Gal4 Driver Transgenic Zebrafish: Powerful Tools to Study Developmental Biology, Organogenesis, and Neuroscience.

Targeted expression by the Gal4-UAS system is a powerful genetic method to analyze the functions of genes and cells in vivo. Although the Gal4-UAS system has been extensively used in genetic studies in Drosophila, it had not been applied to genetic studies in vertebrates until the mid-2000s. This was mainly due to the lack of an efficient transgenesis tool in model vertebrates, such as the P-transposable element of Drosophila, that can create hundreds or thousands of transgene insertions in different loci on the genome and thereby enables the generation of transgenic lines expressing Gal4 in various tissues and cells via enhancer trapping. This situation was revolutionized when a highly efficient transgenesis method using the Tol2 transposable element was developed in the model vertebrate zebrafish. By using the Tol2 transposon system, we and other labs successfully performed gene trap and enhancer trap screens in combination with the Gal4-UAS system. To date, numerous transgenic fish lines that express engineered versions of Gal4 in specific cells, organs, and tissues have been generated and used for various aspects of biological studies. By constructing transgenic fish lines harboring genes of interest downstream of UAS, the Gal4-expressing cells and tissues in those transgenic fish have been visualized and manipulated via the Gal4-UAS system. In this review, we describe how the Gal4-UAS system works in zebrafish and how transgenic zebrafish that express Gal4 in specific cells, tissues, and organs have been used for the study of developmental biology, organogenesis, and neuroscience.

Tol2-mediated transgenesis, gene trapping, enhancer trapping, and Gal4-UAS system.

The Tol2 element is an active transposon that was found from the genome of the Japanese medaka fish. Since the Tol2 transposition system is active in all vertebrate cells tested so far, it has been applied to germ line transgenesis in various model animals including fish, frog, chicken, and mouse, and to gene transfer in culture cells. In zebrafish, the Tol2 system consists of the transposase mRNA and a Tol2 transposon-donor plasmid, and is introduced into fertilized eggs by microinjection. Thus genomic integrations of the Tol2 construct are generated in the germ lineage and transmitted to the offspring very efficiently. By using the Tol2 transposition system, we have developed important genetic methods, such as transgenesis, gene trapping, enhancer trapping, and the Gal4-UAS system in zebrafish and applied to many aspects of biological studies. In this chapter, we describe how these methods are performed.

Anti-factor IXa/X bispecific antibody (ACE910): hemostatic potency against ongoing bleeds in a hemophilia A model and the possibility of routine supplementation.

BACKGROUND: We previously reported that a humanized anti-factor IXa/X bispecific antibody, hBS23, mimics the function of FVIII even in the presence of FVIII inhibitors, and has preventive hemostatic activity against bleeding in an animal model of acquired hemophilia A. After further molecular engineering of hBS23, we recently identified an improved humanized bispecific antibody, ACE910, for clinical investigation. OBJECTIVES: To elucidate the in vivo hemostatic potency of ACE910 by examining its effect against ongoing bleeds, and to determine its pharmacokinetic parameters for discussion of its potency for prophylactic use. METHODS: A non-human primate model of acquired hemophilia A was established by injecting anti-primate FVIII neutralizing antibody. When bleeds emerged following an artificial bleed-inducing procedure, either ACE910 or recombinant porcine FVIII (rpoFVIII) was intravenously administered. rpoFVIII was additionally administered twice daily on the following 2 days. Bleeding symptoms were monitored for 3 days. A pharmacokinetic study and multiple-dosing simulations of ACE910 were also performed. RESULTS: A single bolus of 1 or 3 mg kg-1 ACE910 showed hemostatic activity comparable to that of 10 U kg-1 (twice daily) rpoFVIII against ongoing bleeds. The determined ACE910 pharmacokinetic parameters included a long half-life (3 weeks) and high subcutaneous bioavailability (nearly 100%). The simulation results based on pharmacokinetic parameters indicated that the above hemostatic level could be maintained with once-weekly subcutaneous administration of ACE910, suggesting the possibility of more effective prophylaxis. CONCLUSIONS: ACE910 may offer an alternative on-demand treatment option for patients with hemophilia A, as well as user-friendly and aggressive routine supplementation.

Anti-factor IXa/X bispecific antibody (ACE910): hemostatic potency against ongoing bleeds in a hemophilia A model and the possibility of routine supplementation.

BACKGROUND: We previously reported that a humanized anti-factor IXa/X bispecific antibody, hBS23, mimics the function of FVIII even in the presence of FVIII inhibitors, and has preventive hemostatic activity against bleeding in an animal model of acquired hemophilia A. After further molecular engineering of hBS23, we recently identified an improved humanized bispecific antibody, ACE910, for clinical investigation. OBJECTIVES: To elucidate the in vivo hemostatic potency of ACE910 by examining its effect against ongoing bleeds, and to determine its pharmacokinetic parameters for discussion of its potency for prophylactic use. METHODS: A nonhuman primate model of acquired hemophilia A was established by injecting anti-primate FVIII neutralizing antibody. When bleeds emerged following an artificial bleed-inducing procedure, either ACE910 or recombinant porcine FVIII (rpoFVIII) was intravenously administered. rpoFVIII was additionally administered twice daily on the following 2 days. Bleeding symptoms were monitored for 3 days. A pharmacokinetic study and multiple-dosing simulations of ACE910 were also performed. RESULTS: A single bolus of 1 or 3 mg kg⁻¹ ACE910 showed hemostatic activity comparable to that of 10 U kg⁻¹ (twice daily) rpoFVIII against ongoing bleeds. The determined ACE910 pharmacokinetic parameters included a long half-life (3 weeks) and high subcutaneous bioavailability (nearly 100%). The simulation results based on pharmacokinetic parameters indicated that the above hemostatic level could be maintained with once-weekly subcutaneous administration of ACE910, suggesting the possibility of more effective prophylaxis. CONCLUSIONS: ACE910 may offer an alternative on-demand treatment option for patients with hemophilia A, as well as user-friendly and aggressive routine supplementation.

An affective disorder in zebrafish with mutation of the glucocorticoid receptor.

Upon binding of cortisol, the glucocorticoid receptor (GR) regulates the transcription of specific target genes, including those that encode the stress hormones corticotropin-releasing hormone (CRH) and adrenocorticotropic hormone. Dysregulation of the stress axis is a hallmark of major depression in human patients. However, it is still unclear how glucocorticoid signaling is linked to affective disorders. We identified an adult-viable zebrafish mutant in which the negative feedback on the stress response is disrupted, due to abolition of all transcriptional activity of GR. As a consequence, cortisol is elevated, but unable to signal through GR. When placed into an unfamiliar aquarium ('novel tank'), mutant fish become immobile ('freeze'), show reduced exploratory behavior and do not habituate to this stressor upon repeated exposure. Addition of the antidepressant fluoxetine to the holding water and social interactions restore normal behavior, followed by a delayed correction of cortisol levels. Fluoxetine does not affect the overall transcription of CRH, the mineralocorticoid receptor (MR), the serotonin transporter (Serta) or GR itself. Fluoxetine, however, suppresses the stress-induced upregulation of MR and Serta in both wild-type fish and mutants. Our studies show a conserved, protective function of glucocorticoid signaling in the regulation of emotional behavior and reveal novel molecular aspects of how chronic stress impacts vertebrate brain physiology and behavior. Importantly, the zebrafish model opens up the possibility of high-throughput drug screens in search of new classes of antidepressants.

Transcriptional suppression of BACH2 by the Bcr-Abl oncoprotein is mediated by PAX5.

Bach2 is a lymphoid-specific transcription factor with a prominent role in B-cell development and apoptosis-induction in response to oxidative stress. We previously showed that Bach2 is downregulated in chronic myeloid leukaemia (CML), and here we demonstrate the mechanism by which Bcr-Abl mediates this phenomenon. We have cloned a 3.9 Kb genomic DNA fragment upstream of the transcription initiation site, and delineated the core and proximal BACH2 promoter regions. Transient BCR-ABL expression led to significant reduction in BACH2 promoter activity and this effect was dependent on the kinase function of the oncoprotein. Sequential deletions disclosed several regulatory elements within the promoter region, as well as within BACH2 exonic sequences. Analysis of these elements and transient transfection assays led to the identification of the Pax5 transcription factor as a potent trans-activator of BACH2, whose effect is predominantly mediated through occupation of a binding site on the BACH2 promoter, as demonstrated by both in vitro and in vivo experiments. Overall, our data show that Pax5 functions as an intermediate effector in the Bcr-Abl-mediated transcriptional repression of BACH2. The current results, combined with previous reports, establish Pax5 and Bach2 as transcriptional targets of Bcr-Abl, whose downregulation may contribute to lymphoid blast crisis of CML.

Response and safety of sunitinib in a heavily pre-treated metastatic non-small cell lung carcinoma patient.

BACKGROUND: The activity of sunitinib, a multitargeted tyrosine kinase inhibitor with antiangiogenic and antitumor activities, has been explored in several solid malignancies such as breast, lung, prostate and pancreatic cancer. Currently it is approved for the treatment of metastatic renal cell carcinoma and gastrointestinal stromal tumors. Non-small cell lung cancer usually presents at an advanced or metastatic stage at diagnosis. Treatment options are limited for this disease, therefore symptom palliation and patient's quality of life are primary objectives of therapy. CASE REPORT: We describe the case of a patient (male, 67 years old) with heavily pre-treated metastatic non-small cell lung carcinoma who received sunitinib according to the following 3-week schedule: 50 mg daily for 2 weeks followed by a 1-week rest. The patient completed six months of therapy achieving a major disease response without high-grade toxicities. CONCLUSION: In this case, sunitinib shows promising single-agent activity in pretreated non-small cell lung cancer, with a good toxicity profile and flexible administration schedule.

Expression and localization of vascular endothelial growth factor in normal abdominal aorta and abdominal aortic aneurysm.

AIM: The genesis of abdominal aortic aneurysms is associated with remodeling of the vascular wall by angiogenesis as well as proteolysis. Vascular endothelial growth factor (VEGF) is known to be a regulator of angiogenesis and to simultaneously stimulate elastolytic proteinases. We analyzed the expression and localization of VEGF in human abdominal aortic aneurysms compared to normal human aorta METHODS: Eighteen infrarenal aortic aneurysm samples were collected at the time of abdominal aortic aneurysm surgery, while nine normal aortic samples were obtained from autopsy specimens. Immunohistochemical staining was performed to detect VEGF. Immunoenzyme or immunofluorescent double staining was also used to identify those cells presenting VEGF. RESULTS: VEGF was expressed in 18 (100%) of the 18 abdominal aortic aneurysm samples, while 0 (0%) in the 9 normal abdominal aorta samples. Of the 18 samples of aneurysms, all 18 displayed positive VEGF immunostaining in macrophages, 12 in smooth muscle cells (SMCs), and 9 in endothelial cells (ECs). CONCLUSION: Our study clearly demonstrated the expression of VEGF in ECs, and SMCs, and macrophages of abdominal aortic aneurysms as well as its absence in those cells of normal abdominal aorta, suggesting that VEGF may play an important role in aneurysm formation via its direct and/or indirect actions.

Prostaglandin receptors EP2 and IP are detectable in atherosclerotic arteries and plaques.

AIM: Prostaglandin (PG) receptor agonists are frequently used for the pharmacological treatment of arteriosclerosis obliterans (ASO). In particular, the PG receptors EP2 and IP stimulate vasodilation and inhibit platelet aggregation, biological processes thought to be protective against ASO and important for physiological homeostasis. However it is uncertain whether EP2 and IP exist in diseased arteries, or what their distribution within the artery might be. In this study, we analyzed the distribution of these PG receptors in patients with severe ASO to determine the potential application of stimulation of these receptors as targets for pharmacological treatment. METHODS: We collected segments of atherosclerotic femoral arteries during femoropopliteal bypass surgery and determined the expression levels of EP2 and IP receptors by western blotting. Immunofluorescence was used to observe receptor localization. RESULTS: Findings of western blotting showed an increased Cox-2 expression in patients with ASO. The EP2 as well as IP receptors were each induced approximately 3-fold in comparison to normal samples. The expression of these receptors was increased in the intimal layer as well as the medial layer; their expression was also detectable within the atherosclerotic plaque. CONCLUSION: We observed induction of the PG receptors EP2 and IP in atherosclerotic femoral arteries in the arterial intima, medial layer, as well as the associated atherosclerotic plaque. These results suggest that receptor-selective PG agonists specifically target atherosclerotic arteries and therefore, may find potential application in the pharmacological management of patients with ASO.

Glycogen synthase kinase-3: a new therapeutic target in renal cell carcinoma.

BACKGROUND: Renal cell carcinoma (RCC) is highly resistant to chemotherapy because of a high apoptotic threshold. Recent evidences suggest that GSK-3beta positively regulates human pancreatic cancer and leukaemia cell survival in part through regulation of nuclear factor (NF-kappaB)-mediated expression of anti-apoptotic molecules. Our objectives were to determine the expression pattern of GSK-3beta and to assess the anti-cancer effect of GSK-3beta inhibition in RCC. METHODS: Immunohistochemistry and nuclear/cytosolic fractionation were performed to determine the expression pattern of GSK-3beta in human RCCs. We used small molecule inhibitor, RNA interference, western blotting, quantitative RT-PCR, BrDU incorporation and MTS assays to study the effect of GSK-3beta inactivation on renal cancer cell proliferation and survival. RESULTS: We detected aberrant nuclear accumulation of GSK-3beta in RCC cell lines and in 68 out of 74 (91.89%) human RCCs. We found that pharmacological inhibition of GSK-3 led to a decrease in proliferation and survival of renal cancer cells. We observed that inhibition of GSK-3 results in decreased expression of NF-kappaB target genes Bcl-2 and XIAP and a subsequent increase in renal cancer cell apoptosis. Moreover, we show that GSK-3 inhibitor and Docetaxel synergistically suppress proliferation and survival of renal cancer cells. CONCLUSIONS: Our results show nuclear accumulation of GSK-3beta as a new marker of human RCC, identify that GSK-3 positively regulates RCC cell survival and proliferation and suggest inhibition of GSK-3 as a new promising approach in the treatment of human renal cancer.

Thermoelectric properties and efficiency measurements under large temperature differences.

The maximum efficiency of a thermoelectric generator is determined by the material's dimensionless figure of merit ZT. Real thermoelectric material properties are highly temperature dependent and are often measured individually using multiple measurement tools on different samples. As a result, reported ZT values have large uncertainties. In this work we present an experimental technique that eliminates some of these uncertainties. We measure the Seebeck coefficient, electrical conductivity, and thermal conductivity of a single element or leg, as well as the conversion efficiency, under a large temperature difference of 2-160 degrees C. The advantages of this technique include (1) the thermoelectric leg is mounted only once and all measurements are in the same direction and (2) the measured properties are corroborated by efficiency measurements. The directly measured power and efficiency are compared to the values calculated from the measured properties and agree within 0.4% and 2%, respectively. The realistic testing conditions of this technique make it ideal for material characterization prior to implementation in a real thermoelectric generator.

Hybrid surgical and endovascular therapy in multifocal peripheral TASC D lesions: up to three-year follow-up.

AIM: The authors described their three-year experience with hybrid surgical and endovascular therapy for multifocal peripheral TASC D lesions, involving both the aortoiliac and/or superficial femoral and common femoral arteries. METHODS: From February 2005 to March 2008, 21 lower limbs in 20 patients with multifocal peripheral artery disease, involving the aortoiliac and/or superficial femoral as well as common femoral arteries, were treated by hybrid surgical and endovascular therapy, such as aortoiliac and/or superficial femoral artery stenting as an adjunct to common femoral artery endarterectomy. Technical and hemodynamic success as well as primary and primary assisted patency and limb salvage rates were determined in concordance with the Society for Vascular Surgery guidelines. RESULTS: All lower limbs successfully underwent successful hybrid surgical and endovascular therapy. The average ABPI before and after hybrid therapy significantly increased from 0.50 +/- 0.32 to 0.79 +/- 0.24 (P = 0.0022). The mean duration of follow-up was 357 days (range, 4 to 1400 days). Over all, the primary patency rates were 94%, 70% and 70% at 6, 12, and 24 months, respectively, and the primary assisted patency rates were 94% at 24 months. The limb salvage rate was 100% at 24 months. The survival rates were 95%, 88%, and 88% at 6, 12, and 24 months, respectively. The primary patency rate for intermittent claudication was significantly higher that that for critical limb ischemia, while no significant difference was found in the assisted primary patency and survival rates between intermittent claudication and critical limb ischemia. CONCLUSION: Hybrid surgical and endovascular therapy, such as aortoiliac and/or superficial femoral artery stenting as an adjunct to common femoral artery endarterectomy, can provide a less invasive yet effective and durable option to patients with multifocal peripheral artery disease.

Fate of varicose veins after great saphenous vein stripping alone.

AIM: The aim of this study was to observe prospectively the clinical sequelae of varicose veins after great saphenous vein (GSV) stripping alone, and to examine whether spontaneous varicose vein regression or disappearance continued for a long period (>3 years). METHODS: Thirty-nine consecutive patients (20 males and 19 females; mean age 57.2), who underwent GSV stripping in Fujita Health University (55 limbs) between November 1, 2002 and December 31, 2003 were enrolled. RESULTS: At four to six weeks, varicose veins spontaneously resolved in 50 limbs (91%), in which subsequent sclerotherapy was not necessary. Five limbs subsequently underwent sclerotherapy for residual varicose veins (5%). At more than three years, 49 limbs (89%) completed the follow-up study. The recurrence after GSV stripping alone occurred in four of the 45 limbs (9%), while those of GSV stripping with sclerotherapy was one of the four limbs (25%). CONCLUSIONS: This study definitely demonstrated that spontaneous varicose vein resolution can continue for more than three years after GSV stripping alone, suggesting that varicectomy can be deferred or avoided in many patients.

Stent placement in the superficial femoral artery for patients on chronic hemodialysis with peripheral artery disease.

AIM: Chronic hemodialysis is associated with a high prevalence of peripheral artery disease (PAD), and patients on chronic hemodialysis with PAD have an increased risk of critical limb ischemia. The present study assessed the hemodynamic and clinical outcomes of stent placement in the superficial femoral artery (SFA) for patients on chronic hemodialysis. METHODS: Between February 2005 to August 2008, 43 consecutive lower limbs in 42 patients with SFA lesions that were successfully treated by primary stent placement were included in this study. Those were divided into a dialysis group (18 limbs) and a nondialysis group (25 limbs). Outcome measures included primary patency, assisted primary patency, limb salvage, and survival. RESULTS: Patients were significantly younger and presented with significantly more symptomatic limb ischemia in the dialysis group compared to the nondialysis group, despite comparable TransAtlantic Inter-Society Consensus (TASC) classification scores of SFA lesions between the two groups. The primary patency, primary assisted patency, limb salvage, and survival rates of the dialysis group were similar to those of the nondialysis group. CONCLUSIONS: Stent placement in the SFA is a feasible, safe, and effective procedure in patients on chronic hemodialysis with PAD, and may be offered as a first-choice therapeutic option for these patients.

Molecular targeting of Bcl-2 overcomes prostate cancer cell adaptation to XIAP gene downregulation.

X-linked inhibitor of apoptosis (XIAP) is a suppressor of apoptosis that supports an increased survival and resistance to chemotherapy of human prostate cancer (PCa) cells. Effects of transient (24 h) and chronic (beyond 1 month) downregulation of XIAP in DU145 hormone refractory prostate cancer (HRPC) cells were studied. We found that transient downregulation of XIAP by siRNAs resulted in an increase of apoptosis and a decrease in Bcl-2 levels and sensitized PCa cells to cisplatin. XIAP downregulation by shRNA vector stable transfection led to upregulation of Bcl-2 protein. Our results identify the adaptability of PCa cells to chronic loss of XIAP in part through upregulation of Bcl-2 and indicate that multitargeting approach is the most effective application in the chemotherapy of human HRPC.

Inhibition of Bach1 ameliorates indomethacin-induced intestinal injury in mice.

BTB and CNC homolog 1 (Bach1) is a transcriptional repressor of heme oxygenase-1 (HO-1). It plays an important role in the feedback regulation of HO-1 expression, which protects cells from various insults including oxidative stress and inflammatory cytokines. However, the role of Bach1 in intestinal inflammation remains unclear. In this study, the role of Bach1 in intestinal mucosal injury was elucidated using 8-week-old female C57BL/6 (wild-type) and homozygous Bach1-deficient C57BL/6 mice. Intestinal mucosal injuries induced by a single subcutaneous administration of indomethacin were evaluated macroscopically, histologically, and biochemically. Mucosal protein content and chemokine mRNA levels were determined by real-time PCR. Our results showed that the indomethacin-induced intestinal injury was remarkably improved in Bach1-deficient mice. Histological examination showed that the area of injured lesion was decreased in Bach1-deficient mice compared to wild-type mice. Administration of indomethacin induced expression of inflammatory chemokines such as KC, MIP1alpha and MCP1, which was suppressed in Bach1-deficient mice. Myeloperoxidase activity in the intestinal mucosa was also significantly decreased in Bach1-deficient mice. Additionally, Bach1 deficiency enhanced immunopositivity of HO-1 in the intestinal mucosa after indomethacin administration. Disruption of the Bach1 gene thus caused inhibition of mucosal injury, indicating that inhibition of Bach1 may be a novel therapeutic strategy for treating indomethacin-induced intestinal injury.

Relationship between parameters of air plethysmography and types of superficial venous reflux in patients with primary varicose veins.

AIM: We investigated whether parameters of air plethysmography (APG) were correlated with types of superficial venous reflux as categorized by ascending venography in patients with primary varicose veins. METHODS: Two hundred and eight limbs with primary varicose veins in 135 patients were evaluated by both APG and ascending venography. Venous hemodynamics was assessed with APG. The location of incompetent vein segments was determined based on the results of ascending venography. RESULTS: Seventy-seven limbs had incompetence of the greater saphenous vein (GSV, G group), 36 had incompetence of the lesser saphenous vein (LSV, L group), and 77 had incompetence of the GSV and LSV (GL group). Twenty-five limbs did not have incompetence of the GSV or LSV (N group). The venous filling index (VFI) differed significantly between the N and the G and GL groups, the L group and the G and GL groups, and the G and GL groups. No significant difference was found between the N and L groups. The venous volume, ejection fraction, and residual volume fraction did not differ significantly among all four groups. CONCLUSION: The VFI as measured by APG discriminates well between limbs with incompetence of the GSV and those without incompetence of the GSV or LSV, and between limbs with incompetence of the GSV and those with the LSV in patients with primary varicose veins, suggesting that the hemodynamic severity of superficial venous reflux progresses with involvement from the LSV to the GSV to both saphenous veins.

Protective effect of sivelestat sodium (Eraspol) on postoperative lung dysfunction in patients with type A acute aortic dissection: a pilot study.

AIM: The authors evaluated the protective effect of sivelestat sodium on postoperative lung dysfunction in patients with type A acute aortic dissection who underwent aortic arch surgery with cardiopulmonary bypass (CPB) under deep hypothermia with circulatory arrest (DHCA). METHODS: Twelve patients with type A acute aortic dissection who underwent aortic arch replacement under CPB with DHCA and were pretreated with or without sivelestat sodium (sivelestat group, N.=7 patients; control group, N.=5 patients) were observed. The ratio of arterial oxygen tension to inspired oxygen fraction (P/F ratio) was measured as a parameter of pulmonary function before and after operation. The number of white blood cells was also counted as an index of inflammatory reaction before and after the operation. RESULTS: The P/F ratio decreased significantly after operation in the control group. However, the P/F ratio was unchanged between before and after operation in the sivelestat group. The number of white blood cells tended to increase after operation in the control group, whereas it decreased significantly after operation in the sivelestat group. CONCLUSION: The present study demonstrated the protective effect of sivelestat sodium on postoperative lung injury in patients with acute type A aortic dissection undergoing aortic arch surgery under CPB with DHCA.

Double inhibition of XIAP and Bcl-2 axis is beneficial for retrieving sensitivity of renal cell cancer to apoptosis.

Renal cell carcinoma (RCC) is known to be resistant to chemo- and radiotherapy due to a high apoptotic threshold. Smac and XIAP (X-linked inhibitor of apoptosis protein) proteins were detected in all RCC cell lines and tissue samples examined. We modulated the function of XIAP, either through its constitutional downregulation with an shRNA vector or by applying a Smac-mimicking peptide. Among RCC cell lines, Caki1 expresses the highest levels of XIAP. We transfected Caki1 with XIAP-targeting shRNA vector and generated stable clones. XIAP was knocked down by RNA interference in clone no. 14 by 81.6% and in clone no. 19 by 85.3%. Compared to the parental and mock-transfected cells, neither clone was more sensitive to conventional chemotherapeutic agents, but both clones were more susceptible to Fas stimulation (P<0.0001) and to pharmacological Bcl-2 inhibition (P<0.0001), as well as to a combination of the two (P<0.0001). Mature Smac binds to XIAP via the N-terminal residues, disrupting its interaction with caspases and promoting their activity. We determined that exposure of Caki1 cells to Smac-N7 peptide (AVPIAQK) resulted in a slight but significant decrease in viability (P=0.0031) and potentiated cisplatin's effect (P=0.0027). In contrast with point targeting of XIAP by shRNA, Smac-N7 peptide is active against several IAP (inhibitor of apoptosis protein) family members, which can explain its role in sensitising cells to cisplatin. Our results suggest that multiple targeting of both Bcl-2 and XIAP or, alternatively, of several IAP family members by the Smac-N7 peptide is a potent way to overcome resistance of RCC to apoptosis-triggering treatment modalities, and might be a new tool for molecular targeted therapy.