Mohammad Rahmati

Inhibition of hTERT Gene Expression by Silibinin-Loaded PLGA-PEG-Fe3O4 in T47D Breast Cancer Cell Line.

Introduction : Nowadays, using drug delivery is an essential method to improve cancer therapy through decreasing drug toxicity and increasing efficiency of treatment. Silibinin (C25H22O10), a polyphenolic flavonoid which is isolated from the milk thistle plant, has various applications in cancer therapy but it has hydrophobic structure with low water solubility and bioavailability. To increase the effect of silibinin, silibinin-loaded PLGA-PEG-Fe3O4 was prepared to determine the inhibitory effect of this nanodrug on Telomerase gene expression. Methods : The rate of silibinin loaded into PLGA-PEG-Fe3O4 was measured. Then, the cytotoxic effect of silibinin-loaded PLGA-PEG-Fe3O4 was determined by Methyl Thiazol Tetrazolium (MTT) assay. After that, inhibition of Telomerase gene expression was indicated through Real-time PCR. Results : Data analysis from MTT assay showed that silibinin-loaded PLGA-PEG-Fe3O4 had dose dependent cytotoxic effect on T47D cell line. MTT assay showed no cytotoxic effect of free PLGA-PEG-Fe3O4 on T47D breast cancer cell line. Real Time PCR analysis showed that the level of telomerase gene expression more efficiently decreased with silibinin-loaded PLGA-PEG-Fe3O4 than with free silibinin alone. Conclusion : The present study indicates that this nanodrug causes down-regulation of Telomerase gene expression in cancer cells. Therefore, PLGA-PEG-Fe3O4 could be an appropriate carrier for hydrophobic agents such as silibinin to improve their action in cancer therapy.ABSTRACT Introduction: Nowadays using drug delivery is an essential method to improve cancer therapy through decreasing drug toxicity and increasing efficiency of treatment. Silibinin (C25H22O10), a polyphenolic flavonoid which is isolated from the milk thistle plant, has various applications in cancer therapy but it has hydrophobic structure with low water solubility and bioavailability. To increase the effect of Silibinin, Silibinin-loaded PLGA-PEG-Fe₃O₄ was prepared to determine the inhibitory effect of this nano drug on telomerase gene expression. Methods: The rate of Silibinin loaded into PLGA-PEG-Fe₃O₄ was measured. Then the cytotoxic effect of Silibinin-loaded PLGA-PEG-Fe₃O₄ was estimated via colorimetric cell viability (MTT) assay. After that, inhibition of telomerase gene expression was indicated through Real-time PCR. Results: Data analysis from MTT assay showed that Silibinin-loaded PLGA-PEG-Fe₃O₄ has dose dependent cytotoxic effect on T47D cell line. MTT assay showed no cytotoxic effect of free PLGA-PEG-Fe₃O₄ on T47D breast cancer cell line. Real Time PCR analysis showed, as the amount of Silibinin-loaded PLGA-PEG-Fe₃O₄ increased, the level of Telomerase gene expression dramatically decrease which is even more effective than free drug alone. Conclusion: In conclusion this study indicates this nano drug causes down-regulation of telomerase gene expression in cancer cells. Therefore PLGA-PEG-Fe₃O₄ could be an appropriate carrier for hydrophobic agents such as Silibinin to improve their action in cancer therapy.

Comparison of Inhibitory Effect of 17-DMAG Nanoparticles and Free 17-DMAG in HSP90 Gene Expression in Lung Cancer

BACKGROUND Up-regulation of hsp90 gene expression occurs in numerous cancers such as lung cancer. D,L-lactic-co-glycolic acid-poly ethylene glycol-17-dimethylaminoethylamino-17-demethoxy geldanamycin (PLGA-PEG-17DMAG) complexes and free 17-DMAG may inhibit the expression. The purpose of this study was to examine whether nanocapsulating 17DMAG improves the anti cancer effect over free 17DMAG in the A549 lung cancer cell line. MATERIALS AND METHODS Cells were grown in RPMI 1640 supplemented with 10% FBS. Capsulation of 17DMAG is conducted through double emulsion, then the amount of loaded drug was calculated. Other properties of this copolymer were characterized by Fourier transform infrared spectroscopy and H nuclear magnetic resonance spectroscopy. Assessment of drug cytotoxicity on the grown of lung cancer cell line was carried out through MTT assay. After treatment, RNA was extracted and cDNA was synthesized. In order to assess the amount of hsp90 gene expression, real-time PCR was performed. RESULTS In regard to the amount of the drug load, IC50 was significant decreased in nanocapsulated(NC) 17DMAG in comparison with free 17DMAG. This was confirmed through decrease of HSP90 gene expression by real-time PCR. CONCLUSIONS The results demonstrated that PLGA-PEG-17DMAG complexes can be more effective than free 17DMAG in down-regulating of hsp90 expression by enhancing uptake by cells. Therefore, PLGA-PEG could be a superior carrier for this kind of hydrophobic agent.

Fatty Acid Composition of Tissue Cultured Breast Carcinoma and the Effect of Stearoyl-CoA Desaturase 1 Inhibition

Purpose Stearoyl-CoA desaturase 1 (SCD1) is a novel therapeutic target in various malignancies, including breast cancer. The present study was designed to investigate the effect of the pharmacologic inhibition of SCD1 on fatty acid composition in tissue explant cultures of human breast cancer and to compare these effects with those in adjacent nonneoplastic breast tissue. Methods Paired samples of tumor and adjacent noncancerous tissue were isolated from 12 patients with infiltrating ductal breast cancer. Samples were explant cultured in vitro, exposed to the highly selective SCD1 inhibitor CAY10566, and examined for fatty acid composition by gas liquid chromatography. The cytotoxic and antigrowth effects were evaluated by quantification of lactate dehydrogenase release and by sulforhodamine B (SRB) measurement, respectively. Results Breast cancer tissue samples were found to have higher levels of monounsaturated fatty acids (MUFA) (p<0.001) and arachidonic acid (20:4n-6, p<0.001) and a lower level of linoleic acid (18:2n-6, p=0.02) than the normal-appearing breast tissues. While exhibiting no evident cytotoxicity, treatment with the SCD1 inhibitor, CAY10566 (0.1-1 µM), for 48 hours significantly increased 18:2n-6 levels in both the tumor and adjacent normal-appearing tissue (approximately 1.2 fold, p<0.05). However, the breast cancer tissue samples showed significant increases in the levels of MUFA and 20:4n-6 compared to the normal-appearing breast tissues (p<0.05). The SRB growth assay revealed a higher rate of inhibition with the SCD1 inhibitor in breast cancer tissues than in normal-appearing tissues (p<0.01, 41% vs. 29%). The SCD1 inhibitor also elevated saturated fatty acid (1.46-fold, p=0.001) levels only in the tumor tissue explant. Conclusion The fatty acid composition and response to SCD1 inhibition differed between the explant cultures from breast cancer and the adjacent normal-appearing tissue. Altered fatty acid composition induced by SCD1 inhibition may also, in addition to Δ9 desaturation, modulate other reactions in de novo fatty acid synthesis and lipogenesis, and subsequently affect the overall survival and progression of breast cancer.

Comparison of Inhibitory Effects of 17-AAG Nanoparticles and Free 17-AAG on HSP90 Gene Expression in Breast Cancer

BACKGROUND HSP90 may be overexpressed in cancer cells which are greatly dependent on Hsp90 function. Geldanamycin derivative 17 allylamino-17-demethoxygeldanamycin (17-AAG) inhibits the function and expression of HSP90. 17-AAG has poor water-solubility which is a potential problem for clinical practice. In this study for improving the stability and solubility of molecules in drug delivery systems we used a β-cyclodextrin- 17AAG complex. MATERIALS AND METHODS To assess cytotoxic effects of β-cyclodextrin-17AAG complexes and free 17AAG, colorimetric cell viability (MTT) assays were performed. Cells were treated with equal concentrations of β-cyclodextrin- 17AAG complex and free 17AAG and Hsp90 gene expression levels in the two groups was compared by real-time PCR. RESULTS MTT assay confirmed that β-cyclodextrin- 17AAG complex enhanced 17AAG cytotoxicity and drug delivery in T47D breast cancer cells. The level of Hsp90 gene expression in cells treated with β-cyclodextrin- 17AAG complex was lower than that of cells treated with free 17AAG (P=0.001). CONCLUSIONS The results demonstrated that β-cyclodextrin- 17AAG complexes are more effective than free 17AAG in down-regulating HSP90 expression due to enhanced β-cyclodextrin-17AAG uptake by cells. Therefore, β-cyclodextrin could be superior carrier for this kind of hydrophobic agent.

Real-time PCR detection of 16S rRNA novel mutations associated with Helicobacter pylori tetracycline resistance in Iran.

BACKGROUND Tetracycline is an antibiotic widely used for the treatment of Helicobacter pylori infection, but its effectiveness is decreasing due to increasing bacterial resistance. The aim of this study was to investigate the occurrence of 16S rRNA mutations associated with resistance or reduced susceptibility to tetracycline of Helicobacter pylori by real-time PCR (RT-PCR) assays from culture. MATERIALS AND METHODS Tetracycline susceptibility and minimal inhibition concentration (MIC) was determined by the Epsilometer test (Etest) method. A LightCycler assay developed to detect these mutations was applied to DNA extracted from culture. The 16S rRNA of these isolates was sequenced and resistance-associated mutations were identified. From 104 isolates of H. pylori examined, 11 showed resistance to tetracycline. RESULTS LightCycler assay was applied to DNA extracted from 11 tetracycline-susceptible and 11 tetracycline resistance H. pylori isolates. In our study the sequencing of the H. pylori wild types in 16 s rRNA gene were AGA 926-928 with MIC (0.016 to 0.5 μg/ml), while the sequencing and MIC for resistant were GGA and AGC, (0.75 to 1.5 μg/ml), respectively. Also we found a novel mutation in 2 strains with 84° as their melting temperatures and exhibition of an A939C mutation. CONCLUSIONS We conclude that real-time PCR is an excellent method for determination of H. pylori tetracycline resistance related mutations that could be used directly on biopsy specimens.

Construction, expression, and activity of a novel immunotoxin comprising a humanized antiepidermal growth factor receptor scfv and modified pseudomonas aeruginosa exotoxin A

Overexpression of epidermal growth factor receptor (EGFR) plays a significant role in the development and metastasis of many solid tumors. Strategies based on anti-EGFR immunotoxins have shown promising results in several studies, but immunogenicity of antibody and toxin moieties is a limitation of this type of therapeutics. In the present study, a novel humanized anti-EGFR immunotoxin (huscFv-PE25) was developed by genetic fusing of a humanized anti-EGFR single-chain variable fragment (huscFv) with a modified Pseudomonas aeruginosa exotoxin A (PE25KDEL). The reactivity and toxicity of this immunotoxin with tumor cells were assessed by dot-blot, enzyme-linked immunosorbent assay, and MTT procedures. Results of enzyme-linked immunosorbent assay and dot-blot assay indicated that the immunotoxin recognizes and efficiently binds to EGFR-overexpressing tumor cells. MTT assay showed a specific growth-inhibitory effect of huscFv-PE25 on EGFR-overexpressing A431 cells, without any inhibitory effect on EGFR-negative cells. In conclusion, the results of this study indicated that huscFv-PE25 can recognize and exert an inhibitory effect on EGFR-overexpressing cancer cells, despite its smaller size and lower immunogenicity. This may provide a basis for the development of novel clinical therapeutic agents against EGFR-overexpressing tumor cells.

The effect of Helicobacter pylori infection on oxidative stress status in erosive reflux disease

Background and Objectives: Helicobacter pylori (H. pylori) infection has been associated with increased production of reactive oxygen species, which leads to oxidative stress in the gastrointestinal mucosa. Nevertheless, the association of H. pylori infection and oxidative stress in erosive reflux disease (ERD) is still unclear. We sought to investigate the association between oxidative stress status and H. pylori infection in ERD. Materials and Methods: Eighty-three ERD patients (45 male/ 38 female; mean age: 40.4 ± 14.3 years) who had heartburn and/or regurgitation and erosion(s)-confirmed by endoscopy-in the distal esophagus were enrolled. Two antral biopsies were taken from the patients for rapid urease test. Blood samples were drawn for measurement of oxidative stress parameters, including malondialdehyde (MDA), ferric reducing antioxidant power (FRAP), superoxide dismutase (SOD) activity, and glutathione peroxidase (GPX) activity. Data were compared among H. pylori(+) and H. pylori(-) patients. Results : The overall prevalence of H. pylori infection was 71% (59/83). There was a significant increase in the levels of MDA in H. pylori(+) patients (0.98 ± 0.28 μmol/l) when compared with H. pylori(-) patients (0.84 ± 0.33 μmol/l; P = 0.048). However, the levels of FRAP in the H. pylori-infected patients were significantly lower than in those without infection (941 ± 211.8 vs. 1060.3 ± 216.6 μmol/l, respectively; P = 0.028). There were no significant differences in SOD activity, GPX activity, age, sex, and body mass index (BMI) of H. pylori(+) vs. H. pylori(-) patients (P > 0.05). Conclusion: Increased levels of MDA in H. pylori(+) patients showed association between oxidative stress and H. pylori infection in EDR patients. Also, considerable changes of antioxidant concentrations indicate a compensatory mechanism to cope with the increased oxidant status in H. pylori(+) patients. It may be concluded that oxidative stress increases in the presence of H. pylori in ERD patients, and antioxidant defense mechanisms, try to minimize oxidative stress damage.

Regulation and modulation of PTEN activity

PTEN (Phosphatase and tensin homolog deleted on chromosome ten) is a tumor suppressor that is frequently mutated in most human cancers. PTEN is a lipid and protein phosphatase that antagonizes PI3K/AKT pathway through lipid phosphatase activity at the plasma membrane. More recent studies showed that, in addition to the putative role of PTEN as a PI(3,4,5)P3 3-phosphatase, it is a PI(3,4)P2 3-phosphatase during stimulation of class I PI3K signaling pathway by growth factor. Although PTEN tumor suppressor function via it’s lipid phosphatase activity occurs primarily in the plasma membrane, it can also be found in the nucleus, in cytoplasmic organelles and extracellular space. PTEN has also shown phosphatase independent functions in the nucleus. PTEN can exit from the cell through exosomal export or secretion and has a tumor suppressor function in adjacent cells. PTEN has a critical role in growth, the cell cycle, protein synthesis, survival, DNA repair and migration. Understanding the regulation of PTEN function, activity, stability, localization and its dysregulation outcomes and also the intracellular and extracellular role of PTEN and paracrine role of PTEN-L in tumor cells as an exogenous therapeutic agent can help to improve clinical conceptualization and treatment of cancer.

Retraction Note: Inhibition of hTERT Gene Expression by Silibinin-Loaded PLGA-PEG-Fe₃O₄ in T47D Breast Cancer Cell Line

[This retracts the article DOI: 10.5681/bi.2013.005.].