Impact of primary resistance to front-line targeted therapy in metastatic renal cell carcinoma on subsequent immune-checkpoint-inhibition.

INTRODUCTION: Synergistic effects have been discussed for tyrosine kinase (TKI) and immune checkpoint inhibitors (ICI). Primary resistance to TKI might disturb subsequent ICI effectiveness. The objective was to investigate, if primary resistance to 1st line TKI monotherapy predicts response to ICI in subsequent therapy lines and impacts overall survival (OS) in advanced renal cell carcinoma (aRCC). MATERIALS AND METHODS: Retrospectively, aRCC patients which received front-line TKI from 2016 to 2019 were analyzed for the outcomes primary resistance (1LR), response to sequential ICI therapy, progression free survival (PFS) and overall survival (OS). Kaplan-Meier-estimates, Cox proportional hazards and logistic regression were used. RESULTS: Primary resistance to front-line TKI was observed in 27 (53%) of 51 patients. Groups with disease control (DC) and 1st line TKI resistance (1LR) were not different at baseline with regard to clinicopathological features. Median duration on 1st line therapy was significantly shorter in the 1LR (5.1 months) than in the DC (14.7 months) group (p = 0.01). Sequential therapy was started in 21 (75%) and 12 (52%) patients of 1LR and DC groups using nivolumab in 16 (76%) vs. 11 (92%) cases (p > 0.05). Logistic regression revealed that 1LR status, neutrophil-to-lymphocyte ratio < 3, IMDC favorable prognosis and clear cell histology had no significant impact on responsiveness to ICI in subsequent therapy lines. Cox proportional hazards demonstrated no significant association of 1LR status with PFS and OS in patients who received subsequent ICI treatment. CONCLUSION: Primary TKI resistance of aRCC was neither significantly associated with responsiveness to ICI during sequential therapy nor with PFS and OS. This adds the evidence for ICI based sequential therapy in primary TKI resistant aRCC.

Curcumin combined with exposure to visible light blocks bladder cancer cell adhesion and migration by an integrin dependent mechanism.

OBJECTIVE: Although the natural compound curcumin exerts antitumor properties in vitro, its clinical application is hampered due to rapid metabolism. Light exposure following curcumin application has been demonstrated to improve curcumin's bioavailability. Therefore, this investigation was directed towards evaluating whether light exposure in addition to curcumin application enhances curcumin's efficacy against bladder cancer cell adhesion and migration. MATERIALS AND METHODS: RT112, UMUC3, and TCCSUP cells were incubated with low curcumin concentrations (0.1-0.4 µg/ml) and then exposed to 1.65 J/cm2 visible light for 5 min. Controls remained untreated or were treated with curcumin or light alone. Cell adhesion to Human umbilical vein endothelial cells (HUVECs), to immobilized collagen or fibronectin and chemotactic behavior, integrin α and ß receptor expression with functional relevance, as well as focal adhesion kinase (total and phosphorylated FAK) were evaluated. RESULTS: Curcumin plus light, but neither curcumin nor light alone, significantly altered tumor cell adhesion and suppressed chemotaxis. Integrin α and ß subtypes were dissimilarly modified, depending on the cell line. Suppression of pFAK was noted in RT112 and UMUC3, but not in TCCSUP cells. The integrins α3, α5, and ß1 were involved in curcumin's regulation of adhesion and migration. Blocking studies revealed α3, α5, and ß1 to be associated with TCCSUP adhesion and migration, whereas α5 and ß1, but not α3 contributed to UMUC3 adhesion and migration. Integrin α5 and ß1 controlled RT112 chemotaxis as well, but only α5 was involved in the RT112 adhesion process. CONCLUSIONS: Combining curcumin with light exposure enhances curcumin's anti-tumor potential.

Ethanol, ethyl and sodium pyruvate decrease the inflammatory responses of human lung epithelial cells via Akt and NF-κB in vitro but have a low impact on hepatocellular cells.

Increases in pro-inflammatory cytokine levels and tissue-infiltrating leukocytes have been closely linked to increased systemic and local inflammation, which result in organ injury. Previously, we demonstrated the beneficial and hepatoprotective anti-inflammatory effects of acute ethanol (EtOH) ingestion in an in vivo model of acute inflammation. Due to its undesirable side-effects, however, EtOH does not represent a therapeutic option for treatment of acute inflammation. Therefore, in this study, we compared the effects of acute EtOH exposure with ethyl pyruvate (EtP) as an alternative anti-inflammatory drug in an in vitro model of hepatic and pulmonary inflammation. Human hepatocellular carcinoma cells Huh7 and alveolar epithelial cells A549 were stimulated with either interleukin (IL) IL-1ß (1 ng/ml, 24 h) or tumor necrosis factor (TNF) (10 ng/ml, 4 h), and then treated with EtP (2.5-10 mM), sodium pyruvate (NaP, 10 mM) or EtOH (85-170 mM) for 1 h. IL-6 or IL-8 release from Huh7 or A549 cells, respectively, was measured by an enzyme­linked immunosorbent assay. Neutrophil adhesion to cell monolayers and CD54 expression were also analyzed. Bcl-2 and Bax gene expression was determined by RT-qPCR, and western blot analysis was performed to determine the mechanisms involved. Treating A549 cells with either EtOH or EtP significantly reduced the IL-1ß- or TNF­induced IL-8 release, whereas treating Huh7 cells did not significantly alter IL-6 release. Similarly, neutrophil adhesion to stimulated A549 cells was significantly reduced by EtOH or EtP, whereas for Huh7 cells the tendency for reduced neutrophil adhesion rates by EtOH or EtP was not significant. CD54 expression was noticeably reduced in A549 cells, but this was not the case in Huh7 cells after treatment. The Bax/Bcl-2 ratio was dose­dependently decreased by EtOH and by high-dose EtP in A549 cells, indicating a reduction in apoptosis, whereas this effect was not observed in Huh7 cells. The underlying mechanisms involve reduced phosphorylation of Akt and nuclear factor-κB (NF-κB) p65. We noted that as with EtP, EtOH reduced the inflammatory response in lung epithelial cells under acute inflammatory conditions. However, due to the low impact which EtP and EtOH had on the hepatocellular cells, our data suggest that both substances exerted different effects depending on the cellular entity. The possible underlying mechanisms involved the downregulation of Akt and the transcription factor NF-κB, but further research on this subject is required.

Decreased inflammatory responses of human lung epithelial cells after ethanol exposure are mimicked by ethyl pyruvate.

BACKGROUND AND PURPOSE: Leukocyte migration into alveolar space plays a critical role in pulmonary inflammation resulting in lung injury. Acute ethanol (EtOH) exposure exerts anti-inflammatory effects. The clinical use of EtOH is critical due to its side effects. Here, we compared effects of EtOH and ethyl pyruvate (EtP) on neutrophil adhesion and activation of cultured alveolar epithelial cells (A549). EXPERIMENTAL APPROACH: Time course and dose-dependent release of interleukin- (IL-) 6 and IL-8 from A549 were measured after pretreatment of A549 with EtP (2.5-10 mM), sodium pyruvate (NaP, 10 mM), or EtOH (85-170 mM), and subsequent lipopolysaccharide or IL-1beta stimulation. Neutrophil adhesion to pretreated and stimulated A549 monolayers and CD54 surface expression were determined. KEY RESULTS: Treating A549 with EtOH or EtP reduced substantially the cytokine-induced release of IL-8 and IL-6. EtOH and EtP (but not NaP) reduced the adhesion of neutrophils to monolayers in a dose- and time-dependent fashion. CD54 expression on A549 decreased after EtOH or EtP treatment before IL-1beta stimulation. CONCLUSIONS AND IMPLICATIONS: EtP reduces secretory and adhesive potential of lung epithelial cells under inflammatory conditions. These findings suggest EtP as a potential treatment alternative that mimics the anti-inflammatory effects of EtOH in early inflammatory response in lungs.

Resistance to the mTOR-inhibitor RAD001 elevates integrin α2- and ß1-triggered motility, migration and invasion of prostate cancer cells.

BACKGROUND: Inhibitors of the mammalian target of rapamycin (mTOR) might become a novel tool to treat advanced prostate cancer. However, chronic drug exposure may trigger resistance, limiting the utility of mTOR inhibitors. METHODS: Metastatic potential of PC3 prostate cancer cells, susceptible (PC3(par)) or resistant (PC3(res)) to the mTOR-inhibitor RAD001 was investigated. Adhesion to vascular endothelium or immobilised collagen, fibronectin and laminin was quantified. Motility, migration and invasion were explored by modified Boyden chamber assay. Integrin α and ß subtypes were analysed by flow cytometry, western blotting and real-time PCR. Integrin-related signalling, EGFr, Akt, p70S6kinase and ERK1/2 activation were determined. RESULTS: Adhesion was reduced, whereas motility, migration and invasion were enhanced in PC3(res). The α2 and ß1 integrin subtypes were dramatically elevated, integrins α1 and α6 were lowered, whereas α5 was nearly lost in PC3(res). Activation of the Akt signalling pathway was strongly upregulated in these cells. Treating PC3(par) cells with RAD001 reduced motility, migration and invasion and deactivated Akt signalling. Blocking studies revealed that α2 and ß1 integrins significantly trigger the motile behaviour of the tumour cells. CONCLUSION: Chronic RAD001 treatment caused resistance development characterised by distinct modification of the integrin-expression profile, driving prostate cancer cells towards high motility.

Zoledronic acid influences growth, migration and invasive activity of prostate cancer cells in vitro.

BACKGROUND: The influence of the bisphosphonate zoledronic acid (ZA) on prostate cancer (PC) growth, adhesion and invasive behavior was investigated. METHODS: PC-3, DU-145 and LNCaP cells were treated with ZA, and tumor-cell growth was then investigated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Furthermore, tumor-cell adhesion to vascular endothelium or to immobilized extracellular matrix proteins, as well as migratory properties of the cells, was evaluated. Integrin ß subtypes, integrin-dependent signaling, as well as cell-cycle regulating proteins, were analyzed by western blots. RESULTS: ZA dose-dependently reduced tumor-cell growth but did not impair tumor-endothelium and tumor-matrix interaction. However, ZA significantly inhibited tumor migration and invasive activity. Cyclin E was reduced by ZA in LNCaP and DU-145, and p21 was elevated in LNCaP cells. p27 was upregulated in all tumor cell lines, compared with the controls. ZA elevated ß1-integrin in PC-3 and diminished ß4-integrin in PC-3 and DU-145 cells. CONCLUSIONS: ZA inhibits PC growth and motility but does not influence the mechanical contact between tumor cells and the vascular wall.

Alterations of the gene expression profile in renal cell carcinoma after treatment with the histone deacetylase-inhibitor valproic acid and interferon-alpha.

PURPOSE: Renal cell carcinoma (RCC) is highly resistant to chemotherapy and unresponsive to radio- and immunotherapy. Recently, we have documented that the histone deacetylase (HDAC)-inhibitor valproic acid (VPA) in combination with low-dosed interferon (IFN)-alpha significantly inhibits RCC proliferation and adhesion in vitro and in vivo. The current study investigated the effects of these compounds on gene transcription of metastatic RCC cell line Caki-1 after 3 and 5 days exposure. METHODS: To evaluate the gene expression profiles of the RCC cells, we performed microarray analysis using Affymetrix GeneChip. Selected significant genes were further validated by Real Time PCR. RESULTS: Microarray revealed that VPA altered genes that are involved in cell growth, cell survival, immune response, cell motility and cell adhesion. Combination of VPA with IFN-alpha not only enhanced the effects on gene transcription but also resulted in the expression of novel genes, which were not induced by either VPA or IFN-alpha alone. Among the up-regulated genes were chemokines (CXCL10, CXCL11, CXCL16) and integrins (ITGA2, ITGA4, ITGA5, ITGA6, ITGA7). Genes encoding for adhesion molecules (NCAM1, ICAM1, VCAM1) were also modulated. Real Time PCR approved these findings. CONCLUSION: This data provides insight into the molecular mechanism of action of the combined treatment of VPA and IFN-alpha in RCC. Implications are that the combined application of VPA and IFN-alpha may represent a more efficient alternative to existing therapy options for RCC.

[Gross haematuria as the primary symptom of extramedullary IgM plasmacytoma of the bladder]. / Makrohämaturie als Primärsymptom eines extramedullären IgM-Plasmozytoms der Harnblase.

Haematuria is the most common clinical symptom of bladder cancer. Besides antibiotic treatment of a probably existing urinary tract infection, ultrasonography of the urinary organs, diagnostic cystoscopy (with biopsy if needed), and radiologic evaluation of the upper urinary tract (intravenous urography, computed tomography or magnetic resonance urography, retrograde pyelography) should be done for further evaluation. Atypical manifestations of systemic diseases with bladder infiltration could feign the clinical appearance of chronic cystitis and hinder determination of the correct diagnosis. The case of a 40-year-old man with recurrent gross haematuria due to extremely rare bladder infiltration through an IgM plasmacytoma is presented.

[Preclinical studies on the influence of the tyrosine kinase inhibitor AEE788 on malignant properties of renal cell carcinoma cells]. / Präklinische Studien zum Einfluss des Tyrosinkinaseinhibitors AEE788 auf die Malignität des Nierenzellkarzinoms.

BACKGROUND: Conventional therapeutic approaches to treat advanced renal cell carcinoma (RCC) are of limited benefit. Receptor tyrosine kinase inhibitors (RTKI) may open up novel treatment options. In the present study, the effects of the RTKI AEE788 on the growth and adhesion capacity of RCC cell lines were evaluated in vitro. MATERIALS AND METHODS: RCC cells were treated with AEE788, and alterations of tumor growth and tumor cell interaction with vascular endothelium or extracellular matrix proteins were analyzed. Furthermore, the addition of interferon alpha (IFNalpha) was investigated to see whether it may enhance the anti-tumoral potential of AEE788. RESULTS: AEE788 significantly blocked RCC cell growth and adhesion. Analysis of alpha- and beta-integrins revealed distinct alterations of the receptor expression profile and downregulation of integrin-dependent signaling. Growth-blocking effects were further enhanced when the AEE788-IFNalpha combination protocol was applied. In addition, downregulation of integrin-dependent signaling was more intense in the presence of a combination of AEE788 and IFNalpha than with AEE788 monotherapy. CONCLUSIONS: AEE788 exerts significant anti-tumoral properties, particularly when combined with IFNalpha. AEE788 may therefore be an encouraging compound to treat advanced RCC.

New histone deacetylase inhibitors as potential therapeutic tools for advanced prostate carcinoma.

The anti-epileptic drug valproic acid is also under trial as an anti-cancer agent due to its histone deacetylase (HDAC) inhibitory properties. However, the effects of valproic acid (VPA) are limited and concentrations required for exerting anti-neoplastic effects in vitro may not be reached in tumour patients. In this study, we tested in vitro and in vivo effects of two VPA-derivatives (ACS2, ACS33) on pre-clinical prostate cancer models. PC3 and DU-145 prostate tumour cell lines were treated with various concentrations of ACS2 or ACS33 to perform in vitro cell proliferation 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays and to evaluate tumour cell adhesion to endothelial cell monolayers. Analysis of acetylated histones H3 and H4 protein expression was performed by western blotting. In vivo tumour growth was conducted in subcutaneous xenograft mouse models. Tumour sections were assessed by immunohistochemistry for histone H3 acetylation and proliferation. ACS2 and ACS33 significantly up-regulated histone H3 and H4 acetylation in prostate cancer cell lines. In micromolar concentrations both compounds exerted growth arrest in PC3 and DU-145 cells and prevented tumour cell attachment to endothelium. In vivo, ACS33 inhibited the growth of PC3 in subcutaneous xenografts. Immunohistochemistry and western blotting confirmed increased histone H3 acetylation and reduced proliferation. ACS2 and ACS33 represent novel VPA derivatives with superior anti-tumoural activities, compared to the mother compound. This investigation lends support to the clinical testing of ACS2 or ACS33 for the treatment of prostate cancer.