Cisplatin ve metformin iilaç kombinasyonu uygulamasının prostat hücre hatları proliferasyonu ve GSTP, CYP17A1 ve Hekzokinaz II enzimleri üzerine etkilerinin incelenmesi.

Prostate cancer is the most common cancer type both in developed and developing countries. Since 2015, it has been seen as one of the main cause of death in males, both in the world and in our country. Current therapeutic approaches for prostate cancer generally have variable efficiency, develop metastasis and drug resistance, and have high toxicity to normal tissues. Hence, the searching for more effective strategies with moderate or any adverse effects for the chemopreventive intervention of those cancers remains one of the important issues in cancer research. Prostate cancer incidence and mortality vary dramatically by geographical location. Both are higher in developed countries. Some attribute this to westernized lifestyles of high energy diets and limited physical activity with consequent obesity. This dramatic increase in the incidence of cancer in overweight population suggests that there is a correlation between obesity and cancer. As a matter of fact, epidemiological and experimental research indicates that men with abdominal obesity is a risk factor for prostate cancer. Because in the case of obesity, people store plenty of glucose. Thus, cancer cells are more easily and quickly divide. According to this information, our aim in this study was reducing cell proliferation by reducing the blood glucose amount that passes from hepatic tissues by using metformin, a Type2 diabetes drug, hence provide the induction of cell death by creating oxidative stress in cells. Cisplatin, a cell division and growth inhibiting agent, is widely used in the treatment of prostate cancer. Cisplatin binds to DNA and forms a DNA adduct. This adduct cause the death of cancer cells by inhibiting transcription and or replication. Despite the high efficiency of cisplatin on prostate cancer treatment not only nephrotoxicity, neurotoxicity and gastrointestinal irritability but also azoospermia, oligospermia and infertility on reproductive age restrict the use of cisplatin. In order to reduce the side effects of cisplatin, combination therapy with other agents has been the focus of scientists in recent years. Considering the inducer effect of metformin, a type2 diabetic drug, on antiproliferative and apoptotic pathways it can provide a complementary treatment with antineoplastic agents, including cisplatin. GST family enzymes function in the detoxification of xenobiotics, including drugs. Becide, recently it is understood that GST s also function on cell proliferation and death that controls signal transduction. Carcinogenesis, differentiation, growth and important impact on drug resistance and cell death serve GST s as an important drug targets. GST family is also involved in prostaglandin, steroid and leukotriene biosynthesis. In addition, to determination of GTSP protein and gene expression to investigate the effect of combined treatment on the role of androgen synthesis, expression of CYP17A1 enzyme was determined in the cell lines. Furtermore, the effect of this combined treatment on hexokinase enzyme which plays an important role in the glycolysis pathway was investigated. Dose response of this combined therapy and cytotoxic effect of those drugs were measured by Alamar Blue assay. The effects of cisplatin and metformin on proliferation of cancer cell lines were detected by colony formation assay. Also, the effects of drugs on CYP17A1 GSTP and Hexokinase II enzymes will be detected by protein and mRNA expressions by using Western-Blot and qRTPCR techniques, respectively. IC50 value of cisplatin was determined as 17µM for LNCaP and 30 µM for PC3 cell line. The cell proliferation results showed that, metformin potentiates the antiproliferative effect of cisplatin both in LNCaP and PC3 cell lines. However, according to Western-blot and qPCR analysis, this reduction can not be blamed by GSTP enzymes. Expression of GSTP enzyme increased in direct proportion to dose of metformin. At this point, metformin may antagonize the cisplatin apoptotic effect through suppression of oxidative stress in prostate cancer cells. On the other hand, cisplatinmetformin drug combination significantly decreased the CYP17A1 protein expressions in PC3 cell lines. Furthermore, Hexokinase II mRNA expressions are significantly decrease in a dose dependent manner. In the treatment of cancer, which is frightening diseases of our century, combined therapy of diabetes based drugs with chemotherapeutic drugs will be helpful not only to reduce the toxicity of the of cisplatin, but also to reduce the cost.  M.S. - Master of Scienc

FEN BİLİMLERİ ENSTİTÜSÜ/LİSANSÜSTÜ TEZ PROJESİ

CİPSLATİN DERİVATİVLERİNİN ÇEŞİTLİ KANSER HÜCRE HATLARININ METASTAZI VE KEMOTERAPÖTİK İLAÇLARI METABOLİZE EDEN FAZ I, FAZ II, ENZİMLERİ ÜZERİNE ETKİLERİNİN İNCELENMES

FEN BİLİMLERİ ENSTİTÜSÜ/LİSANSÜSTÜ TEZ PROJESİ

CİSPLATİN DERİVATİVLERİNİN ÇEŞİTLİ KANSER HÜCRE HATLARININ METASTAZI VE KEMOTERAPÖTİK İLAÇLARI METABOLİZE EDEN FAZ I, FAZ II ENZİMLERİ ÜZERİNE ETKİLERİNİN İNCELENMES

CİSPLATİN METFORMİN İLAÇ KOMBİNASYONUNUN ETKİLERİNİN KANSER MODELİ OLUŞTURULMUŞ FARELERE UYGULANMASI VE PROSTAT DULULARINDAKİ ETKİLERİNİN MOLEKÜLER DÜZEYDE İNCELENMESİ

CİSPLATİN METFORMİN İLAÇ KOMBİNASYONUNUN ETKİLERİNİN KANSER MODELİ OLUŞTURULMUŞ FARELERE UYGULANMASI VE PROSTAT DULULARINDAKİ ETKİLERİNİN MOLEKÜLER DÜZEYDE İNCELENMES

Cisplatin-Metformin ilaç kombinasyonu uygulamasının prostat hücre hatlarının proliferasyonu ve apoptoz mekanizmalarında önemli rol oynayan GST ile CYP17A1 ve Hekzokinaz enzimleri üzerine etkilerinin incelenmesi

TÜBİTAK KBAG Proje01.08.2016Prostat kanseri, gelişmiş ve gelişmekte olan ülkelerde en sık görülen kanser türlerinden biri olup günümüzde prostat kanseri tedavisindeki yaklaşımlar metastaz gelişimine, ilaç direnci oluşmasına ve sağlıklı dokularda toksik etkilere sebep olabilmektedir. Bu nedenle tedavide daha etkili ancak daha az yan etkiye sahip kanser önleyici molekülleri tespit etmek kanser araştırmalarının önemli bir kısmını oluşturmaktadır. Cisplatin prostat kanseri tedavisinde sıklıkla kullanılan antineoplastik bir ajandır. Ancak, cisplatinin yüksek etki derecesine karşın yan etkileri ilacın kullanımını kısıtlamaktadır. Bu yan etkilerini azaltmak amacıyla başka ajanlar ile kombine terapiler son yıllarda bilim insanlarının ilgi odağı olmuştur. Antidiyabetik ilaç metforminin anti-proliferatif etkisi göz önünde bulundurulduğunda, antineoplastik ajan cisplatin ile tamamlayıcı bir tedavi sunabileceği açıktır. Tasarlanan çalışmada bu iki ilacın kombine kullanımının hücrede apoptoz ve detoksifikasyon enzimleri olarak bilinen Glutatyon-S Transferaz (GST) izozimleri üzerine etkilerine bakılmıştır. Prostat kanseri tedavisinde birincil amaç hormon (androjen) üretimini durdurmaktır. Bu yüzden cisplatin-metformin kombine kullanımının androjen sentezinde anahtar fonksiyonu olan CYP17A1 enzimi üzerine etkisi incelenmiştir. Bilindiği üzere, kanser hücrelerinin hızlı büyüyüp bölünmesinde glikoliz metabolizmasında rol oynayan Hekzokinaz II regülatör bir enzimidir. Bu durum göz önüne alındığında, yapılan çalışmada ilaç kombinasyonunun Hekzokinaz II enzimi üzerine etkileri de incelenmiştir. Yapılan çalışmada androjen reseptörüne bağımlı LNCaP ve androjen reseptöründen bağımsız PC3 insan prostat kanseri hücre hatları ve burada verilen ilaçların sağlıklı dokular üzerine etkilerini saptamak amacıyla normal prostat hücre hattı olan PNT1A kullanılmıştır. İlaçların hücreler üzerindeki sitotoksik etkileri "alamar blue" analizi ile ölçülmüş olup, ilaç kombinasyonunun belirlenen enzimlerin protein ve mRNA ekspresyonlarına olan etkisi sırasıyla Western-blot ve qRT-PCR teknikleriyle tespit edilmiştir. Yapılan deneyler sonucunda, ilaç kombinasyonunun hücre proliferasyonunu durdurucu özelliği olduğu her iki hücre hattında da ortaya konulmuştur. Ancak bu antiproliferatif etki GSTP1 enzimleri ile ilişkilendirilememişdir. Androjen sentezinde rol oynayan CYP17A1 enzim expresyonunun PC3 hücre hattında anlamlı derecede azaldığı saptanmıştır. Hexokinase II gen expresyonu her iki hücre hattında da azalmıştır. Sonuç olarak hücre proliferasyonunun azalması, HKII geninin ekspresyonundaki düşüş ile ilişkilendirilebilir.Prostate cancer is the most commonly seen cancer type both in developed and developing countries. Current therapeutic approaches for prostate cancer generally have variable side effects. Hence, the searching for more effective strategies remains one of the important issues in cancer research. Cisplatin, an antineoplastic agent, is widely used in the treatment of prostate cancer. Despite the high efficiency of cisplatin on prostate cancer treatment side effects restricts the use of cisplatin. In order to reduce the side effects of cisplatin, combination therapy with other agents has been the focus of scientists in recent years. Considering the inducer effect of antidiabetic drug metformin with antineoplastic agent cisplatin can provide a complementary treatment. In the present study, the effects of metformin-cisplatin combined therapy on GST isozymes, known as detoxification and apoptotic enzymes, were determined. In addition, to determine the effect of this combination on the role of androgen synthesis, expression of CYP17A1 enzyme was examined. Furthermore, the effect of treatment on hexokinase II enzyme which plays an important role in the glycolysis pathway was investigated. Two types of human prostate cell lines; androgen receptor dependent PC3 and androgen receptor independent LNCAP prostate cell lines, and also to determine the effect of those drugs on healthy cells PNT1A cells were used. Dose response and cytotoxic effect of combination treatment were measured by Alamar Blue assay. Also, effects of drugs on CYP17A1, GST’s and Hexokinase II were investigated by protein and mRNA expressions using Western-Blot and qRT-PCR, respectively. The cell proliferation results showed that, metformin potentiates the antiproliferative effect of cisplatin both in LNCaP and PC3 cell lines. But, this effect could not be attirubuted to GSTP enzymes. On the other hand, cisplatin-metformin drug combination significantly decreased the CYP17A1 protein expressions in PC3 cell lines. Furthermore, Hexokinase II mRNA expressions are significantly decrease in a dose dependent manner in both cells

Effect of cisplatin-metformin drug combination on GSTP1 mediated apoptosis

Kllng cancer cells by trggerng cellular apoptoss seems to be the prevalent acton mechansm for cancer drugs. Usng substances that targetng the enzymes partcpatng n the apoptotc pathways that trggers the cellular apoptoss can be a useful treatment opton for cancer. GSTP enzyme, ts newly known role as a modulator of cellular survval and death, can be a good proten molecule for cancer studes. Although, csplatn as one of the most potent alkylatng agent that appled wth success n vast majorty of cancers, ts sde effects are stll lmts the usage of ths drug. Concordantly, to overcome the sde effects, producng less toxc and more effectve drug, combnaton treatment optons are popular research topc n recent years. Metformn s one of the natural product used for dabetes. Recent studes showed that metformn can reduce the rsk of gettng cancer even be able to treat t. In ths respect, the am of ths study s to determne the effects of metformn alone and combnaton wth csplatn on apoptoss n androgen ndependent prostate cancer cell lne (PC3). For ths purpose, cells were treated wth ether metformn alone n the range of 1–10 mM, csplatn alone or a combnaton of these two drugs. Cytotoxcty of drugs were determned wth Alamar Blue Assay and IC50 was calculated. The effect of drugs on proten expresson level of GSTP1 was determned by Western blot technque. In order to valdate our results, apoptoss was examned by usng Annexn V-EGFP kt (BoVson Incorporated CA., USA). Csplatn IC50 value was calculated as 30μM for PC3 cell lne. Both alone or combnaton of drugs were nhbted the prolferaton of PC3 cells n a concentraton dependent manner. GSTP1 proten expressons were sgnficantly down regulated (p=0.05) n metformn/csplatn treated cells compared to control groups. In addton to ths, Annexn V-EGFP levels whch was a sgn of apoptoss, were sgnficantly ncreased by drug combnaton. Ths study suggest that metformn and/or csplatn combnaton may decrease the tumour promoton as well as ncrease the apoptoss n PC3 cells. As a result, ths drug combnaton can be usable for treatment for prostate cancer

Metformin – Cisplatin Combination Treatment Alters mRNA Expression of Hexokinase II Gene in LNCaP and PC3 Prostate Cancer Cell Lines

Metformin is an antidiabetic drug with anticancer properties. Cisplatin is known as one of the most potent chemotherapeutics for treatment of various types of cancer. In order to overcome cisplatin resistance and toxicity, the drug can be combined with other chemotherapeutics that sensitize tumour cells to cisplatin. The ability of metformin to potentiate cisplatin-mediated killing of cancer cells in vitro, makes it a plausible candidate for combination with cisplatin-based therapy. The aim of this study is to examine the combined effect of these drugs on mRNA expression of Hexokinase II gene participating in glycolysis as well as cancer promotion

Metformin-Cisplatin Combination Treatment Alters mRNA Expression of Hexokinase II Gene in LNCaP and PC3 Prostate Cancer Cell Lines

Metformin is an antidiabetic drug with anticancer properties. Cisplatin is known as one of the most potent chemotherapeutics for treatment of various types of cancer. In order to overcome cisplatin resistance and toxicity, the drug can be combined with other chemotherapeutics that sensitize tumour cells to cisplatin. The ability of metformin to potentiate cisplatin-mediated killing of cancer cells in vitro, makes it a plausible candidate for combination with cisplatin-based therapy. The aim of this study is to examine the combined effect of these drugs on mRNA expression of Hexokinase II gene participating in glycolysis as well as cancer promotion

Alteration of protein and gene expressions of Hexokinase II in PC3 cell lines by cisplatinmetformin combination treatment

Metformin is an antidiabetic drug with anticancer properties. Cisplatin is known as one of the most potent chemotherapeutics for treatment of various types of cancer. In order to overcome cisplatin resistance and toxicity, the drug can be combined with other drugs that sensitize tumour cells to cisplatin. The ability of metformin to potentiate cisplatin-mediated killing of cancer cells in vitro, makes it a plausible candidate for combination with cisplatin- based therapy. The aim of this study is to examine the combined effect of these drugs on protein and mRNA expressions of Hexokinase II, a regulatory enzyme participating in glycolysis as well as cancer promotion. The effects of drugs on prostate cancer cell lines were studied using androgen independent PC3 cell line. Cells were treated with either metformin alone in the range of 1–10 mM, cisplatin alone or a combination of these two drugs. Cytotoxicity of drugs were determined with Alamar Blue Assay and IC50 was calculated. The effects of drugs on Hexokinase II mRNA and protein expressions were determined by qRT-PCR and western immunoblotting techniques, respectively. The intensity of each band was analyzed by Image J software program. IC50 value of cisplatin on PC3 cell line was found as 30 lM. Both alone or combination of drugs were inhibited the proliferation of PC3 cells in a concentration dependent manner. Hexokinase II mRNA and protein expressions were significantly downregulated in metformin/cisplatin treated cells compared to control groups. Epidemiological and experimental researches indicates that men with abdominal obesity is a risk factor for prostate cancer. Parallel with our results, the adjuvant role of this type of antidiabetic drug must be investigated for new cancer treatment options