Targeting prolidase. A survey of the literature data to depict a structure-activity relationship frame and to address future studies for drug development.

Prolidase (EC.3.4.13.9) is a Mn+2-dependent dipeptidase that is well known to play a crucial role in several physiological and pathological processes affecting humans. More in particular, this enzyme is involved in the cleavage of proline- and hydroxyproline-containing dipeptides (imidodipeptides), providing a fine regulation of the homeostasis of these two amino acids. Hyperactivity or deficiency of prolidase have been clearly associated to the development and progress of several acute and chronic syndromes (e.g. chronic liver fibrosis, viral and acute hepatitis, cancer, neurological disorders, inflammation, skin diseases, intellectual disability, respiratory infection). Thus, targeting prolidase and modulating its activity is an intriguing field of research with a great therapeutic potential for the next future and for the design of specific and selective drugs. Prolidase can be exploited in two essential ways: as an activator of proline containing prodrugs and by direct interaction. In this latter case, few specific ligands for the title enzyme have been described, but with no reports about their structure-activity relationship. The aim of this comprehensive review is to gather all available information on prolidase targeting so far reported in the literature, to rationalize the observed data and effect into a preliminary structure-relationship picture, to comment about the effectiveness of each reported ligands, and to address future research activities providing new potential and putative natural, semisynthetic, and purely synthetic molecules able to trigger prolidase as the main biological target.

Profibrotic mechanisms of DPP8 and DPP9 highly expressed in the proximal renal tubule epithelial cells.

BACKGROUND: DPP8 and DPP9 have been demonstrated to play important roles in multiple diseases. Evidence for increased gene expression of DPP8 and DPP9 in tubulointerstitium was found to be associated with the decline of kidney function in chronic kidney disease (CKD) patients, which was observed in the Nephroseq human database. To examine the role of DPP8 and DPP9 in the tubulointerstitial injury, we determined the efficacy of DPP8 and DPP9 on epithelial-to-mesenchymal transition (EMT) and tubulointerstitial fibrosis (TIF) as well as the underlying mechanisms. METHODS: We conducted the immunofluorescence of DPP8 and DPP9 in kidney biopsy specimens of CKD patients, established unilateral ureteral obstruction (UUO) animal model, treated with TC-E5007 (a specific inhibitor of both DPP8 and DPP9) or Saxagliptin (positive control) or saline, and HK-2 cells model. RESULTS: We observed the significantly increased expression of DPP8 and DPP9 in the renal proximal tubule epithelial cells of CKD patients compared to the healthy control subjects. DPP8/DPP9 inhibitor TC-E5007 could significantly attenuate the EMT and extracellular matrix (ECM) synthesis in UUO mice, all these effects were mediated via interfering with the TGF-ß1/Smad signaling. TC-E5007 treatment also presented reduced renal inflammation and improved renal function in the UUO mice compared to the placebo-treated UUO group. Furthermore, the siRNA for DPP8 and DPP9, and TC-E5007 treatment decreased EMT- and ECM-related proteins in TGF-ß1-treated HK-2 cells respectively, which could be reversed significantly by transduction with lentivirus-DPP8 and lentivirus-DPP9. CONCLUSION: These data obtained provide evidence that the DPP8 and DPP9 could be potential therapeutic targets against TIF.

Decrease of the pro-inflammatory M1-like response by inhibition of dipeptidyl peptidases 8/9 in THP-1 macrophages - quantitative proteomics of the proteome and secretome.

BACKGROUND: Cellular peptidases are an emerging target of novel pharmacological strategies in inflammatory diseases and cancer. In this context, the dipeptidyl peptidases 8 and 9 (DPP8/9) have gained special attention due to their activities in the immune cells. However, in spite of more than hundred protein substrates identified to date by mass spectrometry-based analysis, the cellular DPP8/9 functions are still elusive. METHODS: We applied the proteomic approach (iTRAQ-2DLC-MS/MS) to comprehensively analyze the role of DPP8/9 in the regulation of macrophage activation by in-depth protein quantitation of THP-1 proteome and secretome. RESULTS: Cells pre-incubated with DPP8/9 inhibitor (1G244) prior activation (LPS or IL-4/IL-13) diminished the expression levels of M1-like response markers, but not M2-like phenotype features. This was accompanied by multiple intra- and extra-cellular protein abundance changes in THP-1 cells, related to cellular metabolism, mitochondria and endoplasmic reticulum function, as well as those engaged with inflammatory and apoptotic processes, including previously reported and novel DPP8/9 targets. CONCLUSIONS: Inhibition of DPP 8/9 had a profound effect on the THP-1 macrophage proteome and secretome, evidencing the decrease of the pro-inflammatory M1-like response. Presented results are to our best knowledge the first which, among others, highlight the metabolic effects of DPP8/9 inhibition in macrophages.

DPP8/9 inhibitors activate the CARD8 inflammasome in resting lymphocytes.

Canonical inflammasomes are innate immune signaling platforms that are formed in response to intracellular pathogen-associated signals and trigger caspase-1-dependent pyroptosis. Inflammasome formation and signaling is thought to mainly occur in myeloid cells, and in particular monocytes and macrophages. Here we show that small molecule inhibitors of dipeptidyl peptidases 8 and 9 (DPP8/9), which activate the related CARD8 and NLRP1 inflammasomes, also activate pyroptosis in human and rodent resting lymphocytes. We found that both CD4+ and CD8+ T cells were particularly sensitive to these inhibitors, although the sensitivity of T cells, like macrophages, varied considerably between species. In human T cells, we show that CARD8 mediates DPP8/9 inhibitor-induced pyroptosis. Intriguingly, although activated human T cells express the key proteins known to be required for CARD8-mediated pyroptosis, these cells were completely resistant to DPP8/9 inhibitors. Overall, these data show that resting lymphoid cells can activate at least one inflammasome, revealing additional cell types and states poised to undergo rapid pyroptotic cell death in response to danger-associated signals.

DPP8 is a novel therapeutic target for multiple myeloma.

Dipeptidyl peptidases (DPPs) are proteolytic enzymes that are ideal therapeutic targets in human diseases. Indeed, DPP4 inhibitors are widely used in clinical practice as anti-diabetic agents. In this paper, we show that DPP4 inhibitors also induced cell death in multiple human myeloma cells. Among five DPP4 inhibitors, only two of them, vildagliptin and saxagliptin, exhibited apparent cytotoxic effects on myeloma cell lines, without any difference in suppression of DPP4 activity. As these two DPP4 inhibitors are known to have off-target effects against DPP8/9, we employed the specific DPP8/9 inhibitor 1G244. 1G244 demonstrated anti-myeloma effects on several cell lines and CD138+ cells from patients as well as in murine xenograft model. Through siRNA silencing approach, we further confirmed that DPP8 but not DPP9 is a key molecule in inducing cell death induced by DPP8/9 inhibition. In fact, the expression of DPP8 in CD38+ cells from myeloma patients was higher than that of healthy volunteers. DPP8/9 inhibition induced apoptosis, as evidenced by activated form of PARP, caspases-3 and was suppressed by the pan-caspase inhibitor Z-VAD-FMK. Taken together, these results indicate that DPP8 is a novel therapeutic target for myeloma treatment.

Dipeptidase-1 Is an Adhesion Receptor for Neutrophil Recruitment in Lungs and Liver.

A hallmark feature of inflammation is the orchestrated recruitment of neutrophils from the bloodstream into inflamed tissue. Although selectins and integrins mediate recruitment in many tissues, they have a minimal role in the lungs and liver. Exploiting an unbiased in vivo functional screen, we identified a lung and liver homing peptide that functionally abrogates neutrophil recruitment to these organs. Using biochemical, genetic, and confocal intravital imaging approaches, we identified dipeptidase-1 (DPEP1) as the target and established its role as a physical adhesion receptor for neutrophil sequestration independent of its enzymatic activity. Importantly, genetic ablation or functional peptide blocking of DPEP1 significantly reduced neutrophil recruitment to the lungs and liver and provided improved survival in models of endotoxemia. Our data establish DPEP1 as a major adhesion receptor on the lung and liver endothelium and identify a therapeutic target for neutrophil-driven inflammatory diseases of the lungs.

Inhibition of Dpp8/9 Activates the Nlrp1b Inflammasome.

Val-boroPro (PT-100, Talabostat) induces powerful anti-tumor immune responses in syngeneic cancer models, but its mechanism of action has not yet been established. Val-boroPro is a non-selective inhibitor of post-proline-cleaving serine proteases, and the inhibition of the highly related cytosolic serine proteases Dpp8 and Dpp9 (Dpp8/9) by Val-boroPro was recently demonstrated to trigger an immunostimulatory form of programmed cell death known as pyroptosis selectively in monocytes and macrophages. Here we show that Dpp8/9 inhibition activates the inflammasome sensor protein Nlrp1b, which in turn activates pro-caspase-1 to mediate pyroptosis. This work reveals a previously unrecognized mechanism for activating an innate immune pattern recognition receptor and suggests that Dpp8/9 serve as an intracellular checkpoint to restrain Nlrp1b and the innate immune system.

Allosteric inhibition of carnosinase (CN1) by inducing a conformational shift.

In humans, low serum carnosinase (CN1) activity protects patients with type 2 diabetes from diabetic nephropathy. We now characterized the interaction of thiol-containing compounds with CN1 cysteine residue at position 102, which is important for CN1 activity. Reduced glutathione (GSH), N-acetylcysteine and cysteine (3.2 ± 0.4, 2.0 ± 0.3, 1.6 ± 0.2 µmol/mg/h/mM; p < .05) lowered dose-dependently recombinant CN1 (rCN1) efficiency (5.2 ± 0.2 µmol/mg/h/mM) and normalized increased CN1 activity renal tissue samples of diabetic mice. Inhibition was allosteric. Substitution of rCN1 cysteine residues at position 102 (Mut1C102S) and 229 (Mut2C229S) revealed that only cysteine-102 is influenced by cysteinylation. Molecular dynamic simulation confirmed a conformational rearrangement of negatively charged residues surrounding the zinc ions causing a partial shift of the carnosine ammonium head and resulting in a less effective pose of the substrate within the catalytic cavity and decreased activity. Cysteine-compounds influence the dynamic behaviour of CN1 and therefore present a promising option for the treatment of diabetes.

DPP8 and DPP9 inhibition induces pro-caspase-1-dependent monocyte and macrophage pyroptosis.

Val-boroPro (Talabostat, PT-100), a nonselective inhibitor of post-proline cleaving serine proteases, stimulates mammalian immune systems through an unknown mechanism of action. Despite this lack of mechanistic understanding, Val-boroPro has attracted substantial interest as a potential anticancer agent, reaching phase 3 trials in humans. Here we show that Val-boroPro stimulates the immune system by triggering a proinflammatory form of cell death in monocytes and macrophages known as pyroptosis. We demonstrate that the inhibition of two serine proteases, DPP8 and DPP9, activates the pro-protein form of caspase-1 independent of the inflammasome adaptor ASC. Activated pro-caspase-1 does not efficiently process itself or IL-1ß but does cleave and activate gasdermin D to induce pyroptosis. Mice lacking caspase-1 do not show immune stimulation after treatment with Val-boroPro. Our data identify what is to our knowledge the first small molecule that induces pyroptosis and reveals a new checkpoint that controls the activation of the innate immune system.

Dehydropeptidase 1 promotes metastasis through regulation of E-cadherin expression in colon cancer.

Dehydropeptidase 1 (DPEP1) is a zinc-dependent metalloproteinase that is expressed aberrantly in several cancers. The role of DPEP1 in cancer remain controversial. In this study, we demonstrate that DPEP1 functions as a positive regulator for colon cancer cell metastasis. The expression of DPEP1 mRNA and proteins were upregulated in colon cancer tissues compared to normal mucosa. Gain-of-function and loss-of-function approaches were used to examine the malignant phenotype of DPEP1-expressing or DPEP1-depleted cells. DPEP1 expression caused a significant increase in colon cancer cell adhesion and invasion in vitro, and metastasis in vivo. In contrast, DPEP1 depletion induced opposite effects. Furthermore, cilastatin, a DPEP1 inhibitor, suppressed the invasion and metastasis of DPEP1-expressing cells. DPEP1 inhibited the leukotriene D4 signaling pathway and increased the expression of E-cadherin. We also show that DPEP1 mediates TGF-ß-induced EMT. TGF-ß transcriptionally repressed DPEP1 expression. TGF-ß treatment decreased E-cadherin expression and promoted cell invasion in DPEP1-expressing colon cancer cell lines, whereas it did not affect these parameters in DPEP1-depleted cell lines. These results suggest that DPEP1 promotes cancer metastasis by regulating E-cadherin plasticity and that it might be a potential therapeutic target for preventing the progression of colon cancer.

In vitro characterization of the bioconversion of pomaglumetad methionil, a novel metabotropic glutamate 2/3 receptor agonist peptide prodrug.

To characterize the hydrolysis of the peptide prodrug pomaglumetad methionil (LY2140023; (1R,4S,5S,6S)-4-(L-methionylamino)-2-thiabicyclo[3.1.0]hexane-4,6-dicarboxylic acid 2,2-dioxide), to the active drug LY404039 [(1R,4S,5S,6S)-4-amino-2-thiabicyclo[3.1.0]hexane-4,6-dicarboxylic acid 2,2-dioxide], a series of in vitro studies were performed in various matrices, including human intestinal, liver, kidney homogenate, and human plasma. The studies were performed to determine the tissue(s) and enzyme(s) responsible for the conversion of the prodrug to the active molecule. This could enable an assessment of the risk for drug interactions, an evaluation of pharmacogenomic implications, as well as the development of a Physiologically Based Pharmacokinetic (PBPK) model for formation of the active drug. Of the matrices examined, hydrolysis of pomaglumetad methionil was observed in intestinal and kidney homogenate preparations and plasma, but not in liver homogenate. Clearance values calculated after applying standard scaling factors suggest the intestine and kidney as primary sites of hydrolysis. Studies with peptidase inhibitors were performed in an attempt to identify the enzyme(s) catalyzing the conversion. Near complete inhibition of LY404039 formation was observed in intestinal and kidney homogenate and human plasma with the selective dehydropeptidase1 (DPEP1) inhibitor cilastatin. Human recombinant DPEP1 was expressed and shown to catalyze the hydrolysis, which was completely inhibited by cilastatin. These studies demonstrate pomaglumetad methionil can be converted to LY404039 via one or multiple enzymes completely inhibited by cilastatin, likely DPEP1, in plasma, the intestine, and the kidney, with the plasma and kidney involved in the clearance of the circulating prodrug. These experiments define a strategy for the characterization of enzymes responsible for the metabolism of other peptide-like compounds.

The SUMO1-E67 interacting loop peptide is an allosteric inhibitor of the dipeptidyl peptidases 8 and 9.

The intracellular peptidases dipeptidyl peptidase (DPP) 8 and DPP9 are involved in multiple cellular pathways including antigen maturation, cellular homeostasis, energy metabolism, and cell viability. Previously we showed that the small ubiquitin-like protein modifier SUMO1 interacts with an armlike structure in DPP9, leading to allosteric activation of the peptidase. Here we demonstrate that the E67-interacting loop (EIL) peptide, which corresponds to the interaction surface of SUMO1 with DPP9, acts as a noncompetitive inhibitor of DPP9. Moreover, by analyzing the sensitivity of DPP9 arm mutants to the EIL peptide, we mapped specific residues in the arm that are important for inhibition by the EIL, suggesting that the peptide acts as an allosteric inhibitor of DPP9. By modifying the EIL peptide, we constructed peptide variants with more than a 1,000-fold selectivity toward DPP8 (147 nM) and DPP9 (170 nM) over DPPIV (200 µM). Furthermore, application of these peptides to cells leads to a clear inhibition of cellular prolyl peptidase activity. Importantly, in line with previous publications, inhibition of DPP9 with these novel allosteric peptide inhibitors leads to an increase in EGF-mediated phosphorylation of Akt. This work highlights the potential use of peptides that mimic interaction surfaces for modulating enzyme activity.

Ferrocenyl bioconjugates of ampicillin and 6-aminopenicillinic acid--synthesis, electrochemistry and biological activity.

We report on the synthesis of ferrocenyl-ampicillin and ferrocenyl-6-aminopenicillinic acid bioconjugates. Title compounds were characterized by (1)H NMR, IR, MS and elemental analysis. These novel ferrocenyl-antibiotic conjugates were also investigated by cyclic voltammetry (CV). Ferrocenyl-ampicillin complexes revealed reversible uncomplicated oxidation whereas ferrocenyl-6-aminopenicillinic acid derivatives were found to exhibit adsorption waves in cathodic scans. Antibacterial activities of our ferrocenyl-antibiotic conjugates against Gram-positive methicillin-sensitive Staphylococcus aureus (MSSA), methicillin-resistant S. aureus (MRSA), vancomycin-resistant S. aureus (VRSA) and Staphylococcus epidermidis bacterial strains were determined. Our experiments show significant antibacterial activity of ferrocenyl-6-aminopenicillinic acid bioconjugates against the bacterial strains tested. Contrary to that ferrocenyl-ampicillin derivatives were inactive. The inhibitory effects on the dd-carboxypeptidase 64-575 II exerted by our ferrocenyl-6-aminopenicillinic acid bioconjugates were established in the low nanomolar range. The tumor cell growth inhibition of representative ferrocenyl-ampicillin and ferrocenyl-6-aminopenicillinic acid bioconjugates against MCF-7 breast adenocarcinoma and HT-29 colon carcinoma cell lines were studied in vitro. Similar to the antibacterial activity tests the assays in tumor cells revealed significant antiproliferative effects exerted by ferrocenyl-6-aminopenicillinic acid bioconjugates.

Cilastatin protects against cisplatin-induced nephrotoxicity without compromising its anticancer efficiency in rats.

Cisplatin is an anticancer agent marred by nephrotoxicity; however, limiting this adverse effect may allow the use of higher doses to improve its efficacy. Cilastatin, a small molecule inhibitor of renal dehydropeptidase I, prevents proximal tubular cells from undergoing cisplatin-induced apoptosis in vitro. Here, we explored the in vivo relevance of these findings and the specificity of protection for kidney cells in cisplatin-treated rats. Cisplatin increased serum blood urea nitrogen and creatinine levels, and the fractional excretion of sodium. Cisplatin decreased the glomerular filtration rate, promoted histological renal injury and the expression of many pro-apoptotic proteins in the renal cortex, increased the Bax/Bcl2 ratio, and oxidative stress in kidney tissue and urine. All these features were decreased by cilastatin, which preserved renal function but did not modify the pharmacokinetics of cisplatin area under the curve. The cisplatin-induced death of cervical, colon, breast, and bladder-derived cancer cell lines was not prevented by cilastatin. Thus, cilastatin has the potential to prevent cisplatin nephrotoxicity without compromising its anticancer efficacy.

An example of designed multiple ligands spanning protein classes: dual MCH-1R antagonists/DPPIV inhibitors.

A ligand-based approach to identify potential starting points for a dual MCH-1R antagonist/DPPIV inhibitor medicinal chemistry program was undertaken. Potential ligand pairs were identified by analysis of MCH-1R and DPPIV in vitro data. A highly targeted synthetic effort lead to the discovery of pyridone 11, a dual MCH-1R antagonist/DPPIV inhibitor with selectivity over DPP8 and DPP9.

A novel role of dipeptidyl peptidase 9 in epidermal growth factor signaling.

Dipeptidyl peptidase IV (DPP4), DPP8, DPP9, and fibroblast activation protein (FAP), the four proteases of the DPP4 gene family, have unique peptidase and extra-enzymatic activities that have been implicated in various diseases including cancers. We report here a novel role of DPP9 in regulating cell survival and proliferation through modulating molecular signaling cascades. Akt (protein kinase B) activation was significantly inhibited by human DPP9 overexpression in human hepatoma cells (HepG2 and Huh7) and human embryonic kidney cells (HEK293T), whereas extracellular signal-regulated kinases (ERK1/2) activity was unaffected, revealing a pathway-specific effect. Interestingly, the inhibitory effect of DPP9 on Akt pathway activation was growth factor dependent. DPP9 overexpression caused apoptosis and significantly less epidermal growth factor (EGF)-mediated Akt activation in HepG2 cells. However, such inhibitory effect was not observed in cells stimulated with other growth factors, including connective tissue growth factor, hepatic growth factor, insulin or platelet-derived growth factor-BB. The effect of DPP9 on Akt did not occur when DPP9 enzyme activity was ablated by either mutagenesis or inhibition. The phosphatidylinositol 3-kinase (PI3K)/Akt pathway is a major downstream effector of Ras. We found that DPP9 and DPP8, but not DPP4 or FAP, associate with H-Ras, a key signal molecule of the EGF receptor signaling pathway. These findings suggest an important signaling role of DPP9 in the regulation of survival and proliferation pathways.

Prolidase-dependent regulation of TGF ß (corrected) and TGF ß receptor expressions in human skin fibroblasts.

Transforming growth factor beta 1 (TGF ß1) is a protein that in most cells control proliferation and differentiation. One of the best characterized functions of TGF ß1 is stimulation of collagen biosynthesis that may lead to tissue fibrosis. Several reports suggest that prolidase, through regulation of expression of growth factors and transcription factors, e.g. vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1 α) may be important in many physiologic and pathophysiologic processes like: wound healing, inflammation and angiogenesis. We found that inhibitors of prolidase activity (N-benzyloxycarbonyl-l-proline, Cbz-Pro and phosphoenolopyruvate, PEP) induced decrease in TGF ß1 and its receptor expressions. On the other hand, products of prolidase catalytic activity, proline (Pro) and hydroxyproline (HyPro) induced increase in the amount of TGF ß1 and TGF ß receptors. Simultaneously, inhibitors of prolidase induced down-regulation of expression of the phospho-AKT. An addition of Pro or HyPro to the cells induced increase in the expression of phospho-AKT. An important transcription factor involved in signal induced by TGF ß receptor is mammalian target of rapamycin (mTOR). We found that prolidase inhibitors induced decrease in the expression of phospho-mTOR, while Pro or HyPro counteracted the effect. Rapamycin (pharmacological inhibitor of mTOR) resulted in decrease in prolidase activity. The down-regulation of phospho-mTOR by rapamycin contributed to down-regulation of prolidase activity suggesting its important role in prolidase-dependent function. It seems, that products of prolidase activity, Pro or HyPro may act as an interface between mTOR and phospho-mTOR in regulation of numerous TGF ß receptor-dependent functions.

Substituted 4-carboxymethylpyroglutamic acid diamides as potent and selective inhibitors of fibroblast activation protein.

Fibroblast activation protein (FAP) belongs to the prolyl peptidase family. FAP inhibition is expected to become a new antitumor target. Most known FAP inhibitors often resemble the dipeptide cleavage products, with a boroproline at the P1 site; however, these inhibitors also inhibit DPP-IV, DPP-II, DPP8, and DPP9. Potent and selective FAP inhibitor is needed in evaluating that FAP as a therapeutic target. Therefore, it is important to develop selective FAP inhibitors for the use of target validation. To achieve this, optimization of the nonselective DPP-IV inhibitor 8 led to the discovery of a new class of substituted 4-carboxymethylpyroglutamic acid diamides as FAP inhibitors. SAR studies resulted in a number of FAP inhibitors having IC(50) of <100 nM with excellent selectivity over DPP-IV, DPP-II, DPP8, and DPP9 (IC(50) > 100 µM). Compounds 18a, 18b, and 19 are the only known potent and selective FAP inhibitors, which prompts us to further study the physiological role of FAP.

Cilastatin attenuates cisplatin-induced proximal tubular cell damage.

A major area in cancer therapy is the search for protective strategies against cisplatin-induced nephrotoxicity. We investigated the protective effect of cilastatin on cisplatin-induced injury to renal proximal tubular cells. Cilastatin is a specific inhibitor of renal dehydrodipeptidase I (DHP-I), which prevents hydrolysis of imipenem and its accumulation in the proximal tubule. Primary cultures of proximal cells were treated with cisplatin (1-30 microM) in the presence or absence of cilastatin (200 microg/ml). Apoptosis and mitochondrial injury were assessed by different techniques. Cisplatin uptake and DNA binding were measured by inductively coupled plasma spectrometry. HeLa cells were used to control the effect of cilastatin on the tumoricidal activity of cisplatin. Cisplatin increased cell death, apoptotic-like morphology, caspase activation, and mitochondrial injury in proximal tubular cells in a dose- and time-dependent way. Concomitant treatment with cilastatin reduced cisplatin-induced changes. Cilastatin also reduced the DNA-bound platinum but did not modify cisplatin-dependent up-regulation of death receptors (Fas) or ligands (tumor necrosis factor alpha, Fas ligand). In contrast, cilastatin did not show any effects on cisplatin-treated HeLa cells. Renal DHP-I was virtually absent in HeLa cells. Cilastatin attenuates cisplatin-induced cell death in proximal tubular cells without reducing the cytotoxic activity of cisplatin in tumor cells. Our findings suggest that the affinity of cilastatin for renal dipeptidase makes this effect specific for proximal tubular cells and may be related to a reduction in intracellular drug accumulation. Therefore, cilastatin administration might represent a novel strategy in the prevention of cisplatin-induced acute renal injury.

Betulinic acid inhibits the expression of hypoxia-inducible factor 1alpha and vascular endothelial growth factor in human endometrial adenocarcinoma cells.

Although betulinic acid (BA) is known to induce apoptosis and antiangiogenic response in tumor cells, the underlying mechanism of its action is unknown. Deregulation of tissue collagen metabolism is one of the consequences of neoplastic transformation. The final step of collagen degradation is mediated by prolidase [E.C.3.4.13.9] which may play a role in angiogenesis. The formation of new blood vessels is regulated by the hypoxia-inducible factor 1 (HIF-1). The expression of HIF-1 correlates with hypoxia-induced angiogenesis as a result of the induction of vascular endothelial cell growth factor (VEGF). Since BA evokes anticancer activity, its effect on collagen biosynthesis, HIF-1alpha and VEGF expressions, as well as prolidase activity and expression was studied in cultured endometrial adenocarcinoma (EA) cells. It was found that BA inhibits collagen biosynthesis in EA cells (5[(3)H] proline incorporation assay). It was accompanied by a parallel decrease in prolidase activity and expression and decrease in expressions of alpha(1) and alpha(2) integrins, HIF-1alpha, and VEGF (western immunoblot analysis) in cultured human EA cells. The data suggest that BA may have anti-angiogenic potential by inhibition of prolidase, HIF-1alpha and VEGF expressions, and inhibition of collagen biosynthesis.