Michael Reiter

HDAC-inhibition counteracts everolimus resistance in renal cell carcinoma in vitro by diminishing cdk2 and cyclin A

BackgroundTargeted therapies have improved therapeutic options of treating renal cell carcinoma (RCC). However, drug response is temporary due to resistance development.MethodsFunctional and molecular changes in RCC Caki-1 cells, after acquired resistance to the mammalian target of rapamycin (mTOR)-inhibitor everolimus (Cakires), were investigated with and without additional application of the histone deacetylase (HDAC)-inhibitor valproic acid (VPA). Cell growth was evaluated by MTT assay, cell cycle progression and apoptosis by flow cytometry. Target molecules of everolimus and VPA, apoptotic and cell cycle regulating proteins were investigated by western blotting. siRNA blockade was performed to evaluate the functional relevance of the proteins.ResultsEverolimus resistance was accompanied by significant increases in the percentage of G2/M-phase cells and in the IC50. Akt and p70S6K, targets of everolimus, were activated in Cakires compared to drug sensitive cells. The most prominent change in Cakires cells was an increase in the cell cycle activating proteins cdk2 and cyclin A. Knock-down of cdk2 and cyclin A caused significant growth inhibition in the Cakires cells. The HDAC-inhibitor, VPA, counteracted everolimus resistance in Cakires, evidenced by a significant decrease in tumor growth and cdk2/cyclin A.ConclusionIt is concluded that non-response to everolimus is characterized by increased cdk2/cyclin A, driving RCC cells into the G2/M-phase. VPA hinders everolimus non-response by diminishing cdk2/cyclin A. Therefore, treatment with HDAC-inhibitors might be an option for patients with advanced renal cell carcinoma and acquired everolimus resistance.

CCL2 Chemokine as a Potential Biomarker for Prostate Cancer: A Pilot Study

Purpose Prostate specific antigen is not reliable in diagnosing prostate cancer (PCa), making the identification of novel, precise diagnostic biomarkers important. Since chemokines are associated with more aggressive disease and poor prognosis in diverse malignancies, we aimed to investigate the diagnostic relevance of chemokines in PCa. Materials and Methods Preoperative and early postoperative serum samples were obtained from 39 consecutive PCa patients undergoing radical prostatectomy. Serum from 15 healthy volunteers served as controls. Concentrations of CXCL12, CXCL13, CX3CL1, CCL2, CCL5, and CCL20 were measured in serum by Luminex. The expression activity of CXCR3, CXCR4, CXCR5, CXCR7, CXCL12, CXCL13, CX3CR1, CXCL1, CCR2, CCR5, CCR6, CCR7, CCL2, and CCL5 mRNA was assessed in tumor and adjacent normal tissue of prostatectomy specimens by quantitative real-time polymerase chain reaction. The associations of these chemokines with clinical and histological parameters were tested. Results The gene expression activity of CCL2 and CCR6 was significantly higher in tumor tissue compared to adjacent normal tissue. CCL2 was also significantly higher in the blood samples of PCa patients, compared to controls. CCL5, CCL20, and CX3CL1 were lower in patient serum, compared to controls. CCR2 tissue mRNA was negatively correlated with the Gleason score and grading. Conclusion Chemokines are significantly modified during tumorigenesis of PCa, and CCL2 is a promising diagnostic biomarker.

Amygdalin blocks bladder cancer cell growth in vitro by diminishing cyclin A and cdk2.

Amygdalin, a natural compound, has been used by many cancer patients as an alternative approach to treat their illness. However, whether or not this substance truly exerts an anti-tumor effect has never been settled. An in vitro study was initiated to investigate the influence of amygdalin (1.25–10 mg/ml) on the growth of a panel of bladder cancer cell lines (UMUC-3, RT112 and TCCSUP). Tumor growth, proliferation, clonal growth and cell cycle progression were investigated. The cell cycle regulating proteins cdk1, cdk2, cdk4, cyclin A, cyclin B, cyclin D1, p19, p27 as well as the mammalian target of rapamycin (mTOR) related signals phosphoAkt, phosphoRaptor and phosphoRictor were examined. Amygdalin dose-dependently reduced growth and proliferation in all three bladder cancer cell lines, reflected in a significant delay in cell cycle progression and G0/G1 arrest. Molecular evaluation revealed diminished phosphoAkt, phosphoRictor and loss of Cdk and cyclin components. Since the most outstanding effects of amygdalin were observed on the cdk2-cyclin A axis, siRNA knock down studies were carried out, revealing a positive correlation between cdk2/cyclin A expression level and tumor growth. Amygdalin, therefore, may block tumor growth by down-modulating cdk2 and cyclin A. In vivo investigation must follow to assess amygdalins practical value as an anti-tumor drug.

Molecular analysis of sunitinib resistant renal cell carcinoma cells after sequential treatment with RAD001 (everolimus) or sorafenib

Sequential application of target drugs is standard procedure after renal cell carcinoma (RCC) patients develop resistance. To optimize the sequence, antitumour effects of the mTOR inhibitor RAD001 or the tyrosine kinase inhibitor (TKI) sorafenib on RCC cells with acquired resistance to the TKI sunitinib was evaluated. RCC cells were exposed to 1 μM sunitinib for 24 hrs (as control) and for 8 weeks (to induce resistance) and then switched to RAD001 (5 nM) or sorafenib (5 μM) for a further 8 weeks. Tumour cell growth, cell cycle progression, cell cycle regulating proteins and intracellular signalling were then investigated. Short‐term application of sunitinib (24 hrs) induced cell growth blockade with accumulation in the G2/M phase. RCC cells became resistant to sunitinib after 8 weeks, demonstrated by accelerated cell growth along with enhanced cdk1, cdk2, loss of p27, activation of Akt, Rictor and Raptor. Switching to sorafenib only slightly reduced growth of the sunitinib resistant RCC cells and molecular analysis indicated distinct cross‐resistance. In contrast, full response was achieved when the cancer cells were treated with RAD001. p19 and p27 strongly increased, phosphorylated Akt, Rictor and Raptor decreased and the tumour cells accumulated in G0/G1. It is concluded that an mTOR‐inhibitor for second‐line therapy could be the strategy of choice after first‐line sunitinib failure.

Cross-communication between histone H3 and H4 acetylation and Akt-mTOR signalling in prostate cancer cells

Molecular tumour targeting has significantly improved anti‐cancer protocols. Still, the addition of molecular targeting to the treatment regime has not led to a curative breakthrough. Combined mammalian target of Rapamycin (mTOR) and histone deacetylase (HDAC) inhibition has been shown not only to enhance anti‐tumour potential, but also to prevent resistance development seen under mono‐drug therapy. This investigation was designed to evaluate whether cross‐communication exists between mTOR signalling and epigenetic events regulated by HDAC. DU‐145 prostate cancer cells were treated with insulin‐like growth factor (IGF) to activate the Akt‐mTOR cascade or with the HDAC‐inhibitor valproic acid (VPA) to induce histone H3 and H4 acetylation (aH3, aH4). Subsequently, mTOR, Rictor, Raptor, p70s6k, Akt (all: total and phosphorylated), H3 and H4 (total and acetylated) were analysed by western blotting. Both techniques revealed a link between mTOR and the epigenetic machinery. IGF activated mTOR, Rictor, Raptor, p70s6k and Akt, but also enhanced aH3 and aH4. Inversely, IGFr blockade and knock‐down blocked the Akt‐mTOR axis, but simultaneously diminished aH3 and aH4. VPA treatment up‐regulated histone acetylation, but also activated mTOR‐Akt signalling. HDAC1 and 2 knock‐down revealed that the interaction with the mTOR system is initiated by histone H3 acetylation. HDAC‐mTOR communication, therefore, is apparent whereby tumour‐promoting (Akt/mTORhigh, aH3/aH4low) and tumour‐suppressing signals (Akt/mTORlow, aH3/aH4high) are activated in parallel. Combined use of an HDAC‐ and mTOR inhibitor might then diminish pro‐tumour effects triggered by the HDAC‐ (Akt/mTORhigh) or mTOR inhibitor (aH3/aH4low) alone.

Resistance to the mTOR Inhibitor Temsirolimus Alters Adhesion and Migration Behavior of Renal Cell Carcinoma Cells through an Integrin α5– and Integrin β3–Dependent Mechanism

Inhibitors of the mammalian target of rapamycin (mTOR) have improved the treatment of renal cell carcinoma (RCC). However, chronic drug exposure may trigger resistance, limiting the utility of these agents. The metastatic behavior of RCC cells, susceptible (RCC(par)) or resistant (RCC(res)) to the mTOR inhibitor temsirolimus, was investigated. Adhesion to vascular endothelium or immobilized collagen and fibronectin was quantified. Chemotactic motility was evaluated with a modified Boyden chamber assay. Integrin α and β subtype receptors were analyzed by flow cytometry and Western blot analysis. The physiological relevance of the integrins was then determined by blocking studies and small interfering RNA knockdown. Adhesion to endothelial cells and to fibronectin (not to collagen) and chemotaxis were enhanced in RCC(res) compared to RCC(par). RCC(res) detached from fibronectin and motile activity further increased under retreatment with low-dosed temsirolimus. α5 integrin was diminished inside the cell and at the cell surface, whereas the β3 subtype was reduced intracellularly but elevated at the plasma membrane. In RCC(par), blocking α5 surface receptors enhanced RCC-collagen but reduced RCC-fibronectin interaction, whereas the opposite was true for RCC(res). Chemotaxis of RCC(par) but not of RCC(res) was strongly diminished by the α5 antibody. Blocking β3 significantly lowered chemotaxis with stronger effects on RCC(res), compared to RCC(par). Importantly, β3 knockdown reduced chemotaxis of RCC(par) but upregulated the motile behavior of RCC(res). Temsirolimus resistance is characterized by quantitative alterations of integrin α5 and β3 expression, coupled to functional changes of the integrin molecules, and forces a switch from RCC adhesion to RCC migration.

Combining Open and Laparoscopic Surgery for Partial Nephrectomy

PURPOSEnWe present a simple and easy to apply surgical approach for partial nephrectomy that combines open and laparoscopic surgery allowing for vascular control in technically challenging renal tumors and for reduction of ischemia time.nnnPATIENTS AND METHODSnFive patients underwent partial nephrectomy using the combined laparoscopic/open approach. After complete laparoscopic mobilization of the kidney and securing of the renal vessels, the kidney is transferred extracorporally through a small pararectal incision for tumor resection.nnnRESULTSnThe technique was feasible in all cases, with no conversion needed. In three cases, no clamping of the renal artery was necessary. The mean operative time was 219 minutes (range 195-260u2009min). Pathologic examination revealed no malignancy in two (40%) cases. The estimated median blood loss was 500u2009mL (range 250-1000u2009mL). Renal function decreased from 84.9u2009mL/min (range 48.0-89.0u2009mL/min) to 78.8u2009mL/min (range 52.6-82.6u2009mL/min). Mean hospital stay was 5 days (range 3-14u2009d).nnnCONCLUSIONnBased on our initial experience, we propose a feasible surgical option for minimally invasive partial nephrectomy, which is, in particular, suitable for technically challenging renal tumors (endophytic and central tumors, large tumors, tumors in solitary kidneys).

Midterm results of robot-assisted sacrocolpopexy.

Introduction and hypothesisRobotic assistance simplifies laparoscopic procedures. We hypothesize that robot-assisted sacrocolpopexy is a rapid and safe procedure with satisfying short-term and midterm functional results.MethodsAfter informed consent, we enrolled 101 consecutive patients undergoing sacrocolpopexy at Alfried Krupp Hospital, Essen, Germany. After a median follow-up of 22xa0months, we assessed midterm functional results as the primary endpoint. Secondary endpoints included surgical duration, blood loss, intraoperative complications, and postoperative complications. We described frequencies as counts (percent) and continuous data as median [interquartile range (Q1–Q3)] or mean [standard deviation (SD)], as appropriate.ResultsWe enrolled 101 patients. The mean age was 69xa0years (SD 11); 75 women (74.3xa0%) had undergone previous abdominal surgery. Among the patients, 95 (94.1xa0%) presented with anterior vaginal wall prolapse Baden-Walker grade 2–3, 74 (73.3xa0%) vaginal vault prolapse, and 9 (8.9xa0%) concomitant rectocele. Fifty (50xa0%) patients underwent a modified Burch procedure in addition to sacrocolpopexy. The median surgical duration was 96xa0min (Q1–Q3 83–130). There were six (5.9xa0%) minor intraoperative complications but no conversions to open surgery. Postoperatively, we registered five (4.9xa0%) Clavien-Dindo grade I complications, three (3.0xa0%) grade II complications, and one (1.0xa0%) grade III complication. After a median follow-up of 22xa0months (Q1–Q3 12–49), the patients reported significant decreased impact of pelvic organ prolapse (POP) on quality of life as well as bother resulting from POP symptoms. The overall success rate, defined as none or minor impact of POP on quality of life, was 75xa0%.ConclusionsIn this single-surgeon study, robot-assisted sacrocolpopexy was a safe and rapidly performed procedure that achieved good medium-term functional results.

Prävention der postoperativen Harninkontinenz

ZusammenfassungEin nicht unerheblicher Anteil der Prävalenz der männlichen Inkontinenz geht auf eine postoperative Belastungsinkontinenz nach operativer Therapie des Prostatakarzinoms zurück. Dabei wird die postoperative Inkontinenzrate nach radikaler Prostatektomie je nach Autor und Patientenkollektiv zwischen 2,9% und 87,0% angegeben. Hierbei spielen patientenbezogene Faktoren, wie „Body-Mass-Index“, Patientenalter, Nebenerkrankungen, Prostatagröße und präoperative Kontinenz, aber auch die Art und Weise der Datenerhebung und Klassifikation der Inkontinenz eine Rolle. Allerdings zeigen Untersuchungen der letzten Jahre, dass die angewendete Operationstechnik einen entscheidenden Einfluss auf die postoperative Kontinenz des Patienten ausübt. Dem Erhalt des muskulären urethralen Sphinkters (Rhabdosphinkter, äußerer Schließmuskel) kommt dabei die entscheidende Rolle zu. Weitere anatomische Strukturen und deren Erhalt tragen allerdings zusätzlich zur postoperativen Kontinenz bei. Dazu gehören v.xa0a. der Blasenhalserhalt, nervenschonende Operationsverfahren und die Rekonstruktion des vesikourethralen Übergangs.In den letzten Jahren wurden diesbezüglich verschiedene Modifikationen der Operationstechnik auf ihre spezifischen Auswirkungen auf die postoperative Kontinenz untersucht. Die Auswertung dieser Studien zeigt v.xa0a. eine deutliche Verbesserung der postoperativen Frühkontinenz und eine deutlich kürzere Zeitspanne bis zum Erreichen der vollen Kontinenz. Die Langzeitergebnisse zeigen dahingegen keine signifikanten Vorteile für die beschriebenen Modifikationen in Bezug auf die Kontinenz gegenüber der Standardoperation.Die bisher vorliegenden Studien zeigen nur geringe Auswirkungen der beschriebenen präventiven Maßnahmen auf die postoperative Kontinenz mit insgesamt großer Bandbreite der publizierten Kontinenzraten. Zur Evaluierung der genauen Auswirkungen der präventiven Modifikationen der Operationstechnik sind weitere, v.xa0a. größere und randomisierte Studien notwendig, um die effektivsten Modifikationen zu identifizieren. Dabei scheint es wahrscheinlich, dass schlussendlich Kombinationen dieser präventiven, operativen Maßnahmen zu einer deutlichen Verbesserung der postoperativen Kontinenzraten führen werden.AbstractPostoperative stress incontinence following operative treatment of prostate cancer represents a considerable percentage of overall male incontinence. Postoperative incontinence following radical prostatectomy ranges between 2.9 and 87% depending on author and patient characteristics. Especially patient-related factors such as body mass index, age, size of prostate, preoperative incontinence, and concomitant diseases as well as classification of incontinence and modality of data collection influence postoperative continence rates. However, recent publications demonstrate the important impact of different operative techniques with regard to postoperative continence.The preservation of the muscular urethral sphincter is of particular importance. Nevertheless, the preservation of further anatomical structures contributes to postoperative continence. Preservation of bladder neck, nerve sparing, and reconstruction of the vesicourethral junction are operative techniques to prevent postoperative incontinence. In the last decade different modifications of the operative technique have been investigated regarding specific effects on postoperative continence. The interpretation of these studies investigating these operative techniques showed improvement in early continence with shorter period of time to continence. Long-term follow-up revealed no significant advantages for these modifications with regard to continence compared to the standard procedure. To evaluate the long-term effects of certain modifications to prevent incontinence, randomized and well powered studies are necessary. It seems to be most likely that a combination of these preventive modifications will lead to improved postoperative continence rates.Postoperative stress incontinence following operative treatment of prostate cancer represents a considerable percentage of overall male incontinence. Postoperative incontinence following radical prostatectomy ranges between 2.9 and 87% depending on author and patient characteristics. Especially patient-related factors such as body mass index, age, size of prostate, preoperative incontinence, and concomitant diseases as well as classification of incontinence and modality of data collection influence postoperative continence rates. However, recent publications demonstrate the important impact of different operative techniques with regard to postoperative continence.The preservation of the muscular urethral sphincter is of particular importance. Nevertheless, the preservation of further anatomical structures contributes to postoperative continence. Preservation of bladder neck, nerve sparing, and reconstruction of the vesicourethral junction are operative techniques to prevent postoperative incontinence. In the last decade different modifications of the operative technique have been investigated regarding specific effects on postoperative continence. The interpretation of these studies investigating these operative techniques showed improvement in early continence with shorter period of time to continence. Long-term follow-up revealed no significant advantages for these modifications with regard to continence compared to the standard procedure. To evaluate the long-term effects of certain modifications to prevent incontinence, randomized and well powered studies are necessary. It seems to be most likely that a combination of these preventive modifications will lead to improved postoperative continence rates.

Smac mimetic sensitizes renal cell carcinoma cells to interferon-α-induced apoptosis.

The prognosis of metastatic or relapsed renal cell carcinoma (RCC) is still very poor, highlighting the need for new treatment strategies. Here, we identify a cooperative antitumor activity of interferon-α (IFNα) together with the Smac mimetic BV6 that antagonizes antiapoptotic IAP proteins. BV6 and IFNα act together to reduce cell viability and to induce apoptosis in various RCC cell lines. Molecular studies revealed that BV6/IFNα co-treatment triggers apoptosis independently of autocrine/paracrine Tumor Necrosis Factor (TNF)α signaling, since the TNFα-blocking antibody Enbrel fails to rescue cell death. Importantly, knockdown of Receptor-Interacting Protein (RIP)1 significantly decreases BV6/IFNα-mediated apoptosis, whereas the RIP1 kinase inhibitor necrostatin-1 (Nec-1) provides no protection. This demonstrates that RIP1 protein is critically required for BV6/IFNα-induced apoptosis, while RIP1 kinase activity is dispensable, pointing to a scaffold function of RIP1. Consistently, BV6 and IFNα cooperate to trigger the interaction of RIP1, Fas-Associated Death Domain protein (FADD) and caspase-8 to form a cytosolic cell death complex that drives caspase activation. Addition of the broad-range caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk) significantly protects RCC cells against BV6/IFNα-induced apoptosis, demonstrating that caspase activity is required for apoptosis. In conclusion, the combination approach of IFNα and BV6 represents a promising strategy for cooperative induction of apoptosis in RCC cells, which warrants further investigation.