Pia Heikkilä

Tumor targeting with a selective gelatinase inhibitor

Several lines of evidence suggest that tumor growth, angiogenesis, and metastasis are dependent on matrix metalloproteinase (MMP) activity. However, the lack of inhibitors specific for the type IV collagenase/gelatinase family of MMPs has thus far prevented the selective targeting of MMP-2 (gelatinase A) and MMP-9 (gelatinase B) for therapeutic intervention in cancer. Here, we describe the isolation of specific gelatinase inhibitors from phage display peptide libraries. We show that cyclic peptides containing the sequence HWGF are potent and selective inhibitors of MMP-2 and MMP-9 but not of several other MMP family members. Our prototype synthetic peptide, CTTHWGFTLC, inhibits the migration of human endothelial cells and tumor cells. Moreover, it prevents tumor growth and invasion in animal models and improves survival of mice bearing human tumors. Finally, we show that CTTHWGFTLC–displaying phage specifically target angiogenic blood vessels in vivo. Selective gelatinase inhibitors may prove useful in tumor targeting and anticancer therapies.

Activation of Type IV Procollagenases by Human Tumor-associated Trypsin-2

Increased production of proteinases, such as matrix metalloproteinases (MMPs), is a characteristic feature of malignant tumors. Some human cancers and cell lines derived from them also express trypsinogen, but the function of the extrapancreatic trypsin has remained unclear. In this study we cloned and sequenced trypsinogen-2 cDNA from human COLO 205 colon carcinoma cells and characterized the ability of the enzyme to activate latent human type IV procollagenases (proMMP-2 and proMMP-9). As shown by cloning and N-terminal amino acid sequencing, the amino acid sequence of tumor-associated trypsin-2 is identical to that of pancreatic trypsin-2. We found that both pancreatic trypsin-2 and tumor cell-derived trypsin-2 are efficient activators of proMMP-9 and are capable of activating proMMP-9 at a molar ratio of 1:1000, the lowest reported so far. Human trypsin-2 was a more efficient activator than widely used bovine trypsin and converted the 92-kDa proMMP-9 to a single 77-kDa product that was not fragmented further. The single peptide bond cleaved by trypsin-2 in proMMP-9 was Arg87-Phe88. The generation of the 77-kDa species coincided with the increase in specific activity of MMP-9. In contrast, trypsin-2 only partially activated proMMP-2. Trypsin-2 cleaved the Arg99-Lys100 peptide bond of proMMP-2 generating 62–65-kDa MMP-2 species. Trypsin-2-induced proMMP-2 and -9 conversions were inhibited by tumor-associated trypsin inhibitor added either prior to or during activation indicating that proMMPs were not activated autocatalytically. Trypsin-2 also activated proMMPs associated with tissue inhibitor of matrix metalloproteinases, the complexes of which are thought to be the major MMP forms in vivo. The ability of human tumor cell-derived trypsin-2 to activate latent MMPs suggests a role for trypsin-2 in initiating the proteinase cascade that mediates tumor invasion and metastasis formation.

MMP Inhibition and Downregulation by Bisphosphonates

ABSTRACT: Bisphosphonates are a group of drugs capable of inhibiting bone resorption, and are thus used for the treatment of bone diseases, such as Pagets disease, osteoporosis, and for bone metastases of malignant tumors. Their primary cellular target is considered to be the osteoclast. The molecular mechanisms responsible for the downregulation of bone resorption by bisphosphonates have remain unclear. We have discovered that various matrix metalloproteinases (MMPs) are inhibited in vitro by several bisphosphonates. This novel finding may, in part, explain the efficacy of bisphosphonates in their current indications in humans. In enzyme activity tests using purified and recombinant enzymes, we have observed the inhibition of MMP‐1, ‐2, ‐3, ‐7, ‐8, ‐9, ‐12, ‐13, and ‐14 by clondronate, alendronate, pamidronate, zolendronate, nedrinate, and clodrinate. The IC50s range from 50 to 150 μM. We have also shown that clodronate can downregulate the expression of MT1‐MMP protein and mRNA in several cell lines. Additionally, several bisphosphonates decrease the degree of invasion of malignant melanoma (C8161) and fibrosarcoma (HT1080) cells through artificial basement membrane (Matrigel) in cell cultures at IC50s of 50‐150 μM and below. Having low toxicity and proven to be well tolerated after several years in human use, bisphosphonates have the potential to become one of the main MMP‐inhibitors for MMP‐related human soft and hard tissue‐destructive diseases in the near future.

Bisphosphonates inhibit stromelysin-1 (MMP-3), matrix metalloelastase (MMP-12), collagenase-3 (MMP-13) and enamelysin (MMP-20), but not urokinase-type plasminogen activator, and diminish invasion and migration of human malignant and endothelial cell lines.

Bisphosphonates (clodronate, alendronate, pamidronate and zoledronate) at therapeutically attainable non-cytotoxic concentrations inhibited MMP-3, -12, -13 and -20 as well as MMP-1, -2, -8 and -9, but not urokinase-type plasminogen activator (uPA), a serine proteinase and a pro-MMP activator. Dose-dependent inhibition was shown by three independent MMP assays. The inhibition was reduced in the presence of an increased concentration of Ca2+ when compared to physiologic Ca2+ concentration. Alendronate inhibited the in vitro invasion (Matrigel) of human HT1080 fibrosarcoma and C8161 melanoma cells, and the random migration of these malignant and endothelial cell lines capable of expressing MMPs and uPA. The concentration of alendronate required to inhibit 50% of the activity (IC50=40–70 μM) of MMPs corresponded to the IC50 of down-regulation of in vitro invasion and migration. The ability of bisphosphonates to down-regulate the in vitro invasion and random migration was comparable or slighty better in relation to the selective gelatinase inhibitor CTTHWGFTLC peptide. Alendronate but not CTTHWGFTLC peptide promoted the adhesion of HT1080 fibrosarcoma and C8161 melanoma cell lines on fibronectin. Bisphosphonates are broad-spectrum MMP inhibitors and this inhibition involves cation chelation. Bisphosphonates further exert antimetastatic, anti-invasive and cell adhesion-promoting properties, which may prevent metastases not only into hard tissues but also to soft tissues.

Human tongue carcinoma growth is inhibited by selective antigelatinolytic peptides

Matrix metalloproteinases (MMP‐2 and MMP‐9, or gelatinases) are involved in tongue SCC invasion, metastasis and angiogenesis. We have recently shown that a novel and selective hydrophobic cyclic CTTHWGFTLC (CTT1) peptide is inhibitor for MMP‐2 and MMP‐9 (Koivunen et al., Nat Biotechnol 1999; 17:768–74). In this study, we demonstrate that both the new hydrophilic derivate GRENYHGCTTHWGFTLC (CTT2) peptide and the CTT1 peptide inhibited specifically the human tongue squamous cell carcinoma (HSC‐3) cell‐derived gelatinolytic activity and in vitro invasion and migration of these cells (p ≤ 0.049). In situ zymography revealed that both peptides also inhibited clearly almost all of the gelatinolytic activity present in the human tongue SCC tissue sections, indicating that MMP‐2 and MMP‐9 are the major gelatinases detected in the tongue carcinomas. However, CTT2 did not inhibit the type I collagen degradation by human collagenases (MMP‐1, MMP‐8 and MMP‐13). Furthermore, CTT2 reduced the blood vessel density (p ≤ 0.043) and clearly improved the survival of the mice bearing human tongue carcinoma xenografts (p ≤ 0.012). Overall, we suggest that CTT1 and CTT2 peptides being selective gelatinase inhibitors with significant anti‐tumor properties could be useful to diminish the invasion and angiogenesis of human tongue carcinomas characterized by enhanced gelatinolytic activity in tumors.

Inhibition of matrix metalloproteinase-14 in osteosarcoma cells by clodronate

BACKGROUND Bisphosphonates reduce the bone metastasis formation and angiogenesis but the exact molecular mechanisms involved are unclear. Progelatinase A (proMMP-2; 78 KDa) is activated up during the tumor spread and metastasis by a cell surface-associated matrix metalloproteinase (membrane-type matrix metalloproteinase [MT1-MMP] or MMP-14). MATERIAL AND METHODS We evaluated the effects of a bisphosphonate (clodronate) on MT1-MMP mRNA expression and protein production, catalytic activity and proteolytic activation of proMMP-2 by cultured human MG-63 osteosarcoma cells. RESULTS Clodronate, at therapeutically attainable noncytotoxic concentrations, dose-dependently inhibited phorbol myristic acetate (PMA)-induced proteolytic activation of proMMP-2 by human MG-63 osteosarcoma cells. Clodronate also downregulated the PMA-induced expression of MT1-MMP mRNA and protein production in human MG-63 osteosarcoma cells, as evidenced by Northern analysis and fluorescent immunohistochemistry. Furthermore, clodronate inhibited directly and dose-dependently MT1-MMP activity, and the MT1-MMP inhibition by clodronate was reduced in the presence of an increased (5 mM) Ca(2+) concentrations when compared to physiological (1 mM) Ca(2+) concentrations. CONCLUSION We conclude that (1) the extracellular/cell-associated mechanism of bisphosphonate involves inhibition of MT1-MMP catalytic activity eventually by chelation, and that (2) intracellular mechanism involves downregulation of induced MT1-MMP mRNA and protein expression. The inhibition and downregulation of MT1-MMP by clodronate can be related to their ability to reduce MG-63 osteosarcoma cell invasion and spread. These findings may, at least in part, explain at molecular level the antitumor and antibone resorption activities of clodronate observed in clinical studies.

CMT-8/clodronate combination therapy synergistically inhibits alveolar bone loss in LPS-induced periodontitis.

Earlier studies have reported that a chemically modified doxycycline (CMT-8) administered as a monotherapy inhibited alveolar bone loss in a lipopolysaccharides(LPS) injected rat periodontitis model. 1,2 Teronen et al. reported that clodronate, a bisphosphonate, also inhibited the activity of MMP-8, the predominant collagenase in inflamed gingival tissue and in gingival crevicular fluid in human adult periodontitis. 2 In the present study the effects of the combination of subtherapeutic levels of chemically modified doxycycline (CMT-8) and a bisphosphonate (clodronate) were investigated in a LPS-induced periodontal disease model. This model, described here, was published by Ramamurthy et al. 1 and involves the injection of E. coli endotoxin into the gingival tissues. Injection of endotoxin into the gingiva produces marked inflammation in the periodontium, pathologically elevated levels of tissuedestructive matrix metalloproteinases (MMPs), leading to severe alveolar bone resorption and bone loss around the affected teeth.

Periodontitis and cancer mortality: Register-based cohort study of 68,273 adults in 10-year follow-up: Periodontitis increases the fatal course of cancer

Periodontitis, a multifactorial infection‐induced low‐grade chronic inflammation, can influence the process of carcinogenesis. We studied with 10 years follow‐up of 68,273 adults‐based cohort the involvement of periodontitis as a risk factor for cancer mortality. Periodontal status was defined based on procedure codes of periodontal treatment. Rate ratios and absolute differences of overall and cancer mortality rates were assessed with respect to periodontal status using multiplicative and additive Poisson regression models, respectively. We adjusted for effect of age, sex, calendar time, socio‐economic status, oral health, dental treatments and diabetes. Data about smoking or alcohol consumption were not available. Altogether 797 cancer deaths occurred during 664,020 person‐years accumulated over a mean 10.1‐year follow‐up. Crude cancer mortality rate per 10,000 person‐years for participants without and with periodontitis was 11.36 (95% CI 10.47–12.31) and 14.45 (95% CI 12.51–16.61), respectively. Crude rate ratios for periodontitis indicated an increased risk of overall (RR 1.27, 95% CI 1.08–1.39) and pancreatic cancer (RR 1.69, 95% CI 1.04–2.76) mortality. After adjustment, the results showed even stronger associations of periodontitis with increased overall (RR 1.33, 95% CI 1.10–1.58) and pancreatic cancer (RR 2.32, 95% CI 1.31–3.98) mortality. A higher pancreatic cancer mortality among individuals with periodontitis contributed considerably to the difference in overall cancer mortality, but this difference was not due to pancreatic cancer deaths alone.