Tissue inhibitor of metalloproteinases-1 induces a pro-tumourigenic increase of miR-210 in lung adenocarcinoma cells and their exosomes.

Tissue inhibitor of metalloproteinases-1 (TIMP-1) recently emerged as a pro-metastatic factor highly associated with poor prognosis in a number of cancers. This correlation seemed paradox as TIMP-1 is best described as an inhibitor of pro-tumourigenic matrix metalloproteinases. Only recently, TIMP-1 has been revealed as a signalling molecule that can regulate cancer progression independent of its inhibitory properties. In the present study, we demonstrate that an increase of both exogenous and endogenous TIMP-1 led to the upregulation of miR-210 in a CD63/PI3K/AKT/HIF-1-dependent pathway in lung adenocarcinoma cells. TIMP-1 induced P110/P85 PI3K-signalling and AKT phosphorylation. It also led to increase of HIF-1α protein levels positively correlating with HIF-1-regulated mRNA expression and upregulation of the microRNA miR-210. Downstream targets of miR-210, namely FGFRL1, E2F3, VMP-1, RAD52 and SDHD, were decreased in the presence of TIMP-1. Upon the overexpression of TIMP-1 in tumour cells, miR-210 was accumulated in exosomes in vitro and in vivo. These exosomes promoted tube formation activity in human umbilical vein endothelial cell (HUVECs), which was reflected in increased angiogenesis in A549L-derived tumour xenografts. Activation and elevation of PI3K, AKT, HIF-1A and miR-210 in tumours additionally confirmed our in vitro data. This new pro-tumourigenic signalling function of TIMP-1 may explain why elevated TIMP-1 levels in lung cancer patients are highly correlated with poor prognosis.

[The resorptive behavior of a low-molecular weight humic substance after a single oral administration to the rat]. / Untersuchungen zum Resorptionsverhalten eines niedermolekularen synthetischen Huminstoffes nach einmaliger oraler Applikation an der Ratte.

By labeling HS 1500 with a radioactive 14C, it was proven that after oral application and under certain circumstances, low amounts of low molecular humic acids are for a short time absorbed from the gastrointestinal tract. But the biological availability of the substance is very low (less than 0.1% of the applied high doses). The plasma concentration curve assumes a first order kinetic for invasion and excretion. After oral application of 500 mg/kg b.w. HS 1500 the half life period was 1.5 hours and maximum plasma concentration was 3 micrograms/ml. The result obtained indicate that HS 1500 is toxicological riskless after oral administration. Taking into account the pharmacokinetic data, residues of the substance in animal tissues can be ruled out with high significance.

Inhibition of HIV-1 in cell culture by synthetic humate analogues derived from hydroquinone: mechanism of inhibition.

Humic acids are natural constituents of soil and ground water and mainly consist of mixtures of polycyclic phenolic compounds. A similar complex of compounds with a mean size of about 1000 Da, designated HS-1500, was synthesized by oxidation of hydroquinone. HS-1500 inhibited HIV-1 infection of MT-2 cells with an IC50 of 50-300 ng/ml and showed a mean cell toxicity of about 600 micrograms/ml. Inhibition of HIV-induced syncytium formation was observed at 10-50 micrograms/ml. Treatment of free and cell-attached HIV with HS-1500 irreversibly reduced its infectivity, whereas the susceptibility of target cells for the virus was not impaired by treatment prior to infection. The HIV envelope protein gp120SU bound to sepharose-coupled HS-1500 and could be eluted by high salt and detergent. HS-1500 interfered with the CD4-induced proteolytic cleavage of the V3 loop of virion gp120SU. Furthermore, binding of V3 loop-specific antibodies was irreversibly inhibited, whereas binding of soluble CD4 to gp120SU on virus and infected cells was not affected. In conclusion, our data suggest, that the synthetic humic acid analogue inhibits the infectivity of HIV particles by interference with a V3 loop-mediated step of virus entry.

[The effect of a low molecular weight synthetic humic substance on selected reproductive parameters of male rats]. / Untersuchungen zum Einfluss eines niedermolekularen synthetischen Huminstoffes auf ausgewählte Reproduktionsparameter männlicher Ratten.

Influences of the low molecular humic substance HS 1500 on parameters of organs (testis weight, epididymis weight), FSH-, and LH-levels in plasma as well as on the spermatogenesis of male rats were studied. After a daily oral application of 1000 mg/kg b.w. and 2000 mg/kg b.w., respectively over a period of 60 days (40th to 100th day of life) only 2 animals of the high dose group were observed with bilateral testicular atrophy. Despite of the two animals with testicular atrophy no significant differences between treated and control rats were observed with respect to testis morphology and spermatogenesis. There is no evidence whether the differences between the hormone levels and the testicular atrophy revealed is regarded to a HS 1500-effect or not. But otherwise a direct influence of HS 1500 can be ruled out with high certainty. Possibly because of the thin covering layer on the gastrointestinal mucous membrane formed by HS 1500, the absorption of nutritive substances is inhibited. Therefore the growth of sensitive organs like testis and epididymis, in the development period, can be decreased. Nevertheless, even after long time application the spermatogenesis is not influenced.

[The effect of a low molecular weight synthetic humic substance on pre- and postnatal development in rats]. / Untersuchungen zum Einfluss eines niedermolekularen synthetischen Huminstoffes auf die prä- und postnatale Entwicklung bei Ratten.

The influence of a low molecular synthetic humic substance (HS 1500) on pre- and postnatal development of rats was investigated. After oral application of 1000 mg/kg and 2000 mg/kg b. w. HS 1500 during the period of organogenesis (6th to 15th day p. c.) and from 16th day p. c. up to weaning neither adverse effects in the mothers nor in the fetuses were reported. The occurrence of a single malformation (gastroschisis) after application of 1000 mg/kg b. w. from day 6 to 15 of pregnancy, few ossifications of phalanges and differences in swimming behaviour are not regarded as the effect of the substance. In conclusion, the oral use of HS 1500 in the treatment of gastrointestinal diseases in animals during pregnancy is regarded to be riskless.

Influence of humate on calcium hydroxyapatite implants.

Implanted bovine apatite is highly osteoconductive, since it serves the host tissue as a "guide-line" for the deposition of newly developing bone tissue. It is well tolerated, but it showed no signs of being resorbed during the course of the experiment. Previous impregnation of the bovine hydroxyapatite with a low molecular humate substance obviously encourages its resorption. This is most easily explained by the known ability of humate to induce the activation of leucocytes. The occasional over-resorption of the apatite is dependent (1) upon the preparation of the implant (granulate) and (2) the local concentration of the humate. Future research is being directed towards the production of a satisfactorily usable form of humate and apatite and the investigation of its HIV blocking action on heterologous cancellous bone.

Humate-induced activation of human granulocytes.

Naturally occurring humic substances are particular chemical compounds which are found in humus. They bind to carbohydrates, amino acids and steroids by means of hydrogen bonds, covalent bonds and epsilon donor-acceptor complexes. Three specimens of low-molecular humic substances were tested (two naturally occurring humates and one synthetically prepared humate). They were all capable of stimulating certain functions of human neutrophils (PMN), such as the respiratory burst which results in the production of toxic oxygen compounds. This PMN stimulation can be demonstrated with the help of chemiluminescence, as well as by cytochemistry and with the electron microscope. The main product of the humate-induced PMN response is H2O2. There was no activation of neutrophilic chemokinesis or chemotaxis. It is suggested that the low-molecular humic substances originating from decaying organic material contain chemical structures which can act as signals to change dormant PMN into activated cells.