RGD-decorated nanoliposomes for combined delivery of arsenic trioxide and curcumin to prostate cancer cells.

Nanotechnology and drug co-delivery offer a novel avenue in drug delivery research liposome-based co-delivery of anticancer drugs targeting the apoptosis pathway as a promising new approach to treat cancer. In this study, a co-delivery system of liposomes (arsenic trioxide/curcumin) modified with RGD peptide was designed to aim for enhancing the treatment of prostate cancer cells (PC3 cell line). Liposomal co-loaded curcumin and arsenic trioxide modified by RGD peptide (NLPs-RGD-Cur-ATO) were prepared by thin-layer lipid hydration techniques for the treatment of prostate cancer. The stability of the NLPs-RGD-Cur-ATO was evaluated by particle size analysis through dynamic light scattering (DLS) analysis and transmission electron microscopy (TEM). The percentage of cytotoxicity and apoptotic effect in PC3 cells treated with NLPs-RGD-Cur-ATO were detected by MTT and Annexin V-FITC (fluorescein isothiocyanate)/PI affinity assay, respectively. The particle size of NLPs-RGD-Cur-ATO was approximately 100 nm, with an encapsulation efficiency of about 99.52% and 70.61%, for ATO and Cur, respectively. Besides, NLPs-RGD-Cur-ATO displayed an enhanced anti-proliferative effect, increased the percentage of apoptotic cells 98 ± 1.85% (p < 0.0001), and significantly reduced EGFR gene expression level (p < 0.001) in the cell line tested. These results indicated that our NLPs-RGD-Cur-ATO co-delivery system was a promising strategy for prostate cancer therapy.

Arsenic trioxide and Erlotinib loaded in RGD-modified nanoliposomes for targeted combination delivery to PC3 and PANC-1 cell lines.

During the past few years, advances in drag delivery have provided many opportunities in the treatment of various diseases and cancer. Arsenic trioxide (ATO) and Erlotinib (Erlo) are two drugs, approved by the United States Food and Drug Administration to treat cancer, but their use is limited in terms of the toxicity of ATO and the low solubility of Erlo. This study aimed to prepare arginine-glycine-aspartic acid (RGD)-decorated nanoliposomes (NLPs) containing Erlo and ATO (NLPs-ATO-Erlo-RGD) to increase the solubility and reduce the toxicity of Erlo and ATO for cancer treatment. The results of transmission electron microscopy and dynamic light scattering showed that NLPs were synthesized uniformly, with spherical shape morphology and particle sizes between 140 and 160 nm. High-performance liquid chromatography and ICP-MS results showed that about 90% of the drug was loaded in the NLPs. In comparison with NLPs-ATO-Erlo, NLPs-ATO-Erlo-RGD demonstrated considerable toxicity against the αvß3 overexpressing PC3 cell line in the MTT experiment. It had no effect on the PANC-1 cell line. In addition, apoptosis assays using Annexin V/PI demonstrated that NLPs-ATO-Erlo-RGD generated the highest apoptotic rates in PC3 cells when compared with NLPs-ATO-Erlo and the combination of free ATO and Erlo. Furthermore, treatment with NLPs-ATO-Erlo-RGD in (p < 0.05) PC3 cell line significantly reduced EGFR level. It is concluded NLPs-ATO-Erlo-RGD as a novel drug delivery system may be a promising platform for the treatment of cancer.

GLI inhibitors overcome Erlotinib resistance in human pancreatic cancer cells by modulating E-cadherin.

Inhibition of hedgehog (Hh) signalling pathway, including its end effector GLI1, can reverse epithelial-to-mesenchymal transition (EMT) which plays an important role in drug resistance of pancreatic cancer cells to Erlotinib (ETB). This study investigated the effect of GLI inhibitors Forskolin (FSK), GANT-61 (GNT), and Arsenic trioxide (ATX) on suppressing the resistance of pancreatic cancer cells to ETB. The effect of GLI inhibitors was evaluated by measuring mRNA expression levels of EMT factors using quantitative RT-PCR. Immunocytochemistry and flow cytometry were used to assess E-cadherin (E-Cad) and GLI1 protein levels. MTT and apoptosis assays were used to evaluate the synergistic effects for the combination treatment of each GLI inhibitor with ETB. Pancreatic cancer cells PANC-1 treated by GNT showed the highest significant reduction in mRNA levels of GLI1 and other EMT pathway genes. Moreover, GNT was able to upregulate E-Cad and downregulate GLI1 proteins, more than FSK, while ATX had no effect. Apoptosis levels of PANC-1 cells following treatment with LD30 concentrations of FSK, GNT, or ATX, showed 57%, 62% and 67%, respectively, in comparison to ETB (∼48%). Importantly, combination treatments of ETB with either FSK, GNT, or ATX demonstrated a significant increase in apoptotic cells reaching 61% (ETB + FSK), 80% (ETB + GNT) or 88% (ETB + ATX). FSK did not have much effect on the drug resistance of PANC-1 cells to ETB. However, GNT, but more effectively ATX, were able to reduce the drug resistance of this cell line to ETB.

The Effect of RGD/NGR Peptide Modification of Melanoma Differentiation-Associated Gene-7/Interleukin-24 on Its Receptor Attachment, an In Silico Analysis.

Melanoma differentiation-associated gene-7 (mda-7/interleukin [IL]-24), a unique tumor suppressor gene, induces selective apoptosis in tumor cells. Secreted IL-24 binds to heterodimeric receptor complexes of IL-20R1/IL-20R2, IL-22R1/IL-20R2, or sigma-1 receptor (Sig1R) that consequently enhances apoptosis. However, this mechanism is not well understood and most likely involves different pathways. Targeting of cytokine by tumor homing peptides (THPs) to the tumor cell surface molecule-like integrin shows to be beneficial in gene immunotherapy approaches. In this study, the in silico targeting of RGD/NGR-modified IL-24 to tumor cells was conducted. In this regard, the sequences of six new synthetic IL-24s that have been modified by RGD (Arg-Gly-Asp) or NGR (CRNGRGPDC) were aligned and their structures were modeled through homology modeling to evaluate their attachment potential to cognate receptor complexes such as IL-20R1/IL-20R2, IL-22R1/IL-20R2, or Sig1R. The results of homology modeling showed that modification of IL-24 with RGD motif in N-terminal and middle of this protein exhibited stronger interaction with cognate receptors. These results also demonstrated that modified IL-24 with RGD motif in the C-terminal has lost native activity. However, the interaction of THP-modified IL-24 with Sig1R would not be affected to that extent, interestingly. Conclusively, in silico analysis showed that modification of IL-24 with THPs needs a more detailed study as these modifications may disrupt native interaction with receptors and reduce apoptosis induction property. This structural analysis gives us a better understanding of mda-7/IL-24 interaction with cognate receptors and helps a more rational design for further cytokine modification.

The Regenerative Effect of Bone Marrow-Derived Stem Cells in Spermatogenesis of Infertile Hamster.

BACKGROUND: Infertility is a serious social problem in advanced nations, with male factor in half of all cases of infertility. This study was conducted to determine the regenerative effect of bone marrow-derived stem cells in spermatogenesis of infertile hamster. METHODS: Twelve adult male hamsters were equally divided into azoospermic and control groups. Busulfan was intraperitoneally used for induction of azoospermia, while the right testis was treated with bone marrow-derived stem cells (106 BM-SCs), labeled with sterile trypan blue, 35 days after busulfan injection. The left testis served as positive control for azoospermia. Sixty days after cell transplantation, the animals were euthanized and both testes were removed and evaluated histologically. RESULTS: BM-SCs were spindle-shaped, adherent to the culture flasks and had positive expression of CD29 and CD73 and negative expression of CD45. Alcian blue staining confirmed differentiation of BM-SCs into chondrocytes. Karyotyping denoted to stability of chromosomes. Treatment with busulfan in seminiferous tubules resulted into distruption of spermatogenesis. After two months in busulfan treatment group, seminiferous tubular atrophy and germinal epitheliums degenerations were noticed with no spermatozoa in epididymis. After treatment of busulfan group with BM-SCs, spermatogonia, primary spermatocytes, spermatids and sperms were present in seminiferous tubules. CONCLUSION: As cell transplantation in seminiferous tubules resulted into a rapid repair of pathological changes, BM-SCs can be recommended an effective treatment measure in azoospermia. It seems that more studies are necessary to confirm the use of this technique in treatment of azoospermia and infertility in human.

Effect of RGD coupled MDA-7/IL-24 on apoptosis induction in a hepatocellular carcinoma cell line.

The melanoma differentiation-associated gene­7 (MDA-7) gene, also termed interleukin­24 (IL­24), is a tumor suppressor gene that induces apoptosis in a broad scope of malignant neoplastic cells. The apoptosis induction capacity of the MDA­7/IL­24 gene is partially associated with adhering to cognate receptors. The current study aimed to enhance the antitumor effect of IL­24. The intrinsic signal sequence of IL-24 replaced with a fused artificial signal (secrecon)-RGD4C sequence and its impact was evaluated in HepG2 cells. The modified SP.RGD.IL­24 and native IL­24 cDNA sequences were cloned into the pcDNA3.1 expression vector. Subsequently, the expression level, secretion efficacy and targeting propensity of the modified SP.RGD.IL­24 product compared with normal IL­24 by were determined by enzyme­linked immunosorbent assay. The constructs were then transfected into HepG2 and LX­2 cells as tumor and normal hepatic cell lines, respectively. The expression level of the pro­apoptotic DNA damage inducible transcript 3 (Gadd153) and BCL2 associated X apoptosis regulator (Bax) genes in the different groups were compared by reverse transcription-quantitative polymerase chain reaction. Additionally, the rate of apoptosis induction of modified and intact IL­24 sequences was compared by flow cytometry analysis of cells following their propidium iodide/annexin V staining. SP.RGD-IL-24 protein was expressed and secreted in a similar manner to native IL­24, however, the modified SP.RGD.IL-24 adhered to tumor cells more efficiently than IL­24 (P<0.05). SP.RGD.IL­24 significantly induced upregulation of Gadd153 and Bax in HepG2 cells compared with native IL­24 (P<0.05). However, neither had a significant impact on the expression level of pro-apoptotic genes in LX­2 cells. Flow cytometry analysis also indicated that modified SP.RGD.IL-24 induced apoptosis more than native IL­24 in HepG2 cells (P<0.05). In conclusion, the novel generated RGD­coupled IL-24 construct exhibited sufficient anticancer activity compared with the native IL­24. The results of the current study provide novel insights for the future of cytokine targeting and indicates its potential capacity as a valuable candidate for gene therapy methods.

Experimental verification of a predicted intronic microRNA in human NGFR gene with a potential pro-apoptotic function.

Neurotrophins (NTs) are a family of secreted growth factor proteins primarily involved in the regulation of survival and appropriate development of neural cells, functioning by binding to their specific (TrkA, TtkB, and TrkC) and/or common NGFR receptor. NGFR is the common receptor of NTs, binding with low-affinity to all members of the family. Among different functions assigned to NGFR, it is also involved in apoptosis induction and tumorigenesis processes. Interestingly, some of the functions of NGFR appear to be ligand-independent, suggesting a probable involvement of non-coding RNA residing within the sequence of the gene. Here, we are reporting the existence of a conserved putative microRNA, named Hsa-mir-6165 [EBI accession#: FR873488]. Transfection of a DNA segment corresponding to the pre-mir-6165 sequence in Hela cell line caused the generation of mature exogenous mir-6165 (a ∼200,000 fold overexpression). Furthermore, using specific primers, we succeeded to detect the endogenous expression of mir-6165 in several glioma cell lines and glioma primary tumors known to express NGFR. Similar to the pro-apoptotic role of NGFR in some cell types, overexpression of pre-mir-6165 in U87 cell line resulted in an elevated rate of apoptosis. Moreover, coordinated with the increased level of mir-6165 in the transfected U87 cell line, two of its predicted target genes (Pkd1 and DAGLA) were significantly down-regulated. The latter findings suggest that some of the previously attributed functions of NGFR could be explained indirectly by co-transcription of mir-6165 in the cells.

Neuroprotective effects of mesenchymal stem cell transplantation in animal model of cerebellar degeneration.

BACKGROUND: The cerebellum has been considered a key structure for the processes involved in sensorimotor integration ultimately leading to motor planning and execution of coordinated movement. Thus, motor deficits and behavioral changes can be associated with cerebellar degeneration. METHODS: Here, the chemical neurotoxin pyridine-2,3-dicarboxylic acid (quinolinic acid, QA) used to create partially cerebellar degeneration in adult Wistar rats suitable for use in stem cell transplantation studies. Stereotaxicaly administration of QA (0.2 mmol) in the right cerebellar hemisphere (folia VI) caused noticeable motor disturbance in all treated animals. Forty-eights hours after causing lesion, rat bone marrow-derived mesenchymal stem cells (MSCs) were transplanted into damaged cerebellar hemisphere. We investigated the role of MSC transplantation in forms of motor and non-motor learning that involves the cerebellum and its neuroprotective effects in Purkinje cells loss. RESULTS: CM-Dil labeling showed that the transplanted MSCs survived and migrated in the cerebellum 6 weeks after transplantation. The MSC-transplanted group showed markedly improved functional performance on the rotating rod test (P≤0.0001) and beam walking test (P≤0.0001) during 6 weeks compared with the controls. For non-motor learning, we used passive avoidance learning test in 3 weeks after transplantation. The results showed that MSC transplantation prevented the development of memory deficit caused by cerebellar degeneration (P≤0.001). Stereological analysis in 6 weeks after transplantation showed that QA significantly decreases Purkinje cells in vehicle-treated rats and MSC transplantation is neuroprotective and decreases Purkinje cell loss in MSC-treated rats (P≤0.0001). CONCLUSION: The results indicate that transplantation of MSCs can significantly reduce the behavioral and neuroanatomical abnormalities of these animals during 6 weeks after engraftment. According to results of this assay, cell therapy by means of bone marrow-derived adult stem cells promises for treatment of cerebellar diseases.

Bone marrow derived mesenchymal stem cell transplantation in cerebellar degeneration: a behavioral study.

In addition to its key role in complex motor function, the cerebellum is increasingly recognized to have a role in cognition. Thus, motor and cognitive deficits can be associated with cerebellar degeneration. After unilateral lesion in cerebellum (folia VI) was caused by Quinolinic acid, CM-DiI labeled mesenchymal stem cells (MSCs), which were isolated and purified from bone marrow, were transplanted into the damaged folium. Motor function was assessed using the cylinder test, rotarod, hanging wire and beam balance during 6 weeks after transplantation. Cognitive function was assessed using the Morris water maze learning paradigm in 3 weeks after transplantation. Six weeks after transplantation surviving MSCs were detectable in QA-treated animals. The MSC-transplanted group showed markedly improved functional performance in spatial memory, motor learning, locomotor asymmetry, dysmetria, abnormality in neuromuscular strength and equilibrium 2-6 weeks compared with the controls. We found that cerebellar lesions produced deficits (folia VI) in motor and cognitive aspects of a spatial task. The results indicate that transplantation of MSCs can significantly reduce the behavioral abnormalities of these animals during six weeks after engraftment. According to results of this assay, cell therapy by means of bone marrow derived adult stem cells promises for treatment of cerebellar diseases.