Green, Black and Rooibos Tea Inhibit Prostaglandin E2 Formation in Human Monocytes by Inhibiting Expression of Enzymes in the Prostaglandin E2 Pathway.

The formation of prostaglandin E2 (PGE2) is associated with adverse inflammatory effects. However, long-term treatment with nonsteroidal anti-inflammatory drugs (NSAIDs) comes with risk of severe side effects. Therefore, alternative ways to inhibit PGE2 are warranted. We have investigated the effects of tea extracts and the polyphenols epigallocatechin gallate (EGCG) and quercetin on PGE2 formation, determined by immunoassay, and protein expression, determined by immunoblotting, of cytosolic phospholipase A2 (cPLA2), cyclooxygenase 2 (COX-2) and microsomal PGE synthase-1 (mPGES-1) in human monocytes. Green and black tea extracts, and with a lower potency, Rooibos tea extract, inhibited lipopolysaccharide (LPS) and calcium ionophore-induced PGE2 formation. In addition, all tea extracts inhibited the LPS-induced expression of mPGES-1, and the green and black tea extracts also inhibited, to a lesser extent, COX-2 expression. The tea extracts only marginally reduced cPLA2 expression and had no effect on COX-1 expression. EGCG, present in green and black tea, and quercetin, present in all three teas, also inhibited PGE2 formation and expression of mPGES-1, COX-2 and cPLA2. Cell-based and cell-free assays were also performed to evaluate direct effects on the enzymatic activity of COX and PGE synthases. Mainly, the cell-free assay demonstrated partial inhibition by the tea extracts and polyphenols. However, the inhibition required higher doses compared to the effects demonstrated on protein expression. In conclusion, green and black tea, and to a lesser extent Rooibos tea, are potent inhibitors of PGE2 formation in human monocytes, and mediate their effects by inhibiting the expression of the enzymes responsible for PGE2 formation, especially mPGES-1.

Cutibacterium acnes Induces the Expression of Immunosuppressive Genes in Macrophages and is Associated with an Increase of Regulatory T-Cells in Prostate Cancer.

Tumors and infectious agents both benefit from an immunosuppressive environment. Cutibacterium acnes (C. acnes) is a bacterium in the normal skin microbiota, which has the ability to survive intracellularly in macrophages and is significantly more common in prostate cancer tissue compared with normal prostate tissue. This study investigated if prostate cancer tissue culture positive for C. acnes has a higher infiltration of regulatory T-cells (Tregs) and if macrophages stimulated with C. acnes induced the expression of immunosuppressive genes that could be linked to an increase of Tregs in prostate cancer. Real-time PCR and enzyme-linked immunosorbent spot assay (ELISA) were used to examine the expression of immunosuppressive genes in human macrophages stimulated in vitro with C. acnes, and associations between the presence of C. acnes and infiltration of Tregs were investigated by statistically analyzing data generated in two previous studies. The in vitro results demonstrated that macrophages stimulated with C. acnes significantly increased their expression of PD-L1, CCL17, and CCL18 mRNA and protein (p <0.05). In the cohort, Tregs in tumor stroma and tumor epithelia were positively associated with the presence of C. acnes (P = 0.0004 and P = 0.046, respectively). Since the macrophages stimulated with C. acnes in vitro increased the expression of immunosuppressive genes, and prostate cancer patients with prostatic C. acnes infection had higher infiltration of Tregs than their noninfected counterparts, we suggest that C. acnes may contribute to an immunosuppressive tumor environment that is vital for prostate cancer progression. IMPORTANCE In an immune suppressive tumor microenvironment constituted by immunosuppressive cells and immunosuppressive mediators, tumors may improve their ability to give rise to a clinically relevant cancer. In the present study, we found that C. acnes might contribute to an immunosuppressive environment by recruiting Tregs and by increasing the expression of immunosuppressive mediators such as PD-L1, CCL17, and CCL18. We believe that our data add support to the hypothesis of a contributing role of C. acnes in prostate cancer development. If established that C. acnes stimulates prostate cancer progression it may open up avenues for targeted prostate cancer treatment.

Effect of macrophages on breast cancer cell proliferation, and on expression of hormone receptors, uPAR and HER-2.

Malignant tumors, including breast cancers, are frequently infiltrated with innate immune cells and tumor-associated macrophages (TAMs) represent the major inflammatory component in stroma of many tumors. In this study, we examined the immunoreactivity of the macrophage markers CD68 and CD163 as well as the hormone receptors estrogen receptor α (ERα), progesterone receptor (PR), estrogen receptor ß1 (ERß1), human epidermal growth factor receptor 2 (HER-2), matrix metalloproteinase 9 (MMP­9), urokinase-type plasminogen activator receptor (uPAR) and the proliferations marker Ki67 in 17 breast cancer biopsies. The quantitative score for CD68+ and CD163+ strongly indicate M2 phenotype dominance in the currently investigated biopsies. We found that an increasing level of macrophages was negatively associated with ERα or PR, whereas a positive association was observed for Ki-67 or uPAR. No significant association could be seen between the level of macrophage and HER-2, ERß1 or MMP-9 expression. Effect of conditioned media (CM) generated from cultured human M1 and M2 macrophage phenotypes were investigated on the proliferation and expression of selected markers in the T47D breast cancer cell line. We found that in contrast to the in vivo situation, in particularly the CM from M1 macrophages decreased the growth and Ki67 expression in T47D, and significantly increased ERß1 mRNA levels. Moreover, in accordance to the in vivo situation the CM from the macrophages decreased the expression of ERα protein as well as ERα or PR mRNA. In conclusion our results show that macrophages alone have the capability to decrease the tumor cell expression of ERα and PR in vitro. In the tumor environment in vivo macrophages also contribute to an increase in tumor cell expression of uPAR and Ki67, suggesting that macrophages are involved in impairing the prognosis for breast cancer patients.

Macrophages of M1 phenotype have properties that influence lung cancer cell progression.

Stromal macrophages of different phenotypes can contribute to the expression of proteins that affects metastasis such as urokinase-type plasminogen activator (uPA), its receptor uPAR, and plasminogen activator inhibitor-1 (PAI-1), but knowledge of how essential their contribution is in comparison to the cancer cells in small cell lung cancer (SCLC) and lung squamous cell carcinoma (SCC) is lacking. The expression of uPA, uPAR, and PAI-1 and of the matrix metalloproteinases (MMP)-2 and MMP-9 were studied in human macrophages of M1 and M2 phenotype and compared to a lung SCC (NCI-H520) and a SCLC (NCI-H69) cell line. Effects of treatment with conditioned media (CM) from M1 and M2 macrophages on the expression of these genes in H520 and H69 cells as well as effects on the cell growth were investigated. In addition, data on the stromal macrophages immunoreactivity of uPAR, MMP-2, and MMP-9 in a few SCC and SCLC biopsies was included. uPAR, MMP-2, and MMP-9 were confirmed in stromal cells including macrophages in the SCC and SCLC biopsies. In vitro, both macrophage phenotypes expressed considerably higher mRNA levels of uPA, uPAR, PAI-1, and MMP-9 compared to the cancer cell lines, and regarding uPAR, the highest level was found in the M1 macrophage phenotype. Furthermore, M1 CM treatment not only induced an upregulation of PAI-1 in both H520 and H69 cells but also inhibited cell growth in both cell lines, giving M1 macrophages both tumor-promoting and tumor-killing potential.

Conditioned media from human macrophages of M1 phenotype attenuate the cytotoxic effect of 5­fluorouracil on the HT­29 colon cancer cell line.

Resistance of tumor cells to chemotherapy, such as 5­fluorouracil (5­FU), is an obstacle for successful treatment of cancer. As a follow­up of a previous study we have investigated the effect of conditioned media (CM) from macrophages of M1 or M2 phenotypes on 5­FU cytotoxicity on the colon cancer cell lines HT­29 and CACO­2. HT­29 cells, but not CACO­2 cells, having been treated with a combination of M1 CM and 5­FU recovered their cell growth to a much larger extent compared to cells having been treated with 5­FU alone when further cultured for 7 days in fresh media. M1 CM treatment of HT­29, but not CACO­2 cells, induced cell cycle arrest in the G0/G1 and G2/M phases. 5­FU treatment induced accumulation of cells in S­phase in both HT­29 and CACO­2 cells. This accumulation of cells in S­phase was attenuated by combined M1 CM and 5­FU treatment in HT­29 cells, but not in CACO­2 cells. The mRNA expression of cell cycle regulatory proteins and 5­FU metabolic enzymes were analyzed in an attempt to find possible mechanisms for the M1 CM induced attenuation of 5­FU cytotoxicity in HT­29. Thymidylate synthetase (TS) and thymidine phosphorylase (TP) were found to be substantially downregulated and upregulated, respectively, in HT­29 cells treated with M1 CM, making them unlikely as mediators of reduced 5­FU cytotoxicity. Among cell cycle regulating proteins, p21 was induced in HT­29 cells, but not in CACO­2 cells, in response to M1 CM treatment. However, small interfering RNA (siRNA) knockdown of p21 had no effect on the M1 CM induced cell cycle arrest seen in HT­29 and neither did it change the growth recovery after combined treatment of HT­29 cells with M1 CM and 5­FU. In conclusion, treatment of HT­29 cells with M1 CM reduces the cytotoxic effect of 5­FU and this is mediated by a M1 CM induced cell cycle arrest in the G0/G1 and G2/M phases. So far, we lack an explanation why this action is absent in the CACO­2 cells. The current findings may be important for optimization of chemotherapy in colon cancer.

Conditioned media from macrophages of M1, but not M2 phenotype, inhibit the proliferation of the colon cancer cell lines HT-29 and CACO-2.

Solid tumors are infiltrated by stroma cells including macrophages and these cells can affect tumor growth, metastasis and angiogenesis. We have investigated the effects of conditioned media (CM) from different macrophages on the proliferation of the colon cancer cell lines HT-29 and CACO-2. CM from THP-1 macrophages and monocyte-derived human macrophages of the M1 phenotype, but not the M2 phenotype, inhibited proliferation of the tumor cells in a dose-dependent manner. Lipopolysaccaharide and interferon γ was used for differentiation of macrophages towards the M1 phenotype and CM were generated both during differentiation (M1DIFF) and after differentiation (M1). M1 and M1DIFF CM as well as THP-1 macrophage CM resulted in cell cycle arrest in HT-29 cells with a decrease of cells in S phase and an increase in G2/M phase. Treatment of HT-29 cells with M1DIFF, but not M1 or THP-1 macrophage CM, resulted in apoptosis of about 20% of the tumor cells and this was accompanied by lack of recovery of cell growth after removal of CM and subsequent culture in fresh media. A protein array was used to identify cytokines released from M1 and M2 macrophages. Among the cytokines released by M1 macrophages, tumor necrosis factor α and CXCL9 were tested by direct addition to HT-29 cells, but neither affected proliferation. Our results indicate that M1 macrophages inhibit colon cancer cell growth and have the potential of contributing to reducing tumor growth in vivo.

Ellagic acid inhibits lipopolysaccharide-induced expression of enzymes involved in the synthesis of prostaglandin E2 in human monocytes.

Ellagic acid, a natural polyphenol found in certain fruits, nuts and vegetables, has in recent years been the subject of intense research within the fields of cancer and inflammation. Pain, fever and swelling, all typical symptoms of inflammation, are ascribed to elevated levels of PGE2. In the present study, we have investigated the effects of ellagic acid on PGE2 release and on prostaglandin-synthesising enzymes in human monocytes. Ellagic acid was found to inhibit Ca ionophore A23187-, phorbol myristate acetate- and opsonised zymosan-induced release of PGE2 from monocytes pre-treated with the inflammatory agent lipopolysaccharide. Ellagic acid suppressed the lipopolysaccharide-induced increase in protein expression of cyclo-oxygenase-2 (COX-2), microsomal PGE synthase-1 (mPGEs-1) and cytosolic phospholipase A2alpha (cPLA2alpha), while it had no effect on the constitutively expressed COX-1 protein. Ellagic acid had no apparent inhibitory effect on these enzymes when the activities were determined in cell-free assays. We conclude that the inhibitory effect of ellagic acid on PGE2 release from monocytes is due to a suppressed expression of COX-2, mPGEs-1 and cPLA2alpha, rather than a direct effect on the activities of these enzymes.