Effect of resveratrol on microrna profile and apoptosis in hl-60 leukemia and raji lymphoma cells

Amaç: Bu çalışmanın amacı; HL-60 (promyelositik lösemi) ve Raji (Burkitt lenfoma) hücrelerinde, resveratrol uygulaması sonrası mikroRNA (miRNA) profillerinin belirlenmesi ve apoptoz oranlarının immünohistokimyasal metodlarla belirlenmesidir. Gereç ve Yöntemler: Deney dizaynı dört hücre kültürü grubu üzerinden yapılmıştır; 1. grup: HL-60 (Kontrol), 2. grup: HL-60 + resveratrol, 3. grup: Raji ve 4. grup: Raji + resveratrol. Ardından iki tekrarlı mikroarray analizi yapılmış, biyoenformatik analiz ile farklı miRNA ekspresyonları saptanmış ve sonrasında eş zamanlı polimeraz zincir reaksiyonu (EZ-PZR) ile validasyon yapılmıştır. Ayrıca TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) yöntemi ve immünohistokimyasal belirteçler ile apoptoz oranı belirlenmiştir. Bulgular: Mikroarray analizi sonrasında HL-60 (kontrol) ve HL-60 + resveratrol grupları arasında farklı eksprese olan sekiz adet miRNA (hsa-miR18a, hsa-let-7d, hsa-let-7b, hsa-miR-1246, hsa-miR-320b, hsa-miR-92a, hsa-miR-609 ve hsa-miR- 337-3p) saptanmıştır. Resveratrol verilen Raji grubunda kontrol grubuna kıyasla bir tane farklı miRNA (hsa-miR-378) eksprese olmuştur. Validasyon amacıyla yapılan EZ-PZR’de dört adet miRNA (miR-18a, miR-92a, miR-320a, miR-378) ile konfirmasyon gösterilmiştir. Kontrol grubuna kıyasla resveratrol verilen HL-60 grubunda apoptoz (TUNEL) oranı 2 kat daha yüksek olarak saptandı. Raji grubu ile resveratrol verilen Raji grubu arasında anlamlı bir fark saptanmamış olmakla birlikte resveratrol verilen grupta apoptoz oranında bir artma eğilimi saptandı. Bununla birlikte gruplar arasında immünohistokimyasal analizde özellikle Fas ve kaspaz belirteçleri daha belirgin olarak eksprese oldu. Sonuç: Bu çalışmada doğal bir biyoaktif madde olan resveratrolün HL-60 ve Raji hücrelerindeki etkisine bakılmış ve farklı miRNA ekspresyonları saptanmıştır. Ayrıca hücre ölümünün de özellikle kaspaz ve Fas mekanizması üzerinden olduğu düşünülmüştür. Bununla birlikte lösemi ve lenfomadaki olası terapötik potansiyelleri sebebiyle ileri fonksiyonel ve organizma çalışmalarının da yapılması gerekmektedir.Objective: The purpose of this study was to determine the microRNA (miRNA) profiles and the apoptosis rates in the HL-60 (promyelocytic leukemia) and Raji (Burkitt’s lymphoma) cell lines after resveratrol administration. Materials and Methods: The experimental design included four cell culture groups: group 1: HL-60 (control), group 2: HL-60 + resveratrol, group 3: Raji, and group 4: Raji + resveratrol. Then, two repeated microarray analyses were performed. The different miRNA expressions were identified by bioinformatic analysis and after that, validation was performed by simultaneous polymerase chain reaction. Besides, apoptosis ratio was determined with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and immunohistochemical markers. Results: By microarray analysis, eight miRNAs (hsa-mir18 to, hsa-let-7d, hsa-let- 7b, hsa-mir-1246, hsa-miR-320b, hsa-miR-92a, hsa-miR-609 and hsa-miR-337-3p) which have different expression levels between the HL-60 (control) and HL60 + resveratrol groups were detected. There was one miRNA (hsa-miR-378) which was differently expressed between Raji (control) and Raji + resveratrol groups. Four miRNAs (miR-18a, miR-92a, miR-320a, miR-378) expression levels were confirmed with simultaneous polymerase chain reaction for validation. The apoptosis (TUNEL) rate of HL-60 + resveratrol group was two times higher compared to the HL-60 control group. There was no significant difference in apoptosis (TUNEL) rate between the groups of Raji control and Raji + resveratrol. However, it showed a tendency to increase in resveratrol-administrated Raji cells. However, in the immunohistochemical analysis, especially Fas and caspase markers were more prominently expressed. Conclusion: In this study; effects of resveratrol, natural bioactive substances, on HL-60 and Raji cells were evaluated and different miRNA expression levels were detected. In addition, cell death was thought to be especially through the caspase and Fas mechanism. However, considering their therapeutic potential in leukemia and lymphoma further functional and organism studies are needed

The role of histological components and differentiation on survival ın Renal Wilms' tumor

Amaç: Wilms tümörü böbreğin çocukluk çağında görülen en sık malign tümörüdür. Prognoz yeni gelişmelerle giderek iyileşmektedir. Bir trifazik embriyonel tümör olan Wilms tümöründe bulunabilen her üç histolojik komponent (blastemal, stromal, epitelial) değişik miktar ve diferansiyasyonlarda gözlenebilmektedir. Bu çalışmanın amacı çocukluk çağı renalWilms tümöründe histolojik komponentlerin ve diferansiyasyon varlığının sağkalım üzerine etkisini araştırmaktır. Yöntem: Dr. Behçet Uz Çocuk Hastanesinde izlenen ve 1989-1997 yıllarında tanı almış 30 renalWilms tümörü olgusu bu çalışmada yer almaktadır. Olguların yaş ortalaması 51 aydır. 13 olgu erkek, 17 olgu kız çocuğudur. 8 olgu kötü prognozludur (exitus). Bulgular: 4 yıllık sağkalım %73.3' dür. Bir monofazik (blastemal), 7 bifazik tümör vardır. 22 olgu trifaziktir. 14 olguda mezenkimal, 9 olguda blastemal, 7 olguda tübüler komponent baskın bulunmuştur. 8 olguda mezenkimal komponentte çizgili kas diferansiyasyonu, 1 olguda kondroid, 1 olguda skuamöz diferansiyasyon gözlenmiştir. Sonuç: ıstatistiksel olarak bu seride diferansiyasyon varlığının, tümörün histolojik komponentleri yüzdesinin ya da baskınlığının sağkalımla ilişkisi saptanmamıştır.Wilms tümöründe bilinen ve yeni prognostik faktörlerin araştırılmasının izlem ve tedaviyi yönlendirmede yararlı olacağı sonucuna varılmıştır.Objective: Wilms' tumor is the most common malignant neoplasm of kidney in childhood. The patients enjoy a better prognosis by new advances in medicine. Histologically, the classic Wilms' tumor has a triphasic appearance, composed of variable mixtures of blastemal, epithelial and stromal elements. Stromal or epithelial elements may show different levels of differantiation. The aim of this study is to evaluate the role of histological components and the presence of differentiation on survival in pediatric renal Wilms' tumors. Materials and methods: This study includes 30 cases of renal Wilms' tumor diagnosed and followed at Dr. Behçet Uz Children's State Hospital between 1989 and 1997. The mean age of patients was 51 months. Thirteen casesweremaleand 17 caseswere female. Poorprognosis (exitus)was observedin 8 cases. Results: The 4 year survival rate was %73,3. One case was monomorphic (blastemal) while 7 cases were biphasic and 22 cases triphasic. The stromal component was dominant in 14 cases, blastemal component in 9 cases and epithelial component in 7 cases. Striated muscle was observed in 8 cases, cartilage in 1 case and squamous tissue in1 case. Conclusion: Statistical analysis showed no relationship between survival rate, histologic components and occurance of differantiation in this series. We conclude that studying the known and new prognostic factors in Wilms' tumor will be useful in the assessment of patients

Effect of Ecklonia Cava Polyphenol Extract in House Ear Institute-Organ of Corti 1 Cells Against Cisplatin Ototoxicity: A Preliminary Study

Objective:Cisplatin is a widely used agent for the treatment of adult and childhood malignancies. Side effects such as nephrotoxicity, neurotoxicity, and ototoxicity lead to dose limitations. Ecklonia cava polyphenol extract (ECP) is a molecule obtained from algae that live in seawater in the Far East. ECP has recently been shown to have protective effects against oxidative stress. The aim of this study was to evaluate the possible protective effects of ECP on cisplatin ototoxicity.Methods:In this study, we investigated the protective effects of ECP against cisplatin-induced cell death in mouse-derived House Ear Institute Organ of Corti (HEI-OC1) cochlear cells. Cisplatin (100 μM) and 1, 10, and 25 μM doses of ECP were administered to the cells, and the protective effects of ECP at 24 and 72 hours were investigated. Cell viability was evaluated by the WST-1 (water soluble tetrazolium salt).Results:Cisplatin (100 μM) reduced cell viability in both the 24th and 72nd hour evaluation. Although the 25 μM dose of ECP showed otoprotective effects in the 24th hour, in the 72nd hour this effect disappeared. Other doses of ECP showed no otoprotective effects in the 24th and 72nd hours.Conclusion:Although ECP showed some protective effects in the 24th hour against cisplatin ototoxicity, these effects disappeared by the 72nd hour. Further studies using recurrent and higher doses of ECP are required

Clinical Significance Of DNA Repair Genes Expressions In Neurofibromatosis Type 1 Cases

AMAÇ: Nörofibromatozis Tip 1 (NF1), çeşitli fenotiplere sahip, sık görülen otozomal dominant genetik bir hastalıktır. NF1 hastalarının klinik çeşitliliğinin genetik nedeni sorgulanmaktadır. DNA onarım genleri DNA' daki hataların onarımından sorumludur. Bu çalışmada DNA onarım genlerinin ekspresyonunu ve onların NF1 hastalarındaki klinik önemini nörofibrom, hamartomatöz lezyon, diğer tümörler ya da ailesel NF1 varlığı ile karşılaştırarak analiz etmek ve gen ekspresyonları ile klinik bulgular arasında ilişki olup olmadığını belirlemek amaçlandı. YÖNTEMLER: NF1' li hastalar ve NF1 ile birlikte malignitesi olan hastalar çalışmaya alındı. Kontrol grubu olarak da benzer yaş grubundaki her hangi bir hastalığı olmayan çocuklar ve NF1 ile ilgisi olmayan maligniteli olgular oluşturdu. Çalışma toplam 46 olgu içermekteydi: 36 NF1 hastası (30 çocuk; 6 ebeveyn), hiç bir hastalığı olmayan 8 kontrol olgusu, rabdomiyosarkomlu NF1 olmayan 2 kontrol olgusu çalışmaya alındı. Her bir hasta ve kontrol grubundan periferik kandan mononükleer hücre izolasyonu yapıldı. RNA izolasyonu ve cDNA dönüşümünden sonra, her bir olguda Real-Time PCR ile DNA onarımı ile ilişkili 84 genin ekspresyonu (standart array, SABiosciences) belirlendi. Ekspresyonların kontrol grubuna göre kat değişiklikleri ve T test ile p değeri karşılaştırmalı gruplarda değerlendirildi. BULGULAR: Araştırma grubunu 36 NF1 hastası, kontrol grubunu ise 8 sağlam çocuk ve 2 adet de NF1 ile ilişkisi olmayan maligniteli olgu (rabdomiyosarkom) oluşturmaktadır. 8 kontrol olgusunun yaş ortalaması 17±7,03 (10-30 yaş) (ortanca 13 yaş) idi. NF1 olgularının 17' si kadın 19' u erkekti. NF1' li olgularımız için tanı anındaki yaş ortalaması 10,08±8,86 (9 ay- 38 yaş) (ortanca 8 yaş) iken hastalarımızın çalışmaya alınan ebeveynlerinin yaş ortalaması 40,50±1,22 (39-42 yaş) (ortanca40 yaş) idi. 36 hastanın, 17' si nörofibromlu, 17' si hamartomatöz lezyonluydu. 1 hastada rabdomiyosarkom (RMS) gözlenmiş, 1 hasta meme kanseri ve 4 hasta da optik gliomluydu. NF1 olgularında, PNKP, RAD18, XAB2, XRCC3, XRCC4 ve XRCC5 genlerinin ekspresyonu kontrol grup ile karşılaştırıldığında azaldı (p<0.05, T test). Nörofibromlu NF 1 olgularında, nörofibromsuz NF 1 olgularıyla karşılaştırıldığında POLB ekspresyonu artarken; ERCC3,LIG1,MGMT, MRE11A, MPG, MSH6, PARP2, PRKDC, RAD51B, RAD52, RPA3, SMUG1, TREX1, UNG ekspresyonu azaldı. RAD18 ailesel NF 1 varlığında ekspresyonu azalmış ve istatistiksel olarak önemli olduğu saptandı. Malign tümör olgularında NF 1' li ya da NF 1' siz gruplar karşılaştırıldığında gen ekspresyonunda kat değişiklikleri vardı. Maligniteli olgularda DDB2, MGMT, MLH1, POLB UNG, XPA ekspresyonları arttı. NF 1'li RMS olgusu ile NF 1' siz RMS olguları karşılaştırıldığında DDB2, MGMT, MLH1, POLB, UNG, XPA olmak üzere 6 genin ekspresyonu 10 kattan fazla artmış saptandı. SONUÇ: Bulgularımız NF 1 olgularındaki klinik bulgulardan tümör gelişimini öngörmek için DNA onarım sistemi ilişkili gen ekspresyon değişikliklerinin rolü olabileceğini göstermiştir. POLB nörofibrom varlığı belirteci, DDB2, MGMT, MLH1, POLB UNG, XPA malign tümör gözlenme belirteci olmaya aday genler olarak saptandı. Bu genlerin ekspresyonlarının daha geniş seri NF1li ve maligniteli olgularda çalışılması uygundur. OBJECTIVE: Neurofibromatosis Type 1 (NF1) is a a common autosomal dominant genetic disorder that has a variable phenotype. The genetical causes of clinical variability of NF1 patients is questioned. DNA repair genes are responsible for proofreading the missing in the DNA. We aimed to analyze expression of DNA repair genes and their clinical significance in NF1 patients; comparing exsistance of neurofibroma or hamartomatous lesions or other tumours or existance of NF1 in the family. The other aim of this study was to determine whether any relationship between gene expressions and clinical findings. METHODS: NF1 patients and malignancy with NF1 pateints were included and in this study. In the control gruop children that they are in the similar age group and they have no disease and no malignacy group were included. This study included total 46 cases. 36 NF1 patients (30 children; 6 parents), 8 control cases without any disease, two control cases with rhabdomyosarcoma without NF1 were included in this study. The mean age of control group was 17±7,03 (10-30 age) (median 13 age). In the NF1 pateints gruop 17 of them are female, and 19 are male. The mean age at diagnosis is 10,08±8,86 (9 months- 38 age)(median age 8) for children and 40,50±1,22 (39-42 age) (median age 40) for parents. Among 36 patients, 17 had neurofibromas, 17 had hamartomatous lesions. Rhabdomyosarcoma (RMS) was observed in one patient, breast cancer in one patient and four patients suffered optic glioma. Peripheral blood was obtained from each cases and mononuclear cells were separated. After RNA isolation and cDNA converting, expressions of 84 genes related with DNA Repair in standard array (SABiosciences) were determined by Real-Time PCR for each case. Fold changes and p values compared with control groups and fold changes evaluated with T test and p value in the comperative groups. RESULTS: 36 NF1 patients, 8 healthy children as a control and 2 cases no NF1 relationship with malignancy (rhabdomyosarcoma) were included in the study group. In NF1 cases PNKP, RAD18, XAB2, XRCC3, XRCC4 and XRCC5 genes were downregulated compared with control group. In NF1 cases having neurofibromas, POLB was over expressed; while ERCC3, LIG1, MGMT, MRE11A, MPG, MSH6, PARP2, PRKDC, RAD51B, RAD52, RPA3, SMUG1, TREX1, UNG were downregulated compared with the NF1 cases without neurofibromas (p<0.05, T test). RAD18 is the downregulated and statistical significant gene for existence of NF1 in the family. There are gene expression fold change differences determined when malign tumor cases with/without NF1 were compared. DDB2, MGMT, MLH1, POLB UNG, XPA are increased.   CONCLUSION: Our results may point toward a role of gene expression changes of DNA repair system to be predictive for clinical manifestations in NF1 cases

Effect of Korean Red Ginseng on Noise-Induced Hearing Loss

Objective:Noise-induced hearing loss (NIHL) is one of the most important problems affecting both social and professional life of patients. There is no treatment method considered to be successful on the hearing loss that has become a permanent nature. Aim of this study is to evaluate protective effect of Korean Red Ginseng (KRG) against NIHL in an animal model.Methods:Twenty-eight rats were separated into four groups [control saline (group I), control KRG (group II), saline + noise (group III), KRG + noise (group IV)]. Rats in the saline and KRG groups were fed via oral gavage with a dose of 200 mg/kg/day throughout for 10 days. Fourteen rats (group III and IV) were exposed to 4 kHz octave band noise at 120 dB SPL for 5 hours. Hearing levels of rats were evaluated by distortion product otoacoustic emissions (DPOAE) and auditory brainstem responses (ABR) at 4, 8, 12, 16 and 32 kHz frequencies prior to and on days 1, 7 and 10 after the noise exposure. Rats were sacrificed on 10th day, after the last audiological test. Cochlea and spiral ganglion tissues were evaluated by light microscopy.Results:Audiological and histological results demonstrated that after noise the group IV showed better results than group III. In the noise exposed groups, the most prominent damage was seen at the 8 kHz frequency region than other regions. After the noise exposure, DPOAE responses were lost in 1st, 7th and 10th measurements in both group III and IV. Thus, we were not able to perform any statistical analyses for DPOAE results.Conclusion:Our findings suggest that KRG seems to be an efficient agent against NIHL. There is need for additional research to find out about the mechanisms of KRG’s protective effect

Future aspects of immunotherapy and gene therapy in neuroblastoma

Immunotherapy against cancer aims at stimulating the immune system or building an immune response against targeted tumor-associated antigens (TAAs). It was proposed theoretically as a potential therapy for cancer over a century ago but it became popular in the past two decades. Gene therapy represents a promising approach for reversing the neoplastic phenotype or driving tumor cells to self-destruction. Although survival rates of neuroblastoma (NB) with biologically favorable disease are greater than 90%, outcomes of patients with high risk disease are less than 40%. Stage 4 metastatic NB cases over 18 months of age are often incurable with multimodality chemotherapy regimens. In this article, translation of immuno-gene therapy strategies into clinical trials for NB are reviewed. Future aspects of immuno-gene therapy are discussed. © 2009 Zerbinis Medical Publications