Curcumin Combined with FOLFOX Chemotherapy Is Safe and Tolerable in Patients with Metastatic Colorectal Cancer in a Randomized Phase IIa Trial.

BACKGROUND: Curcumin is the main active ingredient of the spice turmeric, investigated extensively for putative anticancer properties. OBJECTIVES: This phase IIa open-labelled randomized controlled trial aimed to assess safety, efficacy, quality of life, neurotoxicity, curcuminoids, and C-X-C-motif chemokine ligand 1 (CXCL1) in patients receiving folinic acid/5-fluorouracil/oxaliplatin chemotherapy (FOLFOX) compared with FOLFOX + 2 g oral curcumin/d (CUFOX). METHODS: Twenty-eight patients aged >18 y with a histological diagnosis of metastatic colorectal cancer were randomly assigned (1:2) to receive either FOLFOX or CUFOX. Safety was assessed by Common Toxicity Criteria-Adverse Event reporting, and efficacy via progression-free survival (PFS) and overall survival (OS). Quality of life and neurotoxicity were assessed using questionnaires (European Organization for Research and Treatment of Cancer Quality of Life Questionnaire-C30 and Functional Assessment of Cancer Treatment-Gynecologic Oncology Group-Neurotoxicity). Plasma curcuminoids were determined with liquid chromatography (LC) electrospray ionization tandem mass spectrometry and CXCL1 by ELISA. RESULTS: Addition of daily oral curcumin to FOLFOX chemotherapy was safe and tolerable (primary outcome). Similar adverse event profiles were observed for both arms. In the intention-to-treat population, the HR for PFS was 0.57 (95% CI: 0.24, 1.36; P = 0.2) (median of 171 and 291 d for FOLFOX and CUFOX, respectively) and for OS was 0.34 (95% CI: 0.14, 0.82; P = 0.02) (median of 200 and 502 d for FOLFOX and CUFOX, respectively). There was no significant difference between arms for quality of life (P = 0.248) or neurotoxicity (P = 0.223). Curcumin glucuronide was detectable at concentrations >1.00 pmol/mL in 15 of 18 patients receiving CUFOX. Curcumin did not significantly alter CXCL1 over time (P = 0.712). CONCLUSION: Curcumin is a safe and tolerable adjunct to FOLFOX chemotherapy in patients with metastatic colorectal cancer. This trial was registered at clinicaltrials.gov as NCT01490996 and at www.clinicaltrialsregister.eu as EudraCT 2011-002289-19.

Increased sensitivity of Treg cells from patients with PBC to low dose IL-12 drives their differentiation into IFN-γ secreting cells.

IL-12 is a pro-inflammatory cytokine that induces the production of interferon-γ (IFNγ) and favours the differentiation of T helper 1 (Th1) cells. In the presence of IL-12 human Treg cells acquire a Th1-like phenotype with reduced suppressive activity in vitro. Primary biliary cholangitis (PBC) is an autoimmune cholestatic liver disease characterised by high Th1 and Th17 infiltrating cells, reduced frequencies of Treg cells, and a genetic association with IL-12 signalling. Herein, we sought to evaluate the IL-12 signalling pathway in PBC pathology, by studying human samples from patients with PBC, alongside those with primary Sjögren's syndrome (pSS)(autoimmune disease with IL-12 signalling gene association), primary sclerosing cholangitis (PSC) (cholestatic liver disease without IL-12 gene association) and healthy individuals. Our data revealed that TLR stimulation of PBC (n = 17) and pSS monocytes (n = 6) resulted in significant induction of IL12A mRNA (p < 0.05, p < 0.01, respectively) compared to PSC monocytes (n = 13) and at similar levels to HC monocytes (n = 8). PSC monocytes expressed significantly less IL-12p70 (108 pg/ml, mean) and IL-23 (358 pg/ml) compared to HC (458 pg/ml and 951 pg/ml, respectively) (p < 0.01, p < 0.05). Treg cells from patients with PBC (n = 16) and pSS (n = 3) but not PSC (n = 10) and HC (n = 8) responded to low dose (10 ng/ml) IL-12 stimulation by significant upregulation of IFNγ (mean 277 and 254 pg/ml, respectively) compared to PSC and HC Treg cells (mean 22 and 77 pg/ml, respectively)(p < 0.05). This effect was mediated by the rapid and strong phosphorylation of STAT4 on Treg cells from patients with PBC and pSS (p < 0.05) but not PSC and HC. In the liver of patients with PBC (n = 7) a significantly higher proportion of IL-12Rß2+Tregs (16% on average) was detected (p < 0.05) compared to other liver disease controls (5%)(n = 18) which also showed ex vivo high IFNG and TBET expression. CONCLUSION: Our data show an increased sensitivity of PBC and pSS Treg cells to low dose IL-12 stimulation, providing ongoing support for the importance of the IL12-IL-12Rß2-STAT4 pathway on Treg cells in disease pathogenesis and potentially treatment.

Low-level C-reactive protein levels exert cytoprotective actions on human podocytes.

BACKGROUND: Albuminuria and elevated C-reactive protein (CRP) levels are common manifestations of many inflammatory diseases. Cardiovascular-based drugs, with secondary anti-inflammatory actions, such as angiotensin-converting enzyme-inhibitors are able to reduce both proteinuria and CRP levels, raising the question of whether CRP directly influences the processes that result in proteinuria. As proteinuria is thought to be induced as a result of podocyte dysfunction, we investigated whether there is a pathomechanistic link with CRP. METHODS: Podocytes were analysed for evidence of endogenous CRP production in response to inflammatory agents. In addition, they were incubated in the presence of various concentrations of exogenous CRP and analysed for evidence of a response to treatment. RESULTS: Our results demonstrated that inflammatory agents such as macrophage-conditioned medium and interleukin-1ß induced the expression of CRP messenger RNA in podocytes. However, they were unable to induce CRP protein. Stimulation of podocytes with exogenous CRP demonstrated that 10 µg/mL CRP induced a low but significant level of interleukin-6 secretion. Tumour necrosis factor α, however, was not detected. CRP did up-regulate the expression of the slit diaphragm proteins nephrin and CD2AP, as well as the structural proteins ezrin and podocalyxin-like protein-1, proteins known to be involved in signalling via the phosphotidylinositol-3 (PI-3) kinase pathway. CRP exposure reduced caspase-3 enzyme activity and up-regulated the expression of the anti-apoptotic protein Bcl-2. In the presence of the PI-3 kinase inhibitor LY294002, the ability of CRP to suppress caspase-3 activity was significantly reduced. CONCLUSIONS: Taken together, these data suggest that rather than inducing podocyte damage, CRP may be a survival factor for podocytes by maintaining their structural integrity and initiating a survival cascade, which may facilitate podocyte recovery from injury.

Curcumin inhibits cancer stem cell phenotypes in ex vivo models of colorectal liver metastases, and is clinically safe and tolerable in combination with FOLFOX chemotherapy.

In vitro and pre-clinical studies have suggested that addition of the diet-derived agent curcumin may provide a suitable adjunct to enhance efficacy of chemotherapy in models of colorectal cancer. However, the majority of evidence for this currently derives from established cell lines. Here, we utilised patient-derived colorectal liver metastases (CRLM) to assess whether curcumin may provide added benefit over 5-fluorouracil (5-FU) and oxaliplatin (FOLFOX) in cancer stem cell (CSC) models. Combination of curcumin with FOLFOX chemotherapy was then assessed clinically in a phase I dose escalation study. Curcumin alone and in combination significantly reduced spheroid number in CRLM CSC models, and decreased the number of cells with high aldehyde dehydrogenase activity (ALDH(high)/CD133(-)). Addition of curcumin to oxaliplatin/5-FU enhanced anti-proliferative and pro-apoptotic effects in a proportion of patient-derived explants, whilst reducing expression of stem cell-associated markers ALDH and CD133. The phase I dose escalation study revealed curcumin to be a safe and tolerable adjunct to FOLFOX chemotherapy in patients with CRLM (n = 12) at doses up to 2 grams daily. Curcumin may provide added benefit in subsets of patients when administered with FOLFOX, and is a well-tolerated chemotherapy adjunct.

Perindoprilat modulates the activity of lipoprotein receptor-related protein in human mesangial cells.

Low density lipoprotein receptor-related protein (LRP) is a multifunctional endocytic receptor implicated in the modulation of a number of cellular processes, including the turnover of proteases and the degradation of extracellular matrix proteins. As such, it can play a key role in the control of fibrosis. The aim of this investigation was to ascertain whether the anti-fibrotic effects exerted by the angiotensin-converting enzyme inhibitor (ACE-I) perindoprilat on macrophage-conditioned medium (MPCM)-injured human mesangial cells can be modulated by this receptor. Addition of receptor-associated protein to MPCM-injured mesangial cells with and without ACE-I increased the amount of tissue plasminogen activator protein detected in mesangial cell culture supernatants without affecting the protein levels of plasminogen activator inhibitor-1. The ability of ACE-I to reduce fibronectin was diminished in the presence of receptor-associated protein. ACE-I induced an increase in mesangial cell MMP9 mRNA, but reduced the MMP9 enzyme activity detected in mesangial cell supernatants. Mesangial cell lysates from ACE-I-treated cells were able to bind immobilized fibronectin at higher dilutions than cell lysates from untreated cells. Flow cytometry showed that MPCM induced an increase in LRP surface expression in mesangial cells over that in control cells and that this expression was further increased by ACE-I treatment. The increase in LRP expression in response to ACE-I was also observed by Western blotting. Northern blot analysis of RNA extracted from cells following a 24-h exposure to MPCM with and without ACE-I demonstrated that there was no change in LRP mRNA expression upon ACE-I treatment. In conclusion, we show that ACE-I treatment is able to modulate mesangial cell-surface expression of LRP, providing an additional mechanism whereby ACE-Is can mediate anti-fibrotic actions independent of their hemodynamic actions.

Pharmacological enhancement of the kallikrein-kinin system promotes anti-fibrotic responses in human mesangial cells.

The aim of the present study was to investigate whether pharmacological enhancement of the renal kallikrein-kinin system using the vasopeptidase inhibitor omapatrilat plays a direct role in modulating the fibrotic responses of human mesangial cells to injury. Treatment with 40 micromol/L omapatrilat was able to reduce macrophage-conditioned medium (MPCM)-induced fibronectin levels without affecting mRNA expression. MPCM injury also suppressed kallikrein and low molecular weight kininogen mRNA. Omapatrilat was able to attenuate this suppression. Bradykinin levels in contrast were increased by MPCM and treatment with omapatrilat further augmented levels. Co-incubation with the bradykinin B2 receptor antagonist HOE 140 attenuated the omapatrilat-induced lowering of fibronectin. Moreover, inhibition of cGMP release had a similar effect. Paradoxically, RT-PCR and Southern blotting demonstrated that bradykinin B2 receptor mRNA levels were down regulated in response to omapatrilat. Western blotting supported this data. Supernatant levels of tissue plasminogen activator (tPA), a product of bradykinin stimulation, were decreased by omapatrilat while cell associated tPA levels were increased. Matrix metalloproteinase-9 (MMP-9) mRNA expression was up regulated by omapatrilat treatment, although no difference in active zymogen levels was observed. In conclusion enhancement of kallikrein-kinin system appears to play a direct role in promoting anti-fibrotic responses in MPCM-injured human mesangial cells.

The role of the alpha-1 adrenoceptor in modulating human mesangial cell matrix production.

BACKGROUND: The sympathetic nervous system is frequently activated in hypertension and may modify various aspects of renal function. Whether modulation of the sympathetic nervous system directly influences the development of renal fibrosis is yet to be established. The current study investigates the role of the alpha-1 adrenoceptor on human mesangial cell scarring. METHODS: Human mesangial cells were injured with macrophage-conditioned medium (MPCM) and treated with doxazosin for 1 or 3 days. RESULTS: alpha-1 Adrenoceptor antagonist doxazosin of 2 micromol/l reduced fibronectin protein in MPCM-injured female mesangial cells by 31 +/- 1.03% (P < 0.001) and by 9.5 +/- 0.3% (P = 0.01) in male mesangial cells. The differential response between sexes was significant (P = 0.004). alpha-1B Adrenoceptors were detected in human mesangial cells by reverse transcription-polymerase chain reaction with expression in female cells being 87% higher than in males (P = 0.04). Injury with MPCM reduced alpha-1B adrenoceptor mRNA expression in both cell types. Doxazosin had no effect on the protein levels of transforming growth factor-beta (TGF-beta) or interleukin-1beta (IL-1beta), however, a small reduction in tumour necrosis factor-alpha (TNF-alpha) levels was observed. Doxazosin had no effect on the modulators of matrix turnover matrix metalloproteinases MMP3, MMP9 and tissue inhibitor of matrix metalloproteinases (TIMP-1), although a significant reduction in tissue plasminogen activator (tPA); (36.5 +/- 2.6%, P < 0.001) was observed. Doxazosin caused an up-regulation of kallikrein expression, both at mRNA and protein levels. Co-treatment with the bradykinin B2 receptor antagonist HOE140 was able to attenuate the effects of doxazosin treatment on fibronectin levels. CONCLUSION: These data suggest that inhibition of alpha-1B adrenoceptors in mesangial cells exerts an anti-fibrotic effect in a sex-specific manner via modulation of the kallikrein-kinin/plasminogen activator system.

The role of bradykinin in the antifibrotic actions of perindoprilat on human mesangial cells.

BACKGROUND: Angiotensin-converting enzyme inhibitors (ACE-I) protect against the development of glomerulosclerosis using mechanisms partly dissociated from their systemic antihypertensive action. The aim of the current study was to delineate the mechanism of action underlying the antifibrotic effects of the ACE-I perindoprilat in the context of macrophage-mediated scarring in human mesangial cells. METHODS: Mesangial cells were treated with macrophage-conditioned medium (MPCM) in the presence or absence of the ACE-I perindoprilat. RESULTS: Forty micromol/L perindoprilat reduced MPCM-induced mesangial cell fibronectin levels by 19.4 +/- 0.6% (P < 0.001). Immunoprecipitation of 35S-methionine biosynthetically labeled fibronectin and Northern analysis suggested that the decrease in fibronectin levels was not caused by reduced synthesis. MPCM stimulated the production of matrix metalloproteinases (MMP) 2, 3, and 9 in mesangial cells; however, these were not significantly altered by ACE-I treatment, and neither was production of their tissue inhibitor of metalloproteinases (TIMP-1). Addition of exogenous bradykinin to MPCM-treated mesangial cells resulted in a 22.5 +/- 1.4% (P < 0.02) reduction in secreted fibronectin levels, while semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and Southern blotting demonstrated that bradykinin B2 receptor expression was up regulated by 71 +/- 30% in MPCM-stimulated mesangial cells in response to ACE-I treatment (P= 0.032). Moreover, the bradykinin B2 receptor antagonist HOE 140 attenuated the beneficial effects of perindoprilat. MPCM-stimulated mesangial cell protein expression levels of plasminogen activator system components tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) were altered after treatment with ACE-I. CONCLUSION: These results suggest that ACE-I-induced renoprotection, in the context of macrophage-stimulated mesangial cell scarring, is mediated, at least in part, via the actions of bradykinin.