Staurosporine-induced cleavage of apoptosis-inducing factor in human fibrosarcoma cells is independent of matrix metalloproteinase-2

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein which mediates staurosporine (STS)-induced cell death. AIF cleavage and translocation to the cytosol is thought to be calpain-1-dependent as calpain inhibitors reduced AIF proteolysis. However, many calpain inhibitors also inhibit matrix metalloproteinase-2 (MMP-2) activity, an intracellular and extracellular protease implicated in apoptosis. Here we investigated whether MMP-2 activity is affected in response to STS and if contributes to AIF cleavage. Human fibrosarcoma HT1080 cells were treated with STS (0.1 µM, 0.25-24 hr). A significant increase in cellular MMP-2 activity was seen by gelatin zymography after 6 hr STS treatment, prior to induction of cell necrosis. Western blot showed the time-dependent appearance of two forms of AIF (~60 and 45 kDa) in the cytosol which were significantly increased at 6 hr. Surprisingly, knocking down MMP-2 or inhibiting its activity with MMP-2 preferring inhibitors ARP-100 or ONO-4817, or inhibiting calpain activity with ALLM or PD150606, did not prevent the STS-induced increase in cytosolic AIF. These results show that although STS rapidly increases MMP-2 activity, the cytosolic release of AIF may be independent of the proteolytic activities of MMP-2 or calpain.

Constituents of Aquilaria sinensis Leaves Upregulate the Expression of Matrix Metalloproteases 2 and 9

In this novel study, we isolated 28 compounds from the leaves of Aquilaria sinensis (Lour.) Gilg based on a bioassay-guided procedure and also discovered the possible matrix metalloprotease 2 (MMP-2) and 9 (MMP-9) modulatory effect of pheophorbide A (PA). To evaluate the regulatory activity on MMP-2 and MMP-9, the HT-1080 human fibrosarcoma cells were treated with various concentrations of extracted materials and isolated compounds. PA was extracted by methanol from the leaves of A. sinensis and separated from the fraction of the partitioned ethyl acetate layer. PA is believed to be an active component for MMP expression since it exhibited significant stimulation on MMP-2 and proMMP-9 activity. When treating with 50 μM of PA, the expression of MMP-2 and MMP-9 were increased 1.9-fold and 2.3-fold, respectively. PA also exhibited no cytotoxicity against HT-1080 cells when the cell viability was monitored. Furthermore, no significant MMP activity was observed when five PA analogues were evaluated. This study is the first to demonstrate that C-17 of PA is the deciding factor in determining the bioactivity of the compound. The MMP-2 and proMMP-9 modulatory activity of PA indicate its potential applications for reducing scar formation and comparative medical purposes.

4-Methylumbelliferone administration enhances radiosensitivity of human fibrosarcoma by intercellular communication

Hyaluronan synthesis inhibitor 4-methylumbelliferone (4-MU) is a candidate of radiosensitizers which enables both anti-tumour and anti-metastasis effects in X-ray therapy. The curative effects under such 4-MU administration have been investigated in vitro; however, the radiosensitizing mechanisms remain unclear. Here, we investigated the radiosensitizing effects under 4-MU treatment from cell experiments and model estimations. We generated experimental surviving fractions of human fibrosarcoma cells (HT1080) after 4-MU treatment combined with X-ray irradiation. Meanwhilst, we also modelled the pharmacological effects of 4-MU treatment and theoretically analyzed the synergetic effects between 4-MU treatment and X-ray irradiation. The results show that the enhancement of cell killing by 4-MU treatment is the greatest in the intermediate dose range of around 4 Gy, which can be reproduced by considering intercellular communication (so called non-targeted effects) through the model analysis. As supposed to be the involvement of intercellular communication in radiosensitization, the oxidative stress level associated with reactive oxygen species (ROS), which leads to DNA damage induction, is significantly higher by the combination of 4-MU treatment and irradiation than only by X-ray irradiation, and the radiosensitization by 4-MU can be suppressed by the ROS inhibitors. These findings suggest that the synergetic effects between 4-MU treatment and irradiation are predominantly attributed to intercellular communication and provide more efficient tumour control than conventional X-ray therapy.

4-Methylumbelliferone Administration Enhances Radiosensitivity of Human Fibrosarcoma by Intercellular Communication

Hyaluronan synthesis inhibitor 4-methylumbelliferone (4-MU) is a candidate of radiosensitizers which enables both anti-tumour and anti-metastasis effects in X-ray therapy. The curative effects under such 4-MU administration have been investigated in vitro; however, the radiosensitizing mechanisms remain unclear. Here, we investigated the radiosensitizing effects under 4-MU treatment from cell experiments and model estimations. We generated experimental surviving fractions of human fibrosarcoma cells (HT1080) after 4-MU treatment combined with X-ray irradiation. Meanwhilst, we also modelled the pharmacological effects of 4-MU treatment and theoretically analyzed the synergetic effects between 4-MU treatment and X-ray irradiation. The results show that the enhancement of cell killing by 4-MU treatment is the greatest in the intermediate dose range of around 4 Gy, which can be reproduced by considering intercellular communication (so called non-targeted effects) through the model analysis. As supposed to be the involvement of intercellular communication in radiosensitization, the oxidative stress level associated with reactive oxygen species (ROS), which leads to DNA damage induction, is significantly higher by the combination of 4-MU treatment and irradiation than only by X-ray irradiation, and the radiosensitization by 4-MU can be suppressed by the ROS inhibitors. These findings suggest that the synergetic effects between 4-MU treatment and irradiation are predominantly attributed to intercellular communication and provide more efficient tumour control than conventional X-ray therapy.

Przeciwnowotworowe działanie składników propolisu. Cz. 2. Związki flawonoidowe

The paper presents a review of research on the anticancerogenic activity of flavonoid compounds and their prenylated derivatives occurring in the propolis. In addition to the numerous biological and pharmacological properties of flavonoids, they also have a cytotoxic effect. The presented studies of flavonoids isolated from propolis fractions of various origins were carried out in vitro and in vivo against various human and animal cancer cell lines, e.g. HT-1080 human fibrosarcoma cells, A549 lung adenocarcinoma and Hela cervix, murine L5-26 colon carcinoma and B16-BL6 melanoma, and others. The obtained cytotoxic activity of flavonoid compounds and their prenylated derivatives as IC50 values was in the range of 3.4-10.0 μg/ml, while the ED50 values were in the range of 2.3-205.0 μg/ml. Flavonoids have a multidirectional effect on cancer cells: antioxidant, antiproliferative, blocking the cell cycle, inhibiting angiogenesis, inducing apoptosis, and inactivating carcinogens and reducing the resistance of anti-cancer drugs.

Effect of Lapachol on the Inhibition of Matrix Metalloproteinase Related to the Invasion of Human Fibrosarcoma Cells

Background: Anti-cancer effect of lapachol contained in Tabebuia avellandae has been poorly understood until now. Objective: The aim of this study was to investigate the inhibitory effect of lapachol on MMPs related to cell invasion. Its action mechanism was elucidated by analyzing the activity and the expression of MMPs and the proteins involved in the signalling pathway of cell invasion. Methods: The cytotoxicity of lapachol was evaluated by MTT assay in HT1080 cells. The effects of lapachol on the expression and the activation of MMPs were analyzed by western blot, immunofluorescence staining and gelatin zymography assays. Their gene expression was analyzed by RT-PCR, and metastasis was evaluated by cell invasion assay. Results: Lapachol below 2 µM showed no cytotoxicity. It was observed that lapachol above 0.5 µM inhibited the activation of MMP-2 and MMP-9 stimulated by PMA. In particular, the protein and gene expression levels of MMP-2 stimulated by PMA were remarkably decreased in the presence of lapachol at 1 µM compared with PMA treatment group. In addition, lapachol increased the expression level of TIMP-1 compared with PMA treatment group. Moreover, lapachol decreased the expression level of p-p38 among MAPKs compared with PMA treatment group. It was also found that the expression level of p65, a part of NF-kB, in nuclei was reduced in the presence of lapachol above 0.5 µM compared with PMA treatment group. In addition, lapachol inhibited the invasion of human fibrosarcoma cells stimulated with VEGF. Conclusion: Above results suggest that lapachol could play an important role in the modulation of MMPs related to cell invasion via the increase of TIMP-1 expression as well as the inactivation of p38 through NF-kB transcription factor.

Cathepsin B-Cleavable Cyclopeptidic Chemotherapeutic Prodrugs

Cyclopeptidic chemotherapeutic prodrugs (cPCPs) are macromolecular protease-sensitive doxorubicin (DOX) prodrugs synthesized from a cyclodecapeptidic scaffold, termed Regioselectively Addressable Functionalized Template (RAFT). In order to increase the chemotherapeutic potential of DOX and limit its toxicity, we used a Cathepsin B (Cat B)-sensitive prodrug concept for its targeted release since this enzyme is frequently overexpressed in cancer cells. Copper-free “click” chemistry was used to synthesize cPCPs containing up to four DOX moieties tethered to the upper face of the scaffold through a Cat B-cleavable peptidic linker (GAGRRAAG). On the lower part, PEG 5, 10 and 20 kDa and a fifth peptidyl DOX moiety were grafted in order to improve the solubility, bioavailability and pharmacokinetic profiles of the compound. In vitro results on HT1080 human fibrosarcoma cells showed that cPCPs display a delayed action that consists of a cell cycle arrest in the G2 phase comparable to DOX alone, and increased cell membrane permeability.

Evaluation of MMP Inhibitors Isolated from Ligustrum japonicum Fructus

The current study investigated the ability of two secoiridoids, GL-3 (1) and oleonuezhenide (2), isolated from the fruits of Ligustrum japonicum to inhibit MMP-2 and -9 activity in phorbol 12-myristate 13-acetate (PMA)-induced HT-1080 human fibrosarcoma cells. Both compounds 1 and 2 were able to exert lowered gelatin digestion activity for MMP-2 and -9 tested by gelatin zymography via suppressing the release of MMPs to culture medium according to ELISA results. Treatment with compounds was also able to suppress the expression of both mRNA and protein levels of MMP-2 and -9. Action mechanism behind the MMP inhibitory effect of the compounds was suggested to be via MAPK pathway indicated by decreased levels of phosphorylated p38, ERK and JNK proteins evaluated employing immunoblotting. Compound 1 was shown to be slightly more active to inhibit MMP-2 and -9, however, compound 2 showed more regular dose-dependency during inhibition. In conclusion, this study suggested that GL-3 and oleonuezhenide were notable natural origin potent MMP inhibitors and could serve as lead compounds for development of anti-invasive MMP inhibitors against tumor metastasis.

1-(5-Bromo-2-hydroxy-4-methoxyphenyl)ethanone [SE1] Inhibits MMP-9 Expression by Regulating NF-κB and MAPKs Signaling Pathways in HT1080 Human Fibrosarcoma Cells

Hippocampus is a traditional medicine in China, which can be used for treating tumors, aging, fatigue, thrombosis, inflammation, hypertension, prostatic hyperplasia, and other diseases. 1-(5-Bromo-2-hydroxy-4-methoxyphenyl)ethanone [SE1] from seahorse (Hippocampus kuda Bleeler) has been shown to suppress proinflammatory responses. In the present study, SE1 potently inhibited gelatin digestion by MMP-9 induced by phorbol 12-myristate 13-acetate (PMA) and migration of human fibrosarcoma HT1080 cells in dose-dependent manner. Moreover, western blot analysis and immunofluorescence analysis have been studied on MAPKs (ERK1/2, p38 kinase and JNK) and NF-κB (p65 and IκB), which refer to the clear molecular mechanism. The results indicated that SE1 significantly suppressed the phosphorylation of mitogen-activated protein kinases (MAPK: p38 kinase and JNK) and NF-κB. Finally, molecular docking result showed SE1 interacts with TYR245 and HIS226 of MMP-9 by hydrogen bond and Pi-Pi bond to suppress MMP-9 activity. This data suggested that the SE1 may possess therapeutic and preventive potential for the treatment of MMP-9 related disorders.