tumour of mice treated with either the selective or non-selective cyclo-oxygenase inhibitor. In vitro, cyclo-oxygenase inhibition decreased vascular endothelial growth factor production and increased apoptosis of tumour cells. Our results suggest that cyclo-oxygenase inhibitors will be of value in the treatment of both primary and metastatic breast cancer

Antimetastatic activity of a cyclooxygenase-2 inhibitor

Cyclooxygenase-2 (COX-2) expression is increased in breast cancer and surgery has been shown to increase the growth of metastatic tumours. We investigated the effect of selective COX-2 inhibition on the growth of metastases in either an experimental metastasis model or following excision of a murine primary breast tumour. 50,000 4T1 mammary carcinoma cells were injected into the mammary fat pad of female BALB/c mice. When the mean TD reached 8+/-0.4 mm, tumours were excised and the mice were randomised into two groups (n=12 per group) to receive daily intraperitoneal injections of the selective COX-2 inhibitor, SC-236 or drug vehicle for 14 days. Alternatively, experimental metastases were established by tail-vein injection of 50,000 4T1 cells. Mice received either the selective COX-2 inhibitor, SC-236 or drug vehicle for 14 days (n=12 per group). SC-236 treatment significantly reduced tumour burden, the number and size of spontaneous metastases following primary tumour excision. SC-236 treatment also reduced tumour burden, the number and size of experimental metastases. Immunohistochemical staining demonstrated that COX-2 inhibition reduced microvessel density and increased apoptosis within both spontaneous and experimental metastases. These data clearly demonstrate that the selective COX-2 inhibitor, SC-236, has potent antimetastatic activity against both spontaneous metastases arising following primary tumour excision and experimental metastases.</p

Expression of a protease-resistant insulin-like growth factor-binding protein-4 inhibits tumour growth in a murine model of breast cancer

BACKGROUND: Insulin-like growth factor 1 (IGF1) promotes breast cancer and disease progression. Bioavailability of IGF1 is modulated by IGF-binding proteins (IGFBPs). IGFBP4 inhibits IGF1 activity but cleavage by pregnancy-associated plasma protein-A (PAPP-A) protease releases active IGF1. METHODS: Expression of IGF pathway components and PAPP-A was assessed by western blot or RT-PCR. IGFBP4 (dBP4) resistant to PAPP-A cleavage, but retaining IGF-binding capacity, was used to block IGF activity in vivo. 4T1.2 mouse mammary adenocarcinoma cells transfected with empty vector, vector expressing wild-type IGFBP4 or vector expressing dBP4 were implanted in the mammary fat pad of BALB/c mice and tumour growth was assessed. Tumour angiogenesis and endothelial cell apoptosis were assessed by immunohistochemistry. RESULTS: 4T1.2 cells expressed the IGF1R receptor and IGFBP4. PAPP-A was expressed within mammary tumours but not by 4T1.2 cells. Proliferation and vascular endothelial growth factor (VEGF) production by 4T1.2 cells was increased by IGF1(E3R) (recombinant IGF1 resistant to binding by IGFBPs) but not by wild-type IGF1. IGF1-stimulated microvascular endothelial cell proliferation was blocked by recombinant IGFBP4. 4T1.2 tumours expressing dBP4 grew significantly more slowly than controls or tumours expressing wild-type IGFBP4. Inhibition of tumour growth by dBP4 was accompanied by the increased endothelial cell apoptosis. CONCLUSION: Protease-resistant IGFBP4 blocks IGF activity, tumour growth and angiogenesis

Metadherin Mediates Lipopolysaccharide-Induced Migration and Invasion of Breast Cancer Cells

BACKGROUND: Breast cancer is the most prevalent cancer in women worldwide and metastatic breast cancer has very poor prognosis. Inflammation has been implicated in migration and metastasis of breast cancer, although the exact molecular mechanism remains elusive. PRINCIPAL FINDINGS: We show that the pro-inflammatory endotoxin Lipopolysaccharide (LPS) upregulates the expression of Metadherin (MTDH), a recently identified oncogene, in a number of breast cancer lines. Stable knockdown of MTDH by shRNA in human breast MDA-MB-231 cells abolishes LPS-induced cell migration and invasion as determined by several in vitro assays. In addition, knockdown of MTDH diminishes Nuclear Factor-kappa B (NF-κB) activation by LPS and inhibited LPS-induced IL-8 and MMP-9 production. CONCLUSIONS: These results strongly suggest that MTDH is a pivotal molecule in inflammation-mediated tumor metastasis. Since NF-κB, IL-8 and MMP-9 play roles in LPS-induced invasion or metastasis, the mechanism of MTDH-promoted invasion and metastasis may be through the activation of NF-κB, IL-8 and MMP-9, also suggesting a role of MTDH in regulating both inflammatory responses and inflammation-associated tumor invasion. These findings indicate that MTDH is involved in inflammation-induced tumor progression, and support that MTDH targeting therapy may hold promising prospects in treating breast cancer

TNF autovaccination induces self anti-TNF antibodies and inhibits metastasis in a murine melanoma model

TNF is a proinflammatory cytokine involved in the pathogenesis of chronic inflammatory diseases, but also in metastasis in certain types of cancer. In terms of therapy, TNF is targeted by anti-TNF neutralising monoclonal antibodies or soluble TNF receptors. Recently, a novel strategy based on the generation of self anti-TNF antibodies (TNF autovaccination) has been developed. We have previously shown that TNF autovaccination successfully generates high anti-TNF antibody titres, blocks TNF and ameliorates collagen-induced arthritis in DBA/1 mice. In this study, we examined the ability of TNF autovaccination to generate anti-TNF antibody titres and block metastasis in the murine B16F10 melanoma model. We found that immunisation of C57BL/6 mice with TNF autovaccine produces a 100-fold antibody response to TNF compared to immunisation with phosphate-buffered saline vehicle control and significantly reduces both the number (P&lt;0.01) and size of metastases (P&lt;0.01) of B16F10 melanoma cells. This effect is also observed when an anti-TNF neutralising monoclonal antibody is administered, confirming the essential role TNF plays in metastasis in this model. This study suggests that TNF autovaccination is a cheaper and highly efficient alternative that can block TNF and reduce metastasis in vivo and trials with TNF autovaccination are already underway in patients with metastatic cancer