Christina Körbel

Overcoming Intrinsic Multidrug Resistance in Melanoma by Blocking the Mitochondrial Respiratory Chain of Slow-Cycling JARID1Bhigh Cells

Despite success with BRAFV600E inhibitors, therapeutic responses in patients with metastatic melanoma are short-lived because of the acquisition of drug resistance. We identified a mechanism of intrinsic multidrug resistance based on the survival of a tumor cell subpopulation. Treatment with various drugs, including cisplatin and vemurafenib, uniformly leads to enrichment of slow-cycling, long-term tumor-maintaining melanoma cells expressing the H3K4-demethylase JARID1B/KDM5B/PLU-1. Proteome-profiling revealed an upregulation in enzymes of mitochondrial oxidative-ATP-synthesis (oxidative phosphorylation) in this subpopulation. Inhibition of mitochondrial respiration blocked the emergence of the JARID1B(high) subpopulation and sensitized melanoma cells to therapy, independent of their genotype. Our findings support a two-tiered approach combining anticancer agents that eliminate rapidly proliferating melanoma cells with inhibitors of the drug-resistant slow-cycling subpopulation.

High-Resolution Ultrasound Imaging: A Novel Technique for the Noninvasive in Vivo Analysis of Endometriotic Lesion and Cyst Formation in Small Animal Models

Endometriosis, the presence of endometrial tissue at ectopic sites, is a highly prevalent gynecological disease severely affecting a patients quality of life. To analyze the mechanisms involved in the disease and to identify new molecular targets for effective therapies, small animal models are an important approach. Herein, we report the first use of high-resolution ultrasound imaging for the in vivo analysis of intraperitoneal endometriotic lesions in mice. This noninvasive technology allows for the repetitive quantitative analysis of growth, cyst development, and adhesion formation of endometriotic lesions with a low intra- and interobserver variability. Moreover, it enables one to easily differentiate between endometrial cysts and stroma. Accordingly, volume measurements of both endometrial cysts and stroma indicated that the initial establishment of endometriotic lesions is associated with enhanced cellular proliferation, followed by a phase of increased secretory activity of endometrial glands. Results of ultrasound analysis correlated well with measurements of lesion volumes by caliper and histology. Importantly, ultrasound imaging could be performed repetitively and noninvasively and reflected best the in vivo situation. The technique could further be demonstrated to successfully monitor the significant inhibition of growth of endometriotic lesions after specific estrogen receptor destabilizator treatment. Thus, high-resolution ultrasound imaging represents an important tool for future preclinical small animal studies, which address the pathophysiology of endometriosis and the development of new treatment strategies.

Protein kinase CK2 is a regulator of angiogenesis in endometriotic lesions.

Endometriosis is a frequent gynecological disease, which is crucially dependent on the process of angiogenesis. However, the underlying regulatory mechanisms of blood vessel development are still poorly understood. CK2 is a pleiotropic protein kinase, which is implicated in the regulation of various cellular processes including angiogenesis. Herein we studied for the first time the function of protein kinase CK2 in angiogenesis of endometriotic lesions. For this purpose, we analyzed the anti-angiogenic activity of the CK2 inhibitor quinalizarin in a rat aortic ring assay and its effect on the expression of individual CK2 subunits and on kinase activity in endometrial tissue. Moreover, endometriotic lesions were induced in dorsal skinfold chambers of quinalizarin- and vehicle-treated C57BL/6 mice to study their vascularization and morphology by means of repetitive intravital fluorescence microscopy and histology. Our results demonstrate that quinalizarin dose-dependently inhibits vascular sprouting. In addition, treatment of endometrial tissue with quinalizarin reduces CK2 activity without affecting the expression of the three CK2 subunits α, α′ and β. In the dorsal skinfold chamber model of endometriosis, quinalizarin inhibits the vascularization of endometriotic lesions, which exhibit a significantly decreased vascularized area and functional capillary density when compared to those of vehicle-treated controls. This is associated with a reduced lesion size and histological fraction of endometrial glands. These findings indicate that CK2 is a regulator of angiogenesis in endometriotic lesions. Accordingly, inhibition of CK2 represents a novel option in the development of anti-angiogenic strategies for the treatment of endometriosis.

Combined blockade of angiotensin II type 1 receptor and activation of peroxisome proliferator-activated receptor-γ by telmisartan effectively inhibits vascularization and growth of murine endometriosis-like lesions

STUDY QUESTION Is telmisartan effective in the treatment of endometriosis? SUMMARY ANSWER Combined blockade of angiotensin II type 1 receptor (AT1R) and activation of peroxisome proliferator-activated receptor (PPAR)-γ by telmisartan inhibits vascularization and growth of murine endometriosis-like lesions. WHAT IS KNOWN ALREADY AT1R and PPAR-γ are involved in the regulation of inflammation, proliferation and angiogenesis. These processes are also crucial for the pathogenesis of endometriosis and both receptors are expressed in endometrial tissue. Telmisartan is a partial agonist of PPAR-γ, which additionally blocks AT1R. STUDY DESIGN, SIZE, DURATION This was a randomized study in the mouse dorsal skinfold chamber and peritoneal model of endometriosis. Endometriosis-like lesions were induced in dorsal skinfold chambers of 21 female C57BL/6 mice, and in the peritoneal cavity of 15 additional animals, which were daily treated with an i.p. injection of pioglitazone (10 mg/kg, n = 12), telmisartan (10 mg/kg, n = 12) or vehicle (5% dimethyl sulfoxide (DMSO), n = 12) throughout an observation period of 14 and 28 days, respectively. PARTICIPANTS/MATERIALS, SETTING, METHODS The anti-angiogenic actions of pioglitazone, a full PPAR-γ agonist, and telmisartan were firstly assessed in vitro by an aortic ring assay. Endometriosis-like lesions were induced in the dorsal skinfold chamber or peritoneal cavity and the effects of telmisartan and pioglitazone on their vascularization, immune cell content and growth were studied by intravital fluorescence microscopy, high-resolution ultrasound imaging as well as histological, immunohistochemical and immunofluorescent analyses. Additional quantitative real-time polymerase chain reaction (qRT-PCR) arrays served for gene expression profiling of the lesions. To limit the role of chance, the experiments were conducted under standardized laboratory conditions with appropriate vehicle-treated controls. Statistical significance was accepted for a value of P < 0.05. MAIN RESULTS AND THE ROLE OF CHANCE Telmisartan inhibited vascular sprout formation of aortic rings more effectively than pioglitazone. Accordingly, endometriosis-like lesions in dorsal skinfold chambers of telmisartan-treated animals exhibited a markedly lower functional microvessel density and blood perfusion. High-resolution ultrasound analyses of peritoneal endometriosis-like lesions revealed that the compound inhibited the stromal tissue growth, resulting in a significantly reduced final lesion volume. In contrast, the development of cysts did not differ between the groups. Moreover, telmisartan induced an up-regulation of PPAR-γ and a down-regulation of AT1R proteins in endometriosis-like lesions, which was associated with a decreased density of CD31-positive microvessels, a reduced immune cell content and a lower number of Ki67-positive proliferating cells. qRT-PCR arrays further demonstrated an inhibitory action of telmisartan on the expression of several angiogenic and inflammatory genes. LIMITATIONS, REASONS FOR CAUTION Endometriosis-like lesions were induced by syngeneic tissue transplantation into recipient mice without the use of pathological endometriotic tissue of human nature. Therefore, the results obtained in this study may not fully relate to human patients with endometriosis. WIDER IMPLICATIONS OF THE FINDINGS This study demonstrates that telmisartan inhibits vascularization, immune cell content and growth of endometriosis-like lesions. Accordingly, the combined blockade of AT1R and activation of PPAR-γ represents a promising new concept in the development of novel compounds for the treatment of endometriosis. STUDY FUNDING/COMPETING INTEREST(S) There was no specific funding of this study. The authors have no conflicts of interest to declare.

Xanthohumol inhibits growth and vascularization of developing endometriotic lesions

BACKGROUND Xanthohumol is a prenylated flavonoid isolated from hops, which is known to act as a pleiotropic cancer chemopreventive agent owing to its anti-proliferative, anti-inflammatory and anti-angiogenic properties. In the present study, we analyzed, for the first time, whether this dietary compound may also be used for the treatment of endometriosis. METHODS Peritoneal and mesenteric endometriotic lesions were surgically induced in BALB/c mice by uterine tissue transplantation into the abdominal cavity. The animals were treated daily with 100 µM xanthohumol (n= 8) or vehicle (control, n= 8) via the drinking water, starting 3 days before tissue transplantations. Lesion growth, cyst formation and vascularization were subsequently analyzed by means of high-resolution ultrasound imaging (at Day 0 and then once per week for 28 days), caliper measurements, western blotting, histology and immunohistochemistry over 4 weeks. RESULTS In the treatment and control groups, uterine grafts developed typical endometriotic lesions with cyst-like dilated glands surrounded by a vascularized endometrial stroma. However, xanthohumol efficiently decreased the size of these lesions at Day 28, independent of their localization within the peritoneal cavity, compared with control (peritoneal: P =0.041; mesenteric: P =0.038). This was associated with a reduced level of phosphoinositide 3-kinase protein. Moreover, vascularization of xanthohumol-treated lesions was suppressed, as indicated by a significantly lower microvessel density at Day 28 when compared with vehicle-treated controls (peritoneal: P =0.026; mesenteric: P =0.004). Additional analyses revealed that treatment with xanthohumol did not affect the histomorphology, proliferation and vascularization of the uterine horns and ovaries. CONCLUSIONS Taken together, these experimental findings suggest that xanthohumol inhibits the development of endometriotic lesions in mice without inducing serious side effects in the reproductive organs. Thus, xanthohumol represents a promising dietary phytochemical that, after further testing, may be considered for the use in the selective treatment of endometriotic lesions.

Size and spatial orientation of uterine tissue transplants on the peritoneum crucially determine the growth and cyst formation of endometriosis-like lesions in mice

BACKGROUND In many studies in rodents, intraperitoneal endometriosis-like lesions are surgically induced by syngeneic or autologous transplantation of uterine tissue samples, which are sutured to the abdominal wall. However, until now the surgical techniques have not been standardized, and we address this issue here. METHODS Uterine tissue samples were transplanted to the peritoneum of C57BL/6 mice (four study groups, n = 7 each). Using non-invasive high-resolution ultrasound imaging over a period of 4 weeks, we analyzed growth characteristics and cyst formation of the endometriosis-like lesions which developed, in relation to mode of transplantation (syngeneic versus autologous), type of tissue fixed adjacent to the peritoneum (endometrium versus perimetrium), and size of tissue transplanted (2 versus 3 mm). Immunohistochemical analysis was also performed. RESULTS When the perimetrium, with underlying myometrium, was sutured next to the host peritoneum the endometriosis-like lesions which developed exhibited a higher growth rate (P< 0.05 versus endometrium), and contained more proliferating cell nuclear antigen (PCNA)-positive cells and an increased microvessel density (both P< 0.05 versus endometrium). In the group with 3 mm uterine tissue grafts, lesion growth was significantly decreased when compared with 2 mm samples (P< 0.05). However, the larger grafts developed more cysts throughout the observation period than the smaller ones. There was no difference between syngeneic and autologous endometriosis-like lesions. CONCLUSIONS Our study demonstrates that size and spatial orientation of peritoneally fixed uterine tissue samples crucially determine growth and cyst formation of endometriotic lesions in mice. These findings should improve the standardization and reliability of future studies, performed in the frequently used mouse model of surgically induced endometriosis.

Experimental orthotopic prostate tumor in nude mice: Techniques for local cell inoculation and three-dimensional ultrasound monitoring

OBJECTIVES Orthotopic prostate cancer models are of great importance for cancer research. Orthotopic models in mice have been described previously. However, these studies lack a detailed methodological description and fail to define standards for local cell inoculation. Herein, we studied the effect of different protocols on tumor growth and report for the first time the use of high resolution ultrasound for monitoring of tumor growth. MATERIALS AND METHODS Orthotopic inoculation of DU 145 MN1 prostate cancer cells was performed in 30 nude mice varying (1) the amount of cells (5 × 10(5) vs. 5 × 10(4)), (2) the number of puncture sites, and (3) the addition of matrigel. Surgical complications such as recoil of cells through the injection canal and rupture of the prostatic capsule were monitored. Animals were tracked by ultrasound imaging after 4, 5, and 6 weeks. Autopsy and histology confirmed local tumor growth. RESULTS A take rate of 27/30 (90%) was observed. Growth of orthotopic prostate tumors was increased after inoculation of a large amount of cells under the capsule of 1 dorsal prostate lobe, but inoculation of small amounts of cells still induced local tumors. Noninvasive ultrasound examination allowed to identify orthotopic tumor formation and to monitor tumor growth in vivo. Addition of matrigel did not accelerate tumor growth. Complications like recoil (6.8%) or rupture of the prostate capsule (1.4%) were rare. CONCLUSIONS Inoculation of DU 145 MN1 cells under the prostate capsule with a defined procedure results in very high take rates. Ultrasound screening is feasible to repetitively monitor tumor growth.

C-kit(+) resident cardiac stem cells improve left ventricular fibrosis in pressure overload.

To investigate the effect of resident cardiac stem cells (RCSC) on myocardial remodeling, c-kit(+) RCSC were isolated from hearts of C57Bl/6-Tg (ACTb-EGFP)1Osb/J mice expressing green fluorescent protein and expanded in vitro. C57/Bl6N wildtype mice were subjected to transverse aortic constriction (TAC, 360 μm) or sham-operation. 5 × 10(5) c-kit(+) RCSC or c-kit(-) cardiac cells or cell buffer were infused intravenously 24 h post-surgery (n = 11-24 per group). Hypoxia-inducible factor-1α-mRNA in left ventricles of TAC mice was enhanced 24 h after transplantation. 35 days post-TAC, the density of c-kit(+) RCSC in the myocardium was increased by two-fold. Infusion of c-kit(+) resident cardiac stem cells post-TAC markedly reduced myocardial fibrosis and the expression of collagen Iα2 and connective tissue growth factor. Infusion of c-kit(-) cardiac cells did not ameliorate cardiac fibrosis. In parallel, expression of pro-angiogenic mediators (FGFb, IL-4, IL-6, TGFß, leptin) and the density of CD31(+) and CD31(+) GFP(+) endothelial cells were increased. Transplantation reduced brain- and atrial natriuretic peptides and the cardiomyocyte cross-sectional area. Infusion of c-kit(+) resident cardiac stem reduced the rate of apoptosis and oxidative stress in cardiomyocytes and in non-cardiomyocyte cells.

Tubeimoside-1 suppresses tumor angiogenesis by stimulation of proteasomal VEGFR2 and Tie2 degradation in a non-small cell lung cancer xenograft model

Tubeimoside-1 (TBMS1) is a potent anti-tumor phytochemical. Its functional and molecular mode of action, however, remains elusive so far. Since angiogenesis is essential for tumor progression and metastasis, we herein investigated the anti-angiogenic effects of the compound. In a non-small cell lung cancer (NSCLC) xenograft model we found that treatment of CD1 nu/nu mice with TBMS1 (5mg/kg) significantly suppressed the growth and vascularization of NCI-H460 flank tumors. Moreover, TBMS1 dose-dependently reduced vascular sprouting in a rat aortic ring assay. In vitro, TBMS1 induced endothelial cell apoptosis without decreasing the viability of NSCLC tumor cells and inhibited the migration of endothelial cells by disturbing their actin filament organization. TBMS1 further stimulated the proteasomal degradation of vascular endothelial growth factor receptor-2 (VEGFR2) and Tie2 in endothelial cells, which down-regulated AKT/mTOR signaling. These findings indicate that TBMS1 represents a novel phytochemical for anti-angiogenic treatment of cancer and other angiogenesis-related diseases.

Orthotopic tumorgrafts in nude mice: A new method to study human prostate cancer.

In vivo model systems in prostate cancer research that authentically reproduce tumor growth are still sparse. While orthotopic implantation is technically difficult, particularly in the mouse, most models favor subcutaneous tumor growth. This however provides little information about natural tumor growth behavior and tumor stroma interaction. Furthermore, established prostate cancer cell lines grown as in vivo xenografts are not able to reflect the variety of tumor specific growth patterns and growth behavior in men. Primary cell cultures are difficult to handle and an induction of orthotopic tumors has not been successful yet. Therefore, a tumorgraft model using tumor tissue from prostatectomy specimens was developed.