Nuclear PKR in retinal neurons in the early stage of diabetic retinopathy in streptozotocin­induced diabetic rats.

Retinal neuron apoptosis is a key component of diabetic retinopathy (DR), one of the most common complications of diabetes. Stress due to persistent hyperglycaemia and corresponding glucotoxicity represents one of the primary pathogenic mechanisms of diabetes and its complications. Apoptosis of retinal neurons serves a critical role in the pathogenesis of DR observed in patients with diabetes and streptozotocin (STZ)­induced diabetic rats. Retinal neuron apoptosis occurs one month after STZ injection, which is considered the early stage of DR. The molecular mechanism involved in the suppression of retinal neuron apoptosis during the early stage of DR remains unclear. RNA­dependent protein kinase (PKR) is a stress­sensitive pro­apoptotic kinase. Our previous study indicated that PKR­associated protein X, a stress­sensitive activator of PKR, is upregulated in the early stage of STZ­induced diabetes. In order to assess the role of PKR in DR prior to apoptosis of retinal neurons, immunofluorescence and western blotting were performed to investigate the cellular localization and expression of PKR in the retina in the early stage of STZ­induced diabetes in rats. PKR activity was indirectly assessed by expression levels of phosphorylated eukaryotic translation initiation factor 2α (p­eIF2­α) and the presence of apoptotic cells in the retina was investigated by TUNEL assay. The findings revealed that PKR was localized in the nucleus of retinal ganglion and inner nuclear layer cells from normal and diabetic rats. To the best of our knowledge, the present study is the first to demonstrate nuclear localization of PKR in retinal neurons. Immunofluorescence analysis demonstrated that PKR was expressed in the nuclei of retinal neurons at 3 and 6 days and its expression was decreased at 15 days after STZ treatment. In addition, p­eIF2­α expression and cellular localization followed the trend of PKR, suggesting that this pro­apoptotic kinase was active in the nuclei of retinal neurons. These findings are consistent with the hypothesis that nuclear translocation of PKR may be a mechanism to sequester active PKR, thus preventing upregulation of cytosolic signalling pathways that induce apoptosis in retinal neurons. Apoptotic cells were not detected in the retina in the early stage of DR. A model was proposed to explain the mechanism by which apoptosis of retinal neurons by PKR is suppressed in the early stage of DR. The possible role of mitochondrial RNA (mtRNA) and Alu RNA in this phenomenon is also discussed since it was demonstrated that the cellular stress due to prolonged hyperglycaemia induces the release of mtRNA and transcription of Alu RNA. Moreover, it mtRNA activates PKR, whereas Alu RNA inhibits the activation of this protein kinase.

[Corrigendum] Intratumoral injection of PKR shRNA expressing plasmid inhibits B16-F10 melanoma growth.

It has been brought to our attention that there was an important omission of the word 'shRNA', which should have been featured in a couple of places in the above article. The authors incorrectly claimed that the RNA­dependent protein kinase (PKR) acts as a tumor suppressor; in fact, it is the PKR shRNA that would fulfil the role of being a tumor suppressor. Therefore, the text in the Abstract on p. 2267, line 14, should have read "...these findings suggested that PKR shRNA acts as a tumor suppressor"; likewise, on p. 2272, towards the end of the final sentence of the second paragraph of the Discussion, the text should have read "The results show that PKR silencing inhibits tumor growth, suggesting that PKR shRNA acts as a tumor supressor...". The authors regret that this error was not corrected before the article went to print, and they thank the reader concerned for drawing the matter to our attention. [the original article was published in the Oncology Reports 32: 2267­2273, 2014; DOI: 10.3892/or.2014.3410].

Knockdown of chemokine receptor CXCR4 gene by RNA interference: Effects on the B16-F10 melanoma growth.

The incidence of malignant melanoma has increased greatly in recent decades presenting a high mortality rate despite intensive efforts in this area of research. Recent studies indicate that the chemokine receptor 4 (CXCR4) plays a critical role in cancer. Thus, it has been reported that CXCL12 binding to CXCR4 initiates various downstream signaling pathways that result in a plethora of responses involved in cell proliferation and metastasis. Recently, we demonstrated that CXCR4 silencing by RNA interference (RNAi) significantly reduced the number of pulmonary metastatic nodules. In the present study, we examined the effect of the intratumoral injection of CXCR4 short hairpin (shRNA) expressing plasmids on the growth of B16­F10 melanoma in mice. In vitro transfection of these tumor cells with CXCR4 shRNA expressing plasmid (CXCR4 shRNA) significantly reduced the levels of CXCR4 mRNA (85%) and CXCR4 protein (70%) compared with the control. We showed that the tumor growth was significantly reduced (66%) in mice inoculated with transfected B16­F10 melanoma cells when compared with the control group. We also found that the intratumoral injection of CXCR4 shRNA expressing plasmids results in a significant inhibition (70%) of B16­F10 melanoma growth. This finding supports the hypothesis that a direct administration of RNAi­based therapeutics into the target tumor is a promising approach for overcoming the hurdles of systemic delivery. The present study is the first demonstration that CXCR4 plays a critical role in B16-F10 melanoma growth. Currently there is great interest in the development of antagonists for therapeutic targeting CXCR4 expression. Considering our results and the fact that CXCR4 is highly conserved between human and mouse, this experimental model of cancer may be useful for the discovery of new CXCR4 antagonists with clinical implications.

In vivo knockdown of CXCR4 using jetPEI/CXCR4 shRNA nanoparticles inhibits the pulmonary metastatic potential of B16­F10 melanoma cells.

Metastasis is a key factor that limits survival in the majority of patients with cancer. Thus, numerous efforts have been made to elucidate the molecular mechanisms involved in this phenomenon. B16­F10 melanoma cells have been demonstrated to be highly metastatic to the lungs in mice. The aim of the current study was to investigate the role of CXC motif chemokine receptor 4 (CXCR4) in the metastatic potential of B16­F10 melanoma cells in mice. In vitro transfection of B16­F10 tumor cells with CXCR4 short hairpin RNA (shRNA) expressing plasmids (CXCR4 shRNA) significantly reduced the expression levels of CXCR4 mRNA (80%) and protein (68%), compared with the control. In addition, these results demonstrated that pulmonary metastasis was significantly inhibited (85%) in mice inoculated with CXCR4 shRNA­transfected B16­F10 melanoma cells. The polycation­based nanoparticle (jetPEI) was used to investigate the effect of CXCR4 knockdown in vivo on the metastatic potential of B16­F10 melanoma cells. The number of pulmonary metastatic nodules was significantly reduced (50%) in animals that received a retro­orbital injection of jetPEI­CXCR4­1 shRNA. The current study demonstrated that CXCR4 serves a role in the metastatic potential of B16­F10 melanoma cells. Currently there is a great interest in the development of antagonists for the therapeutic targeting of CXCR4 expression. Taking the results of the current study and the fact that CXCR4 is highly conserved between humans and mice into account, this experimental model of metastasis with B16­F10 melanoma cells may aid in the discovery of CXCR4 antagonists with clinical implications.

Intratumoral injection of PKR shRNA expressing plasmid inhibits B16-F10 melanoma growth.

The RNA-dependent protein kinase (PKR) is a serine/threonine kinase that is involved in the regulation of important cell processes such as apoptosis, signal transduction, cell proliferation and differentiation. However, the role played by PKR in cancer remains controversial. RNA interference (RNAi) has currently become an important technique in understanding gene function. Previously, we showed that PKR shRNA downregulates PKR expression in B16-F10 melanoma cells and reduces the metastatic potential of these tumor cells. In the present study, we examined the effect of the intratumoral injection of PKR shRNA­expressing plasmid on the growth of B16-F10 melanoma in mice. The results showed that this treatment significantly reduced tumor growth. Thus, these findings suggested that PKR acts as a tumor suppressor, a finding that is consistent with our previous study on the experimental model of metastasis. Moreover, the results suggested that this effect may be mediated by the transcription factor NF-κB. The present study confirmed the hypothesis that the direct administration of RNAi-based therapeutics in the target tumor is a promising approach for overcoming the obstacles of systemic delivery. The results also suggested that the intratumoral injection of PKR shRNA­expressing vector is a novel therapeutic approach for human solid tumors such as cutaneous melanoma and breast cancer, since PKR is overexpressed in these tumors.

Activation of the kinin B1 receptor attenuates melanoma tumor growth and metastasis.

Melanoma is a very aggressive tumor that does not respond well to standard therapeutic approaches, such as radio- and chemotherapies. Furthermore, acquiring the ability to metastasize in melanoma and many other tumor types is directly related to incurable disease. The B1 kinin receptor participates in a variety of cancer-related pathophysiological events, such as inflammation and angiogenesis. Therefore, we investigated whether this G protein-coupled receptor plays a role in tumor progression. We used a murine melanoma cell line that expresses the kinin B1 receptor and does not express the kinin B2 receptor to investigate the precise contribution of activation of the B1 receptor in tumor progression and correlated events using various in vitro and in vivo approaches. Activation of the kinin B1 receptor in the absence of B2 receptor inhibits cell migration in vitro and decreases tumor formation in vivo. Moreover, tumors formed from cells stimulated with B1-specific agonist showed several features of decreased aggressiveness, such as smaller size and infiltration of inflammatory cells within the tumor area, higher levels of pro-inflammatory cytokines implicated in the host anti-tumor immune response, lower number of cells undergoing mitosis, a poorer vascular network, no signs of invasion of surrounding tissues or metastasis and increased animal survival. Our findings reveal that activation of the kinin B1 receptor has a host protective role during murine melanoma tumor progression, suggesting that the B1 receptor could be a new anti-tumor GPCR and provide new opportunities for therapeutic targeting.

Expression of noncoding mRNA in human blood cells activated with synthetic peptide of HIV.

T cells activation includes several steps such as translational events, activation of transcription for different genes, expression of surface molecules, secretion of cytokines, effectors functions. Knowledge has been accumulated on various nontranslatable RNA transcripts that are synthesized. In this context, a member of T cell noncoding transcripts (NTT) has been identified. It has been known that this gene is selectively expressed in activated T cells, as a 17-kb transcript. In this study, we investigate cell activation using RT-PCR to detect NTT. We investigated the expression of IFNgamma mRNA, a cytokine produced by activated blood mononuclear cells treated with HLA-A2 restricted synthetic peptide of HIV (p9) by RT-PCR detecting a fragment of 300 bp. This finding demonstrated that human HLA-A2 blood mononuclear cells have been activated in the presence of synthetic peptide of HIV (p9) and can induce expression of mRNA of NTT and IFNgamma which was confirmed by direct sequencing. For the first time, we have demonstrated an endogenous noncoding human RNA molecule, NTT mRNA, suggesting its implication in the cellular immune response.

Molecular investigation of the stromal cell-derived factor-1 chemokine in lymphoid leukemia and lymphoma patients from Brazil.

The stromal cell-derived factor-1 (SDF-1) gene contains a common polymorphism, termed SDF1-3'A, in an evolutionarily conserved segment of the 3'-untranslated region (3'-UTR). We compared SDF-1 genotypes in patients diagnosed with lymphoid leukemias and lymphomas. Since the SDF1-3'A variant deletes the MspI restriction site, PCR-restriction fragment length polymorphism (RFLP) analysis was used for identification of genotypes. We identified the heterozygous genotype (3'A/wt) in 38.8% (24/62) of lymphoma patients and in 26.2% (11/42) of lymphoid leukemias. The percentage of 3'A carriers was significantly higher in lymphomas (43.5%) than in lymphoid leukemias (26.2%; P < 0.05). Our study indicates that lymphoma patients from Brazil are more likely to carry the 3'A gene than patients with lymphoid leukemias, suggesting that this polymorphism may be a differential determinant of lymphomas and lymphoid leukemia.

Antitumor activity induced by regulatory RNA: possible role of RNA-dependent protein kinase and nuclear factor-kappaB.

Regulatory RNAs are noncoding RNAs that can regulate gene expression. Our previous results showed that regulatory RNAs can induce the production of interleukin-1, interleukin-2, interleukin-8, tumor necrosis factor-alpha (TNF-alpha), interferon-gamma, and Fas ligand (FasL). These cytokines and FasL are involved in host defense mechanisms against tumors. B16-F10 melanoma cells are highly metastatic to the lungs and we showed that lymphocytes treated with the regulatory B16-RNA reduce significantly the number of metastatic nodules. We also found that B16-RNA activates RNA-dependent protein kinase (PKR) and the active B16-RNA fraction is polyadenylated with a sedimentation coefficient of 18S. Our findings suggest that the antitumor activity of B16-RNA is mediated by PKR through activation of the transcription factor NF-kappaB. Thus, B16-RNA may act as a regulatory RNA and may regulate gene expression at transcriptional level. This study provides the rationale for the use of B16-RNA as an immunomodulator in melanoma.