Combination of Quercetin or/and siRNA-loaded DDAB-mPEG-PCL hybrid nanoparticles reverse resistance to Regorafenib in colon cancer cells.

BACKGROUND: Colorectal cancer (CRC) is the second leading cause of cancer death. Although Regorafenib showed survival benefits in patients with CRC, reports imply the recurrence of malignant phenotype resulting from chemotherapy. Evidence demonstrated that a5ß1 integrin plays an important role in the Regorafenib treatment, which, may be led to resistance. In this study, the effects of /siRNA or/ and Quercetin loaded DDAB-mPEG-PCLnanoparticles could reverse this resistance phenotype in colon cancer cells in vitro. METHODS: Regorafenib-resistant Ls-180 colon cancer cell line was developed by long-term exposure to Regorafenib. Quercetin and Regorafenib were separately encapsulated into mPEG-PCL micelles through the nano-precipitation method and characterized by DLS. Optimized doses of Quercetin and Regorafenib were used for combination therapy of resistant cells followed cytotoxicity study using MTT. Gene expression levels of the ß1 subunit of integrin were determined by the real-time method of RT-PCR. RESULTS: Developed Regorafenib resistant LS-180 showed to have Regorafenib IC50 of 38.96 ± 1.72 µM whereas IC50 in non-resistant cells were 8.51 ± 0.29 µM, which meaningful was lower statistically compared to that of a resistant one. The ß1 mRNA level of whole α5ß1 integrin was significantly higher in the resistant cells compared to those of non-resistant ones. Gene expression levels in each siRNA-loaded nanoparticle and Quercetin-loaded one were lower than that in mock experiments. Finally, when these two types of nanoparticles were used to treat resistant cells, gene expression decrease of integrin indicated a greater effect that could be capable of reverse resistancy. CONCLUSION: Results of this study demonstrated another confirmation of involving integrins in cancer resistance following chemotherapy using Regorafenib. Also, it indicated how using siRNA targeting integrin could enhance the plant derivatives like Quercetin effects to reverse resistance in vitro.

Betanin alleviates oxidative stress through the Nrf2 signaling pathway in the liver of STZ-induced diabetic rats.

BACKGROUND: Continuing hyperglycemia causes and exacerbate oxidative stress. Betanin as the principal pigment of red beet root has antioxidant, anti-inflammatory, and anti-diabetic properties. The purpose of this study was to investigate the potency of betanin on antioxidant defense in STZ-induced diabetic rats' livers. METHODS: STZ at a single dose of 60 mg/kg body weight was intraperitoneally injected and betanin (10, 20, and 40 mg/kg/day) was administered orally for 28 days. Malondialdehyde (MDA), total antioxidant capacity (TAC), protein carbonyl (PC) levels, and the enzyme activity of superoxide dismutase (SOD), catalases and glutathione peroxidases (GPx) were evaluated in the liver. Furthermore, gene expression of Nrf2 and mentioned antioxidant enzymes were measured by Real-time PCR. RESULTS: Betanin (10 and 20 mg/kg) significantly reduced PC levels and increased antioxidant enzyme activity in diabetic rats compared to the control diabetic group (P < 0.01). In comparison to the diabetic control group, all studied genes expression in diabetic rats were increased significantly with betanin at doses of 10 and 20 mg/kg (P < 0.02). The increase in gene expression at 20 mg/kg of betanin was significantly stronger than others (P < 0.015) except for the catalase (P = 0.201), that was almost the same. Moreover, treatment of diabetic rats with 20 mg/kg of betanin could significantly increase TAC levels (P < 0.05) and decrease MDA levels (P < 0.001) compared to diabetic control group. CONCLUSIONS: Betanin could increase the antioxidant capacity of liver tissue associated with the Nrf2-mediated pathway in a dose-dependent manner.

Co-delivery of siRNA and lycopene encapsulated hybrid lipid nanoparticles for dual silencing of insulin-like growth factor 1 receptor in MCF-7 breast cancer cell line.

Insulin-like growth factor-1 receptor (IGF-1R) is expressed in malignant and normal breast tissue, and its intermittent activation by multiple IGF-1 signaling pathways leads to neoplasm cell proliferation, impaired apoptosis, increased survival, and resistance to cytotoxic therapeutic agents. Therefore, simultaneous suppression of the receptor and its cognate ligand would be a powerful promising strategy inhibiting malignant phenotypes of breast cancer cells. In the present study, Methoxypoly(ethylene glycol) - Poly(caprolactone) was hybridized with Dimethyldioctadecylammonium bromide (DDAB) cationic lipid (mPEG-PCL-DDAB) nanoparticles (NPs) and used as a carrier for simultaneous delivery of lycopene and insulin-like growth factor 1 receptor-specific lycopene encapsulated-mPEG-PCL-DDAB nanoparticle/siRNA to MCF-7 breast cancer cells. Then, the antitumor effects of this construct were evaluated in vitro. The results demonstrated that the synthesized mPEG-PCL-DDAB nanoparticle had suitable physicochemical properties. The use of mPEG-PCL-DDAB nanoparticle-loaded anti-insulin-like growth factor 1 receptor-siRNA and lycopene dramatically induced the process of apoptosis and arrested cell cycle in the MCF-7 tumor cell lines. In general, the findings of this study demonstrated the potency of mPEG-PCL-DDAB nanoparticles for dual delivery of siRNA, and lycopene in breast cancer cell lines followed the induction of apoptosis.

Apoptosis induction by siRNA targeting integrin-ß1 and regorafenib/DDAB-mPEG-PCL hybrid nanoparticles in regorafenib-resistant colon cancer cells.

Colorectal cancer (CRC) is regarded as the third most common cancer worldwide. Although Regorafenib as a receptor tyrosine kinase inhibitor (RTKI) disrupts tumor growth and angiogenesis in metastatic CRC (mCRC) patients, drug resistance leads to poor prognosis and survival. Integrin-ß1 overexpression has been proposed to be the major player in this regard. Herein, the Regorafenib-resistant human colon cancer cell line (SW-48) was induced, and the Integrin-ß1 gene expression, as well as apoptosis, was assessed through the combination of small interfering RNA (siRNA) targeting Integrin-ß1 and Regorafenib/Dimethyldioctadecylammonium bromide (DDAB)-methoxy poly (ethylene glycol) (mPEG)-poly-ε-caprolactone (PCL) hybrid nanoparticles (HNPs). In the current study, Regorafenib-resistant SW-48 cell line was generated in which the Regorafenib half-maximal inhibitory concentration (IC50) for non-resistant and resistant cells was 13.5±1.5 µM and 55.1±0.8 µM, respectively. The results of DLS also demonstrated that the size and the charge of the HNPs were equal to 66.56±0.5 nm and +29.5±1.2 mv, respectively. In addition, the Integrin-ß1 gene expression was significantly higher in resistant cells than in non-resistant ones (P<0.05). The siRNA/HNP complexes in combination with Regorafenib/HNPs were accordingly identified as the most effective treatment to decrease the Integrin-ß1 gene expression and to enhance the apoptosis rate in resistant cells (P<0.001). Overall, the study indicated that combination therapy using siRNA/HNP and Regorafenib/HNPs complex could down-regulate the Integrin-ß1 gene expression and consequently trigger apoptosis, and this may potentially induce drug sensitivity.

Clinicopathological, Immunohistochemical, and PMS2 Gene Expression Profiling of Patients with Sporadic Colorectal Cancer.

BACKGROUND: The DNA mismatch repair (MMR) system is one of the molecular pathways involved in colorectal cancer (CRC) carcinogenesis that consists of several genes, including MLH1 (MutL homolog 1), MSH6 (MutS homolog 6), MSH2 (MutS homolog 2), and MSH3 (MutS homolog 3). The protein encoded by PMS2 (post-meiotic segregation 2) is also essential for MMR. Here, we address the correlation between immunohistochemical and transcriptional expression of PMS2 with the tumor grade and clinical stage of non-hereditary/sporadic CRC disease. METHODS: This study retrospectively analyzed 67 colorectal resections performed for 38 male and 29 female patients. Random biopsies were taken by a gastroenterologist from patients referring to three hospitals in the cities of Zanjan, Urmia and Qazvin (Iran) during 2017-2019. All specimens were examined and classified for localization of tumor, pathological stage and grade. The PMS2 protein expression was studied immunohistochemically and analysis of mRNA expression was performed in the same tissue sections. RESULTS: Immunohistochemistry and quantitative real-time polymerase chain reaction (PCR) analysis showed a decrease in PMS2 expression compared with paracancerous tissue (P<0.001), which correlated with tumor stage. In addition, reduced PMS2 expression was correlated with the tumor differentiation grade, underlining a connection between downregulation of PMS2 and progression of CRC. Comparing the PMS2 mRNA levels in different groups showed the following results: 0.92 ± 0.18 in patients with Stage I CRC tumor, 0.86 ± 0.38 in Stage Ⅱ, 0.50 ± 0.29 in Stage Ⅲ, and 0.47 ± 0.23 in Stage Ⅳ. CONCLUSION: These findings suggest that PMS2 may provide a potential reliable biomarker for CRC classification by combined immunohistochemical and mRNA analysis.

Application of decoy oligodeoxynucleotides strategy for inhibition of cell growth and reduction of metastatic properties in nonresistant and erlotinib-resistant SW480 cell line.

Signal transducer and activator of transcription 3 (STAT3) is a critical regulator for angiogenesis, cell cycle progression, apoptosis, and drug resistance. Resistance toward EGF receptor (EGFR) inhibitors is a significant clinical concern for metastatic colon cancer patients. The present study aimed to evaluate the blocking influences of STAT3 decoy oligodeoxynucleotides (ODNs) on the STAT3 survival signaling pathway in nonresistant and erlotinib-resistant SW480 colon cancer cells. First, STAT3 decoy and scramble ODNs were designed according to STAT3 elements in the promoter region of MYCT1 gene and tested for the interaction of STAT3 protein with designed ODNs via in silico molecular docking study. Then, the efficiency of transfection and subcellular localization of ODNs were assessed using flow cytometry and fluorescence microscopy, respectively. Cell viability, cell cycle, and apoptosis tests, scratch and colony formation assays, and real-time PCR were also used to study the cancerous properties of cells. A considerable decrease in proliferation of colon cancer cells was observed with blockade of STAT3 signaling due to cell cycle arrest and induced apoptosis via downregulation of cyclin D1 and Bcl-XL, respectively. Furthermore, upon transfecting STAT3 decoy ODNs, colony formation potential and migration activity in both SW480 colon cancer cell lines were decreased compared to the control groups. From this study, it could be concluded that STAT3 is critical for cell growth inhibition and metastatic properties reduction of resistant SW480 colon cancer cells; therefore, STAT3 decoy ODNs could be considered as potential therapeutics along with current remedies for treating drug-resistant colon cancer.

Determination of carcinoembryonic antigen as a tumor marker using a novel graphene-based label-free electrochemical immunosensor.

In this work, a simple label-free electrochemical immunosensor for the detection of carcinoembryonic antigen (CEA) was developed. At first, the GC electrode was coated with partially reduced graphene oxide (rGO) to form a platform to bind the antibody. After activating the carboxyl groups of rGO through the EDC/NHS linker, the electrode surface was covered with the antibody. Then, the electrochemical behavior of the antibody-modified electrode and the parameters of the interactions of antibody-antigen immune complexes were investigated using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). This immune-complex layer was found to attenuate the electrochemical current which can be used as a good signal to determine the antigen concentration. The proposed immunosensor exhibited a good amperometric response to CEA within a concentration range of 0.1-5 ng mL-1 with a detection limit of 0.05 ng ml-1. Furthermore, the developed method was evaluated for the detection of CEA in the real sample (human blood serum), and the results were comparable with the reference values obtained by the standard enzyme-linked immunosorbent assay (ELISA). Our findings suggest the present immunosensor as a good candidate for application in clinical screening.

Oxidative toxic stress and p53 level in healthy subjects occupationally exposed to outdoor air Pollution - a cross-sectional study in Iran.

INTRODUCTION: It is suggested that air pollution exposure induces oxidative stress in the body and causes diseases. However, current evidence regarding the association of outdoor air pollution with some oxidative toxic stress (OTS) biomarkers in areas with different pollutant concentrations is equivocal. OBJECTIVE: The aim of study was to investigate the adverse effects of outdoor air pollution on human health, by evaluating potential oxidative and anti-oxidative biomarkers and p53 protein levels in subjects exposed to different outdoor air pollution from two polluted and less polluted cities of Iran. MATERIAL AND METHODS: In this cross-sectional study, a total of 203 healthy working men were selected from two cities. The activities of superoxide dismutase (SOD), catalase (CAT) and γ-glutamyltransferase (GGT) and the levels of malondialdehyde (MDA), total antioxidant capacity (TAC), and total oxidant status (TOS), were measured by the colorimetric method. The levels of p53 were measured by an ELISA method. RESULTS: The results showed a significant increase in the levels of p53 and MDA in the exposure group compared to the control group, while the activity of SOD and TAC was significantly decreased in the exposure group. No significant differences were found in activities of CAT and GGT, and levels of TOS between the two groups. CONCLUSIONS: The findings obtained confirmed the implication of air pollution in the development of OTS, and suggested useful biomarkers to evaluate the air pollution-induced harmful effects on human health in the polluted areas.

Circulating Irisin Levels and Redox Status Markers in Patients with Gastric Cancer: A Case-Control Study.

OBJECTIVE: Irisin, mostly known as an exercise-induced fat browning myokine, has been recently detected in several cancer cells, and its potential for being utilized as a biomarker for early diagnosis of some cancers, such as Gastric cancer (GC), is the subject of speculation. The present study aims to compare serum irisin levels in GC patients and healthy controls and assess the interrelation between irisin and oxidative stress markers. METHODS: In this case-control study, 22 newly diagnosed GC patients and 29 healthy controls were recruited based on the inclusion criteria. Serum levels of irisin were quantified in duplicates by ELISA. Oxidative stress indices, including total antioxidant power in sera, thiol group, malondialdehyde, and superoxide dismutase concentrations, were also measured in both groups. An independent-sample t-test was used to compare the means between the two studied groups. RESULTS: Serum levels of irisin were significantly higher in the GC group compared with those of their healthy counterparts (p =0.032). No significant differences were observed between the two groups in terms of the serum total antioxidant power or the oxidative stress marker, including MDA, thiol groups, and SOD concentration in sera. Furthermore, there was no significant association between irisin, FRAP, the Thiol group, and the SOD activity. CONCLUSION: According to the finding, the increased serum levels of irisin in GC patients can play a potential role in the early diagnosis of the GC patients; hence, this peptide can be employed as a new diagnostic indicator of GC.
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Crosstalk between Epidermal Growth Factor Receptors (EGFR) and integrins in resistance to EGFR tyrosine kinase inhibitors (TKIs) in solid tumors.

Cell adhesion to the extracellular matrix (ECM) is important in a variety of physiological and pathologic processes, including development, tumor invasion, and metastasis. Integrin-mediated attachment to ECM proteins has emerged to cue events primitively important for the transformed phenotype of human cancer cells. Cross-talk between integrins and growth factor receptors takes an increasingly prominent role in defining adhesion, motility, and cell growth. This functional interaction has expanded beyond to link integrins with resistance to Tyrosine kinase inhibitors (TKIs) of Epidermal Growth Factor Receptors (EGFRs). In this regard, integrin-mediated adhesion has two separate functions one as a clear collaborator with growth factor receptor signaling and the second as a basic mechanism contributing in Epithelial to Mesenchymal Transition (EMT) which affects response to chemotherapy. This review provides an overview of these mechanisms and describes treatment options for selectively targeting and disrupting integrin interaction to EGFR for cancer therapy.

Preliminary study showing no association between G238A (rs361525) tumor necrosis factor-α (TNF-α) gene polymorphism and its serum level, hormonal and biochemical aspects of polycystic ovary syndrome.

BACKGROUND: Polycystic ovary syndrome (PCOS) is the main cause of female infertility. Interactions among genetic, biochemical, and immunological factors can affect the pathogenesis of PCOS. As a proinflammatory cytokine, tumor necrosis factor-α (TNF-α) plays an important role in this regard. The present study aimed to evaluate the association of the rs361525 gene single-nucleotide polymorphism (SNP) and TNF-α serum levels with the hormonal and biochemical characteristics of PCOS in Iranian individuals. METHODS: The SNP rs361525 in the TNF-α gene was analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in a total of 111 PCOS patients and 105 healthy females. Serum levels of TNF-α, lipid and hormone profiles, and biochemical factors were measured using enzyme-linked immunosorbent assay (ELISA) and calorimetric methods, as appropriate. RESULTS: The TNF-α serum level was higher in women with PCOS compared with the control group (p <  0.0001), and it was significantly correlated with the homeostasis model assessment (HOMA) factor (r = 0.138, p <  0.05). No significant differences were found in the genotype and allelic frequencies between the two groups (p >  0.05). Higher levels and significant differences were found for the HOMA factor, luteinizing hormone/follicle-stimulating hormone (LH/FSH), testosterone, and body mass index (BMI) in the PCOS group compared with the control group (p <  0.0001). High LH/FSH ratios (odds ratio [OR] = 1.98, 95% confidence interval [CI] = 1.20-3.28, p <  0.01), and high HOMA factor (OR = 5.04, 95% CI = 2.82-9.01, p <  0.001) were significantly associated with an increased risk of PCOS. CONCLUSIONS: Despite the lack of significant difference between rs361525 polymorphism of the TNF-α gene and PCOS, the serum level of TNF-α was increased in PCOS patients and positively correlated with the HOMA factor. Elevation of the LH/FSH ratio and HOMA for insulin resistance (HOMA-IR) increased the risk of PCOS. Therefore, TNF-α could indirectly contribute to PCOS progression.

Curcumin mediated down-regulation of αV ß3 integrin and up-regulation of pyruvate dehydrogenase kinase 4 (PDK4) in Erlotinib resistant SW480 colon cancer cells.

Erlotinib is a potent, selective, and orally active inhibitor of the epidermal growth factor receptor, but the development of erlotinib resistance during chemotherapy can lead to treatment failure. To shed light on the erlotinib-resistant pathway, this study investigated the effect of combination therapy using curcumin- and erlotinib-loaded nanoparticles on the expression of αv ß3 integrin and pyruvate dehydrogenase kinase 4 (PDK4) in an erlotinib-resistant SW480 colon cancer cell line. An erlotinib-resistant SW480 colon cancer cell line was produced by long-term exposure to erlotinib. Curcumin-loaded Methoxy poly ethylene glycol Poly caprolactone (cur/mPEG-PCL) and erlotinib-loaded mPEG-PCL (erl/mPEG-PCL) micelles were provided using a single step nanoprecipitation method and used as combination therapy of resistant SW480 cancer cells. After that, gene expression levels of PDK4, αv, and ß3 mRNA were determined by the semiquantitative reverse transcription-polymerase chain reaction. Protein levels of whole αv ß3 integrin were evaluated using the enzyme-linked immunosorbent assay method. In SW480 cell line, the IC50 of nonresistant and resistant cells was 87.6 ± 1.2 nM and 19.1 ± 0.14 µM, for erlotinib and it was about 21.8 and 30 µM for curcumin, respectively. Although PDK4 expression was not significantly different in resistant and nonresistant cells, its expression was up regulated (1.4 fold) in resistant cells by a combination therapy of cur/mPEG-PCL at a dose of 3 µM and erl/mPEG-PCL at a dose of 5 µM. ß3 mRNA and the protein level of whole αv ß3 integrin was significantly higher in resistant SW480 cells as compared with those in nonresistant cells. In terms of treatment, a combination of 6-µM cur/mPEG-PCL and 5-µM erl/mPEG-PCL down regulated ß3 gene expression 6.6-fold in resistant cells as compared with nonresistant cells. At the protein level, a combination of 3-µM-cur/mPEG-PCL and 10-µM erl/mPEG-PCL reduced αv ß3 protein in resistant cells. The results indicated that combination therapy using cur/mPEG-PCL and erl/mPEG-PCL could decrease αv ß3 integrin expression and increase PDK4 gene expression in resistant colon cancer cells, which may have effects on drug resistance signaling pathways.

Evaluation of the Level of Zinc and Malondialdehyde in Basal Cell Carcinoma.

BACKGROUND: Basal Cell Carcinoma (BCC) is one of the most common skin cancers in the world and that use to lifestyle, increasing chemical pollutions, environmental factors and poor nutrition. The most important cause of this cancer is oxidative stress and free radicals so antioxidant activities for the body are so important. The aim of this study was to determine the variation of zinc and (Malondialdehyde) MDA in BCC patients. METHODS: This study has been performed on case and control patients from 2013 to 2014. The samples were collected from cell carcinoma patients at Razi Hospital in Tehran, Iran. We evaluated the level of zinc with the use of Atomic Absorption Spectroscopy (AAS) method. Besides, we evaluated MDA with colorimetric assay. RESULTS: The concentration of MDA was significantly higher in case group in comparison to control group (P=0.001). In addition, case group had lower concentration of zinc than the control group (P=0.000). There was no correlation between MDA and body mass index (BMI) and between zinc and BMI. CONCLUSION: All the patients with BCC showed a significant MDA serum in comparison with control group. However, significant decrease in zinc serum of the patients was seen that is because of consuming zinc during oxidative stress process so topical use of zinc in the form of 2+ ions could be effective on antioxidant protection against the sun UV radiation.

Evaluation of hypoxia inducible factor-1 alpha gene expression in colorectal cancer stages of Iranian patients.

AIM OF STUDY: Colorectal cancer (CRC) is the fourth most prevalent cancer globally. Several factors have roles in cancer establishment. One of the most important factors is hypoxia that induces hypoxia inducible factor-1 (HIF-1). The HIF-1 alpha overexpressed in hypoxia conditions and plays a pivotal role in carcinogenesis features. In this study, we aimed to examine the efficiency of HIF-1 alpha gene expression at mRNA and protein's level for CRC diagnosing and staging. MATERIALS AND METHODS: In this study, the cases included into 75 cancer specimens in different stages (Group 2 = Stage 1, Group 3 = Stage 2, and Group 4 = Stage 3, 4) and ten normal specimens as control (Group 1). Real-time reverse transcription-polymerase chain reaction and immunohistochemistry (IHC) were performed for measuring gene expression at RNA and protein's level, respectively. The raw data were analyzed in the SPSS20 software. RESULTS: HIF-1 alpha gene expression rate (2-ΔΔCT) and ΔCT values were significantly higher increased in Group 4 in compare to control (P < 0.001). Other cancer groups (2 and 3) had greater ΔCT values than control, but it was not statistically significant. Moreover, the rate of HIF-1 alpha gene expression (2-ΔΔCT) was increased with cancer stages. According to the IHC results, there was a positive relationship between CRC stages and HIF-1 alpha protein expression (P < 0.05). CONCLUSIONS: HIF-1 alpha gene expression increased in earlier up to metastasis stages of CRC, but the assessment of HIF-1 alpha gene expression has not important role in the diagnosis of cancer in early stages and classification of carcinoma because the increasing of HIF-1 alpha gene expression is not significant in early cancer stages.

Synthesis of a stabilized 177Lu-siRNA complex and evaluation of its stability and RNAi activity.

PURPOSE: Serum and intracellular instability limits the therapeutic applications of short interfering RNA (siRNA) as a radiopharmaceutical. Chemical modifications like phosphorothioate (PS) substitution and 2'-O-methoxy (2'-O-Me) modifications could eliminate such limitations. In this study, the effects of PS and 2'-O-Me modifications at the backbone of siRNA on serum stability and RNA interference activity were investigated. MATERIALS AND METHODS: Fully PS and 2'-O-Me-modified type 1 insulin-like growth factor receptor (IGF-1R) siRNA was radiolabeled with lutetium-177 ((177)Lu) through p-SCN-Bn-DTPA as a chelator. After purification with Vivaspin and PD-10 columns, the radiolabs were examined for stability in serum by instant thin-layer chromatography and polyacrylamide gel electrophoresis. The level of IGF-1R in response to the modified and labeled IGF-1R siRNA was examined using RT-PCR and ELISA assay in colon cancer cells. The effects of such siRNA on the prevention of proliferation of colon cancer cells and its apoptosis were investigated using MTT assay and Annexin-V/propidium iodide double staining, respectively. Cellular accumulation quantities of the labeled and modified IGF-1R siRNA were determined using a γ-counter by taking advantage of (177)Lu as a γ-emitter. RESULTS: Both the modified (177)Lu-siRNA complex and the modified nonlabeled siRNA showed significant stability in serum. The levels of IGF-1R mRNA and protein significantly decreased with both the labeled and nonlabeled IGF-1R siRNAs, but no such reduction in IGF-1R was observed with luciferase siRNA (P<0.01). Proliferation decreased significantly and apoptosis increased in the cells treated with modified (177)Lu-IGF-1R siRNA in comparison with either (177)Lu or labeled luciferase siRNA (P<0.001). CONCLUSION: Uniform chemically modified siRNAs can form stable complexes with Lu that pronounce its cytotoxic effect through apoptosis in colon cancer cells.

Demonstration of dose dependent cytotoxic activity in SW480 colon cancer cells by ¹77Lu-labeled siRNA targeting IGF-1R.

PURPOSE: The radiolabeling of targeting biomolecules with gamma emitter radionuclides for tracing and beta emitters for therapy involves the conjugation of such biomolecules to the chelating agents to form complexes with the radionuclide of interest. In this study, radioconjugate of IGF-1R siRNA with lutetium-177 ((177)Lu) was produced, and the anti-proliferation and apoptosis effects elicited by this (177)Lu-siRNA complex in the SW480 colon cancer cells were evaluated. METHODS: IGF-1R and Luciferase siRNAs were conjugated with p-SCN-Bn-DTPA, and then radiolabeled with (177)Lu. The effects of labeled and non-labeled IGF-1R siRNAs on IGF-1R expression were assessed with RT-PCR analysis and ELISA assay. IGF-1R siRNAs induced cell death and apoptosis were investigated using MTT assay and Annexin-V/propidium iodide (PI) double staining, respectively. RESULTS: Combined purification using Vivaspin and PD-10 columns resulted in a radiochemical purity of 97.32% ± 1.97%. Knockdown effect of the labeled IGF-1R siRNA was not significantly different from the non-labeled duplex of the same sequence (P<0.05), but it was significant compared to the Luciferase siRNAs (P<0.001). Proliferation decreased significantly, but apoptosis increased in the cells treated with the (177)Lu-IGF-1R siRNA in comparison with either (177)Lu or IGF-1R siRNA (P<0.001). CONCLUSION: Radioconjugate of IGF-1R siRNA, p-SCN-Bn-DTPA and (177)Lu was successfully produced and characterized as radiopharmaceutical. The present study demonstrates the involvement of (177)Lu-labeled IGF-1R siRNA in the inhibition of cell growth and induction of apoptosis in colon cancer cells.

Adenosine induces cell cycle arrest and apoptosis via cyclinD1/Cdk4 and Bcl-2/Bax pathways in human ovarian cancer cell line OVCAR-3.

Adenosine is a regulatory molecule with widespread physiological effects in almost every cells and acts as a potent regulator of cell growth. Adenosine has been shown to inhibit cell growth and induce apoptosis in the several cancer cells via caspase activation and Bcl-2/Bax pathway. The present study was designed to understand the mechanism underlying adenosine-induced apoptosis in the OVCAR-3 human ovarian cancer cells. MTT viability, BrdU and cell counting assays were used to study the cell proliferation effect of adenosine in presence of adenosine deaminase inhibitor and the nucleoside transporter inhibitor. Cell cycle analysis, propidium iodide and annexin V staining, caspase-3 activity assay, cyclinD1, Cdk4, Bcl-2 and Bax protein expressions were assessed to detect apoptosis. Adenosine significantly inhibited cell proliferation in a concentration-dependent manner in OVCAR-3 cell line. Adenosine induced cell cycle arrest in G0/G1 phase via Cdk4/cyclinD1-mediated pathway. Adenosine induced apoptosis, which was determined by Annexin V-FITC staining and increased sub-G1 population. Moreover, down-regulation of Bcl-2 protein expression, up-regulation of Bax protein expression and activation of caspase-3 were observed in response to adenosine treatment. The results of this study suggest that extracellular adenosine induced G1 cell cycle arrest and apoptosis in ovarian cancer cells via cyclinD1/ Cdk4 and Bcl-2/Bax pathways and caspase-3 activation. These data might suggest that adenosine could be used as an agent for the treatment of ovarian cancer.