Isolation of ketomycin from Actinomycetes as an inhibitor of 2D and 3D cancer cell invasion.

Inhibitors of cancer cell migration and invasion should be useful to inhibit metastasis. Then, we have screened microbial culture filtrates for the inhibitors of cancer cell migration. As a result, we isolated an antibiotic ketomycin from a culture filtrate of Actinomycetes SF2912 as an inhibitor of cancer cell migration. It is a known antibiotic, but its biological activity on mammalian cells has not been reported. Ketomycin inhibited cellular migration and invasion in human breast carcinoma MDA-MB-231 and MCF-7 cells at the non-toxic concentrations. Ketomycin decreased the expressions of MMP-9 and MMP-11 in MDA-MB-231 cells. Knockdown of each gene by siRNA inhibited the cellular migration and invasion. Ketomycin was then found to inhibit the cellular NF-κB activity that may be involved in the upstream signaling. For the mechanism of NF-κB inhibition, ketomycin inhibited autophosphorylation of IKK-α/IKK-ß. Ketomycin also inhibited the 3D-invasion of MDA-MB-231 cells at the non-toxic concentrations. Thus, ketomycin having a comparatively simple structure may become a seed of anti-metastasis agent.

NF­κB inhibitor DHMEQ inhibits titanium dioxide nanoparticle­induced interleukin­1ß production: Inhibition of the PM2.5­induced inflammation model.

PM2.5 is a particle with a diameter <2.5 µm that is often involved in air pollution. Nanoparticles <100 nm are thought to invade the trachea and lungs to cause inflammation, possibly through the activation of macrophages. On the other hand, titanium dioxide (TiO2) particles can be used in models of nano­micro­sized particles, as one can prepare the particles with such sizes. TiO2 particles are classified into Rutile, Anatase, and Brookite types by their crystal structure. Among them, Anatase­type TiO2 particles with a primary diameter of 50 nm (A50) were reported to induce interleukin (IL)­1ß production and secretion effectively in phorbol 12­myristate 13­acetate­treated human monocytic leukemia THP­1 cells (THP­1 macrophages). We previously designed and synthesized dehydroxymethyl­epoxyqinomicin (DHMEQ) as an inhibitor of NF­κB. The present study investigated whether the NF­κB inhibitor DHMEQ inhibits TiO2 nanoparticle­induced IL­1ß production in THP­1 macrophages, and determined the mechanism. As a result, DHMEQ inhibited A50­induced IL­1ß secretion in ELISA assays at nontoxic concentrations. It decreased the expression of IL­1ß mRNA, which was dependent on NF­κB. Although NLR family pyrin domain containing 3 (NLRP3)­inflammasome­caspase­1 activation is required for the maturation of IL­1ß, and DHMEQ reduced the NLRP3 mRNA expression and caspase­1 activity; a caspase­1 inhibitor did not influence the A50­induced IL­1ß production. Therefore, it is likely that inhibition of pro­IL­1ß expression by DHMEQ may be sufficient to inhibit mature IL­1ß production. Thus, DHMEQ may be useful for the amelioration of inflammation in the trachea and lungs caused by inhalation of PM2.5.

Inhibition of Late and Early Phases of Cancer Metastasis by the NF-κB Inhibitor DHMEQ Derived from Microbial Bioactive Metabolite Epoxyquinomicin: A Review.

We previously designed and synthesized dehydroxyepoxyquinomicin (DHMEQ) as an inhibitor of NF-κB based on the structure of microbial secondary metabolite epoxyquinomicin C. DHMEQ showed anti-inflammatory and anticancer activity in various in vivo disease models without toxicity. On the other hand, the process of cancer metastasis consists of cell detachment from the primary tumor, invasion, transportation by blood or lymphatic vessels, invasion, attachment, and formation of secondary tumor. Cell detachment from the primary tumor and subsequent invasion are considered to be early phases of metastasis, while tumor cell attachment to the tissue and secondary tumor formation the late phases. The assay system for the latter phase was set up with intra-portal-vein injection of pancreatic cancer cells. Intraperitoneal administration of DHMEQ was found to inhibit liver metastasis possibly by decreasing the expression of MMP-9 and IL-8. Also, when the pancreatic cancer cells treated with DHMEQ were inoculated into the peritoneal cavity of mice, the metastatic foci formation was inhibited. These results indicate that DHMEQ is likely to inhibit the late phase of metastasis. Meanwhile, we have recently employed three-dimensional (3D) culture of breast cancer cells for the model of early phase metastasis, since the 3D invasion just includes cell detachment and invasion into the matrix. DHMEQ inhibited the 3D invasion of breast cancer cells at 3D-nontoxic concentrations. In this way, DHMEQ was shown to inhibit the late and early phases of metastasis. Thus, DHMEQ is likely to be useful for the suppression of cancer metastasis.

Inhibition of MMP-2-mediated cellular invasion by NF-κB inhibitor DHMEQ in 3D culture of breast carcinoma MDA-MB-231 cells: A model for early phase of metastasis.

The three-dimensional (3D) culture of cancer cells provides an environmental condition closely related to the condition in vivo. It would especially be an ideal model for the early phase of metastasis, including the detachment and invasion of cancer cells from the primary tumor. In one hand, dehydroxymethylepoxyquinomicin (DHMEQ), an NF-κB inhibitor, is known to inhibit cancer progression and late phase metastasis in animal experiments. In the present research, we studied the inhibitory activity on the 3D invasion of breast carcinoma cells. Breast carcinoma MDA-MB-231 cells showed the most active invasion from spheroid among the cell lines tested. DHMEQ inhibited the 3D invasion of cells at the 3D-nontoxic concentrations. The PCR array analysis using RNA isolated from the 3D on-top cultured cells indicated that matrix metalloproteinase (MMP)-2 expression is lowered by DHMEQ. Knockdown of MMP-2 and an MMP inhibitor, GM6001, both inhibited the invasion. DHMEQ was shown to inhibit the promoter activity of MMP-2 in the reporter assay. Thus, DHMEQ was shown to inhibit NF-κB/MMP-2-dependent cellular invasion in 3D-cultured MDA-MB-231 cells, suggesting that DHMEQ would inhibit the early phase of metastasis.

Inhibition of IGF-1-Mediated Cellular Migration and Invasion by Migracin A in Ovarian Clear Cell Carcinoma Cells.

Previously we isolated migracin A from a Streptomyces culture filtrate as an inhibitor of cancer cell migration. In the present research, we found that migracin A inhibited migration and invasion of ovarian clear cell carcinoma ES-2 cells. In the course of our mechanistic study, migracin A was shown to enhance vasohibin-1 expression in an angiogenesis array. We also confirmed that it increased the mRNA expression of this protein. Moreover, overexpression of vasohibin-1 lowered the migration but not the invasion of ES-2 cells. Then, we looked for another target protein employing a motility array, and found that migracin A lowered the IGF-1 expression. Knockdown of IGF-1 by siRNA decreased the migration and invasion of ES-2 cells. Migracin A also decreased Akt phosphorylation involved in the downstream signaling. Crosstalk analysis indicated that overexpression of vasohibin-1 decreased the IGF-1 expression. On the other hand, it showed no direct anticancer activity in terms of the ES-2 growth in agar. Migracin A inhibited the migration and IGF-1 expression in not only ES-2 but also another ovarian clear cell carcinoma JHOC-5 cells. In addition, it also inhibited capillary tube formation of human umbilical vein endothelial cells. Since its cytotoxicity is very low, migracin A may be a candidate for an anti-metastasis agent not exhibiting prominent toxicity.

Novel approaches to target NF-κB and other signaling pathways in cancer stem cells.

Recently cancer tissue is considered to consist of large number of balk cancer cells and a small number of cancer stem cells. After surgery, radiotherapy, or chemotherapy, most cancer cells are removed, but if there are still very small number of cancer stem cells left. They may form the similar tumor again. So removal of cancer stem cells is considered to be important for future cancer therapy. In one hand, NF-κB is the transcription factor that promotes expressions of various inflammatory cytokines and apoptosis inhibitory proteins. Cancer cells often possess constitutively activated NF-κB that often provides excess survival and therapeutic resistance in cancer cells. We have discovered DHMEQ as a specific inhibitor of NF-κB. This compound was found to be more active in cancer stem cells than in balk cancer cells. In breast cancer cells both PI3K-Akt and NF-κB pathways appear in the survival of cancer stem cells.

Inhibition of receptor activator of nuclear factor-κB ligand- or lipopolysaccharide-induced osteoclast formation by conophylline through downregulation of CREB.

The effect of conophylline (CNP) on the receptor activator of nuclear factor-κB ligand (RANKL) or lipopolysaccharide (LPS)-induced osteoclast formation was studied in vitro using bone marrow-derived macrophages (BMMs) or the mouse macrophage-like cell line RAW 264.7. CNP inhibited RANKL-induced formation of osteoclasts identified as tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells in a culture of BMMs. It also inhibited RANKL- or LPS-induced osteoclast formation in RAW 264.7 cells. CNP lowered the osteoclast maturation markers such as calcitonin receptor, MMP9 and cathepsin K in BMMs, suggesting that CNP would inhibit the process of osteoclast differentiation. CNP inhibited the RANKL-induced expressions of c-Fos and nuclear factor of activated T cells (NFATc1), key transcription factors for osteoclastogenesis. On the other hand, CNP did not inhibit the signaling pathway of NF-κB and mitogen-activated protein kinases (MAPKs) in RANKL-stimulated BMMs. Interestingly, CNP inhibited RANKL-induced CREB activation that can mediate c-Fos and NFATc1. CNP also inhibited RANKL- or LPS-induced CREB, c-Fos and NFATc1 activation in RAW 264.7 cells. We have previously found that CNP directly binds to ADP-ribosylation-like factor-6 interacting protein (ARL6ip), although its role in osteoclastogenesis is not clear. Gene knockdown of ARL6ip by siRNA inhibited RANKL-induced c-Fos expression, suggesting that inactivation of ARL6ip may be involved in an inhibitory effect of CNP. Taken together, CNP was shown to inhibit osteoclast formation possibly via CREB inactivation following a decrease in c-Fos and NFATc1 expression.

Involvement of conserved tryptophan residues for secretion of TIMP-2.

Tissue inhibitor of metalloproteinases (TIMPs) are endogenous inhibitor proteins of matrix metalloproteinases and contain 12 cysteine residues that are conserved among TIMPs, and which are important for their activity and structure. In the present study, three tryptophan residues conserved among TIMPs were revealed to be important for the secretion of TIMP-2. Replacement of conserved tryptophan residues in TIMP-2 with alanine led to a decrease in extracellular TIMP-2 levels and an increase in intracellular TIMP-2 levels. Furthermore, wild-type TIMP-2 and TIMP-2 mutated at unconserved tryptophan residues mainly localized in the Golgi apparatus, while TIMP-2 proteins mutated at conserved tryptophan were mainly observed in the endoplasmic reticulum (ER). This indicated that conserved tryptophan is essential for transporting TIMP-2 from the ER to Golgi apparatus. These observations suggested that conserved tryptophan residues among the TIMP family of proteins have critical roles for ER-Golgi transport and subsequent secretion of TIMP-2.

Migracins A and B, new inhibitors of cancer cell migration, produced by Streptomyces sp.

In the course of screening for breast cancer cell migration inhibitors, we isolated two novel compounds, migracins A and B from the culture broth of Streptomyces sp. MI264-NF2. Their structures are related to those of luminacins previously isolated from Streptomyces. Migracins A and B inhibited breast cancer cell migration, monitored by wound healing assay with IC50 values of 1.31 and 1.99 µg ml(-1), respectively, in human breast carcinoma MDA-MB-231 cells without showing any cytotoxicity. Migracins also inhibited the migration of human lung adenocarcinoma A549 cells and human fibrosarcoma HT-1080 cells. Therefore, migracins may become new cancer metastasis inhibitors.