Mogens Winkel Madsen

Protein Expression of TNF-α in Psoriatic Skin Is Regulated at a Posttranscriptional Level by MAPK-Activated Protein Kinase 2

Alterations in specific signal transduction pathways may explain the increased expression of proinflammatory cytokines seen in inflammatory diseases such as psoriasis. We reveal increased TNF-α protein expression, but similar TNF-α mRNA levels, in lesional compared with nonlesional psoriatic skin, demonstrating for the first time that TNF-α expression in lesional psoriatic skin is regulated posttranscriptionally. Increased levels of activated MAPK-activated protein kinase 2 (MK2) together with increased MK2 kinase activity were found in lesional compared with nonlesional psoriatic skin. Immunohistochemical analysis showed that activated MK2 was located in the basal layers of the psoriatic epidermis, whereas no positive staining was seen in nonlesional psoriatic skin. In vitro experiments demonstrated that both anisomycin and IL-1β caused a significant activation of p38 MAPK and MK2 in cultured normal human keratinocytes. In addition, TNF-α protein levels were significantly up-regulated in keratinocytes stimulated with anisomycin or IL-1β. This increase in TNF-α protein expression was completely blocked by the p38 inhibitor, SB202190. Transfection of cultured keratinocytes with MK2-specific small interfering RNA led to a significant decrease in MK2 expression and a subsequent significant reduction in the protein expression of the proinflammatory cytokines TNF-α, IL-6, and IL-8, whereas no change in the expression of the anti-inflammatory cytokine IL-10 was seen. This is the first time that MK2 expression and activity have been investigated in an inflammatory disease such as psoriasis. The results strongly suggest that increased activation of MK2 is responsible for the elevated and posttranscriptionally regulated TNF-α protein expression in psoriatic skin, making MK2 a potential target in the treatment of psoriasis.

Anticancer agent CHS 828 suppresses nuclear factor-κB activity in cancer cells through downregulation of IKK activity

CHS 828, a pyridyl cyanoguanidine, has been shown to exert a significant antitumor effect in preclinical tests in vitro and in vivo, and CHS 828 is in phase I/II clinical trials. We have investigated the effect of CHS 828 on the nuclear factor‐κB (NF‐κB) because of its well‐known role in the control of cell division and apoptosis. CHS 828 is able to inhibit the lipopolysaccharide (LPS)‐induced nuclear localization as well as the transcriptional activity of NF‐κB in human THP‐1 leukemia cells. Moreover, CHS 828 has also been shown to inhibit the LPS‐induced degradation of the IκBα and IκBβ in THP‐1 cells, leading us to identify the IκB kinase complex as a molecular target of CHS 828. The IKK activity is inhibited by CHS 828 with an IC50 of 8 nM. The inhibition of the IKK activity by different CHS 828 analogues correlates well with the inhibition of NYH small cell lung cancer cell proliferation in vitro and in vivo. Moreover, the inhibition of NF‐κB transcriptional activity in different cancer cell lines by CHS 828 correlates to some extent with the reduction by CHS 828 of the size of the corresponding xenografts. Activation of NF‐κB has been shown to induce expression of antiapoptotic proteins, and cancer cells have been shown to have high levels of constitutively active NF‐κB. Therefore, we hypothesize that the anticancer activity of CHS 828 is due to inhibition of the IKK activity by which the antiapoptotic protection of NF‐κB is removed, leading to the promotion of apoptosis.

Resistance of human breast-cancer cells to the pure steroidal anti-estrogen ICI 182,780 is not associated with a general loss of estrogen-receptor expression or lack of estrogen responsiveness

To elucidate the mechanisms responsible for the development of anti‐estrogen resistance, we have cloned and established 3 stable ICI‐182,780‐resistant sub‐lines, MCF‐7/182R‐1, MCF‐7/182R‐6 and MCF‐7/182R‐7 from the estrogen‐receptor(ER)‐positive and estrogen‐responsive human breast‐cancer MCF‐7 cell line by long‐term treatment with 10−7 M ICI 182,780. The ICI‐182,780‐resistant MCF‐7 sub‐lines express ER, but compared with MCF‐7 cells the level is significantly lower in all 3 sub‐lines. In the MCF‐7 cell line we find that ER expression is regulated by estrogen and anti‐estrogens at the transcriptional and post‐transcriptional level. This is in contrast to the ICI‐182,780‐resistant sub‐lines, in which we find very little hormonal effects on the ER mRNA expression level. The resistant sub‐lines also deviate from parent characteristics by the complete lack of expression of progesterone receptor even when grown in the presence of estradiol. All 3 resistant sub‐lines have a lower basal expression of cathepsin‐D mRNA comparable with the lower ER expression, but, in contrast, they have higher basal expression of the pS2 mRNA than the parent MCF‐7 cell line. Although there are different basal expression levels of the pS2 and cathepsin‐D genes, the resistant sub‐lines behave like the parent MCF‐7 cell line with respect to the hormonal regulation of both genes. The estrogen receptors in the resistant sub‐lines have also maintained wild‐type characteristics with respect to estrogen and anti‐estrogen regulation of the estrogen‐regulated proteins procathepsin D, α1‐anti‐trypsin and a 42‐kDa protein. The resistant cells require estrogen for growth in athymic nude mice. Our results clearly demonstrate that the ER in the resistant sub‐lines have a normal function for most parameters investigated, supporting our earlier observation that only wild‐type ER protein is expressed in these cells. The few observed differences in ER function between the parent MCF‐7 cell line and the resistant sub‐lines are not likely to be responsible for the ICI‐182,780‐resistant phenotype. Int. J. Cancer 72:1129–1136, 1997.

MCF‐7/VDR: A new vitamin D resistant cell line

Several in vitro and in vivo experiments have demonstrated potent cell regulatory effects of vitamin D compounds in cancer cells. Moreover, a promising phase I study with the vitamin D analogue Seocalcitol (EB 1089) in patients with advanced breast and colon cancer has already been carried out and more clinical trials evaluating the clinical effectiveness of EB 1089 in other cancer types are in progress (Mørk Hansen et al. [ 2000a ]). However, only little is known about the mechanisms underlying the actions of vitamin D or about the possible development of drug resistance in the patients. Therefore, in an attempt to gain more insight into these aspects, we have developed the MCF‐7/VDR cell line, a stable subclone of the human MCF‐7 breast cancer cell line, which is resistant to the growth inhibitory and apoptosis inducing effects of 1α,25(OH)2D3. Despite this characteristic, receptor studies on the VDR have clearly demonstrated that the MCF‐7/VDR cells contain fully functional VDRs, although in a lower number than seen with the parental MCF‐7 cells. The regulation of the 24‐hydroxylase enzyme appeared to be intact in the MCF‐7/VDR cells and no differences with regard to growth rate and morphological appearance between the MCF‐7/VDR cells and the parental MCF‐7 cells were observed. Interestingly, however, the sensitivity of the MCF‐7/VDR cells to the pure anti‐estrogen ICI 182,780 was found to be increased. The MCF‐7/VDR cell line shows characteristics different from those of previously described vitamin D resistant breast cancer cell lines but also some similarities. Together such vitamin D resistant cell lines therefore serve as a useful tool for studying the exact mechanism of action of vitamin D and the development of vitamin D resistance. J. Cell. Biochem. 82:422–436, 2001.

Sensitivity to growth suppression by 1α,25-dihydroxyvitamin D3 among MCF-7 clones correlates with Vitamin D receptor protein induction

The antiproliferative effect of 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) has been studied for a decade in diverse model systems, but the signalling pathways linking 1alpha,25(OH)(2)D(3) to cell cycle arrest remains unclear. In our attempt to establish a model system which would allow further identification of important players in the process of the 1alpha,25(OH)(2)D(3) imposed cell cycle arrest, we have isolated derivatives of the human breast cancer cell line MCF-7 and chosen two nearly 1alpha,25(OH)(2)D(3) resistant and two hypersensitive sub-clones. Investigation of cell cycle proteins regulated by 1alpha,25(OH)(2)D(3) in these clones indicates that activation of one component/pathway is responsible for the linkage between 1alpha,25(OH)(2)D(3) and growth arrest. Protein levels of the Vitamin D receptor (VDR) were elevated in sensitive cells upon 1alpha,25(OH)(2)D(3) treatment, whereas resistant clones were unable to induce VDR upon 1alpha,25(OH)(2)D(3) treatment. Our data show that VDR protein levels and the ability of a cell to induce VDR upon 1alpha,25(OH)(2)D(3) treatment correlate with the antiproliferative effects of 1alpha,25(OH)(2)D(3), and suggest that the level of VDR in cancer cells might serve as a prognostic marker for treatment of cancer with 1alpha,25(OH)(2)D(3) analogues.

Down-regulation of Laminin-Binding Integrins by 1α,25=Dihydroxyvitamin D3 in Human Melanoma Cells in Vitro

In the present investigation the effect of 1 alpha,25(OH)2D3 on the expression of the integrin laminin receptor on the melanoma cell line SK-MEL-28 has been examined. The SK-MEL-28 cells were shown to contain high-affinity receptors for 1 alpha,25(OH)2D3 and cell proliferation was found to be inhibited in a dose-dependent manner in response to the hormone. Using monoclonal antibodies against the alpha 6-sub-unit of the integrin laminin receptor, immunocytochemistry demonstrated that exposure of cells to 1 alpha,25(OH)2D3 for 5 days caused a reduced staining intensity. This observation was further confirmed by dot blot analysis, where a dose-dependent decline of alpha 6 expression was obtained after treatment of the cells with 1 alpha,25(OH)2D3 for 6 days. FACS-analysis was performed in order to quantify this decline, and it was found that the level of alpha 6-subunits on the cell surface was reduced by more than 40%. Additional investigations including Northern blot analyses of poly(A)+RNA extracts also showed a dose-dependent reduction of alpha 6 mRNA. Interestingly, the decrease of alpha 6 expression on the surface of SK-MEL-28 melanoma cells was accompanied by a reduced ability of the cells to adhere to an artificial basement membrane. In conclusion, the present investigation shows that besides having an antiproliferative effect on the SK-MEL-28 melanoma cells, 1 alpha,25(OH)2D3 is also able to inhibit the surface expression of the alpha 6-subunit of the integrin laminin receptor. Moreover, the results strongly indicate that 1 alpha,25(OH)2D3 exerts its regulatory effect on the alpha 6-subunit at the transcriptional level rather than at the protein level.

Abstract CT154: Phase I dose-escalating PoC study to evaluate the safety and tolerability of LiPlaCis (liposomal cisplatin formulation) in patients with advanced or refractory tumors

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA LiPlaCis is a novel liposomal formulation of cisplatin, designed to be specifically degraded by secretory phospholipase A2 (sPLA2) which is over-expressed in tumor tissue. sPLA2 has been shown to be present in a number of different tumor tissues - e.g. prostate, lung, ovarian, breast etc. ( T. Abe 1997 ). Thus, LiPlaCis is intended to improve the therapeutic index due to an improved therapeutic efficacy and possibly also an improved safety and tolerability profile. Objectives: The primary objectives were safety and determination of MTD. Secondary objectives were evaluation of PK, clinical activity, and PD. This included two PoC cohorts to study platinum-DNA adducts in tumor compared to normal tissue following administration of LiPlaCis. Methods: A standard 3+3 design was used to include patients with advanced solid tumors and PS 0-1. LiPlaCis was administered weekly on day 1, day 8 and possibly day 15 every 3 wks. CTCAE v4.03 and RECIST were used to assess safety and tumor activity. Paired tumor and normal tissue biopsies obtained pre-treatment and on day 2 were used to assess sPLA2-IIA protein levels and platinum-DNA adducts (by 32P-postlabeling assay) in two PoC cohorts of 60 and 90 mg LiPlaCis. Results: A total of 16 patients were included at dose levels of 60, 90 and 120 mg. At 120 mg two DLTs were observed and an intermediate dose level of 90 mg Day 1 and 8 and 45 mg Day 15 was explored. After 1 DLT, this dose level was halted and the PoC cohorts were initiated sequentially. The observed DLTs included renal toxicity and infusion reactions. Most frequent AEs of all grades were fatigue, hypomagnesemia and vomiting. The most common (>10%) grade 3-4 AEs were hypomagnesemia, hypokalemia and anemia. Clinical activity was observed in a patient with HN 60 mg (PR), SCC skin;120 mg (PR), BC; 60 mg (SD-ongoing), CRC; 90 mg (SD -18 wks) and gastric cancer; 90 mg (SD-21 wks). In the 60 mg PoC cohort of 3 pts the tumor- to normal tissue ratio of platinum-DNA adducts was from 5.7 to 8.3 fold. Conclusion: MTD has not yet been determined, but ongoing PoC cohorts indicate that LiPlaCis is preferably released in tumor by sPLA2 compared to normal tissue which potentially could improve the therapeutic index of cisplatin. Citation Format: Ulrik Lassen, Morten Mau-Sorensen, Ulla Hald Buhl, Mogens W. Madsen, Eva Balslev, Dick Pluim, Jan H. M. Schellens, Steen Knudsen, Peter B. Jensen. Phase I dose-escalating PoC study to evaluate the safety and tolerability of LiPlaCis (liposomal cisplatin formulation) in patients with advanced or refractory tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr CT154.