Biologic and binding activities of IFN-alpha subtypes in ACHN human renal cell carcinoma cells and Daudi Burkitt's lymphoma cells.

Nine interferon-alpha subtypes, IFN-alpha1, IFN-alpha2, IFN-alpha5, IFN-alpha7, IFN-alpha8, IFN-alpha10, IFN-alpha14, IFN-alpha17, and IFN-alpha21, were separated from purified human lymphoblastoid IFN. We tested their inhibitory effects on cell growth and replication of Semliki Forest virus (SFV) and vesicular stomatitis virus (VSV) and their induction of 2',5'-oligoadenylate synthetase (2', 5'-OAS) in ACHN renal cell carcinoma cells. In terms of all three activities, the nine subtypes had similar relative activities, with IFN-alpha10 the most active and IFN-alpha1 the least. Their relative effects on cell growth were similar in two other human cell lines, SK-LU-1 lung cancer cells and KU-2 renal cell carcinoma cells, whereas cells of the Daudi Burkitt lymphoma line behaved quite differently, being highly sensitive to all the nine subtypes. The relative effects with ACHN cells correlated well with their relative binding affinities. However, each of the subtypes bound to both ACHN and Daudi cells to almost the same extent. This suggests that their profound inhibitory effects on the growth of Daudi cells are amplified at some stage in the signal transduction pathway or in the expression of genes that results from binding to the IFN-alpha receptor.     

Inhibition of γ-secretase by retinoic acid chalcone (RAC) induces G2/M arrest and triggers apoptosis in renal cell carcinoma

The present study was devised to investigate the effect of RAC on inhibition of cell proliferation and apoptosis of renal carcinoma cells. MTT assay and flow cytometry analysis were used to determine cell proliferation and apoptosis along with cell cycle examination. Western blot analysis and immunohistochemistry were used for the detection of expression levels of Notch1 and Jagged1 in renal cell carcinoma (RCC) and normal kidney tissues. The results revealed a significant inhibition of cell proliferation, G2/M phase cell cycle arrest and cell apoptosis at 30 μM concentration of RAC after 72 h. In ACHN and 769-P cells, the population in G2/M phase was increased to 45.27, and 54.23% respectively on treatment with 30 μM RAC for 72 h. In 769-P and ACHN renal carcinoma cells treatment with 30 μM RAC caused 69.71 and 59.27% of the cells to undergo apoptosis compared to 5.23 and 4.93% respectively in control cells. The positive staining rates of Notch1 and Jagged1 in renal carcinoma tissues were 95.3 and 93.0% compared to normal kidney tissues 36.4 and 42.4% respectively. Treatment of renal carcinoma tissues caused a significant decrease in staining rates of Notch1 and Jagged1 after 96 h. Thus RAC can be a potent agent in the treatment of renal cell carcinoma.     

Growth and metastatic behavior of human tumor cells implanted into nude and beige nude mice

The growth and metastatic behavior of several human tumor lines grown in adult nude mice, nude mice pretreated with antiserum against asialo GM1 glycoprotein, and beige nude mice were studied. The cell lines were all injected s.c. and i.v. A human colon carcinoma line was also injected into the spleen, and two human renal carcinoma lines were injected into renal subcapsule. All the tumor lines grew as fast or faster in adult nude mice compared with beige nude mice. There were no discernible differences in the production of experimental lung metastasis among the three groups of mice, but human colorectal carcinoma cells and human renal carcinoma cells produced more metastases in nude mice than in beige nude mice after intrasplenic or renal subcapsule injection, respectively. In vitro cytotoxicity assays confirmed that adult nude mice had high levels of natural killer (NK) cell activity whereas nude mice pretreated with anti-asialo GM1 serum and beige nude mice did not. The in vitro NK cell activity of nude mice was demonstrable against mouse lymphoma cells but not against human leukemia cells which were sensitive to lysis by human NK cells. These results suggest that the implantation of human tumor cells into beige nude mice, which are deficient in NK cell activity does not provide an advantageous model for the study of growth and metastasis of human neoplasms.Abbreviations NK natural killer - HCC human colon carcinoma - HRCC human renal cell carcinoma - anti-asGM1 anti-asialo GM1 - i.s. intrasplenic - RSC renal subcapsule - HBSS Ca²⁺- and Mg²⁺-free Hanks' balanced salt solution     

Thymoquinone ameliorates oxidative damage and histopathological changes of developing brain neurotoxicity

ABSTRACT

Lead (Pb) toxicity is known to be a chief environmental health issue, especially for pregnant women and young children. Today, the use of medicinal herbs in the treatment of many diseases and different toxic agents has become highly accepted due to their effectiveness and lower costs. Thymoquinone (TQ), which is extracted from Nigella sativa seeds, is a potent antioxidant and anti-inflammatory agent. This study was designed to explore the optional protectivity of TQ against maternal and fetal oxidative stress and brain damage induced by Pb administration. Pregnant rats were distributed into seven groups: control group, TQ group, DMSO group, two groups Pb-treated (160 and 320 ppm), and two groups Pb-treated (160 and 320 ppm) co-treated with TQ. Administration started from gestation day 1 (GD1) to day 20 (GD20) through oral gavage once daily. Lead administration caused a dose-dependent toxicity for both mothers and fetuses. Also, the histopathological assessment of the brains from Pb-treated groups showed marked alterations. Co-treatment of with TQ and Pb caused a significant decrease in Pb levels as compared with those treated with Pb alone and amelioration of histopathological changes in the brains. It was concluded that co-treatment of TQ along with gestational Pb exposure could mitigate the effects against Pb-induced maternal and fetal neurotoxicity.     

Effects of suramin on metastatic ability,proliferation, and production of urokinase-type plasminogen activator and plasminogen activator inhibitor type 2 in human renal cell carcinoma cell line SN12C-PM6

Effects of suramin, a polysulfonated naphthylurea compound, on metastatic ability, proliferation, and production of plasminogen activators and plasminogen activator inhibitors were studied using the highly metastatic human renal cell carcinoma cell line, SN12C-PM6. After renal subscapular implantation of tumor cells in nude mice, suramin significantly inhibited metastasis of tumor cells to the lungs and liver. In vitro growth of tumour cells was inhibited by suramin in a dose-dependent manner, at relatively low doses (ID₅₀ = 105 µg/ml). Plasminogen activator inhibitor type 2 (PAI-2) production by tumor cells was enhanced by suramin (100 µg/ml), whereas urokinase-type plasminogen activator (uPA) production was suppressed. Thus, the increase in PAI-2 and the decrease in uPA production correlated with the inhibitory effects on tumour growth and metastasis by suramin. Therefore suramin may be beneficial for the treatment of patients with an early stage of renal cancer with potential risk of metastasis.     

PEGylated-thymoquinone-nanoparticle mediated retardation of breast cancer cell migration by deregulation of cytoskeletal actin polymerization through miR-34a

Thymoquinone (TQ), a major active constituent of black seeds of Nigella sativa, has potential medical applications including spectrum of therapeutic properties against different cancers. However, little is known about their effect on breast cancer cell migration, which is the cause of over 90% of deaths worldwide. Herein, we have synthesized TQ-encapsulated nanoparticles using biodegradable, hydrophilic polymers like polyvinylpyrrolidone (PVP) and polyethyleneglycol (PEG) to overcome TQ's poor aqueous solubility, thermal and light sensitivity as well as consequently, minimal systemic bioavailability which can greatly improve the cancer treatment efficiency. Sizes of synthesized TQ-Nps were found to be below 50 nm and they were mostly spherical in shape with smooth surface texture. Estimation of the zeta potential also revealed that all the three TQ-Nps were negatively charged which also facilitated their cellular uptake. In the present investigation, we provide direct evidence that TQ-Nps showed more efficiency in killing cancer cells as well as proved to be less toxic to normal cells at a significantly lower dose than TQ. Interestingly, evaluation of the anti-migratory effect of the TQ-Nps, revealed that PEG₄₀₀₀-TQ-Nps showed much potent anti-migratory properties than the other types. Further studies indicated that PEG₄₀₀₀-TQ-Nps could significantly increase the expression of miR-34a through p53. Moreover, NPs mediated miR-34a up-regulation directly down-regulated Rac1 expression followed by actin depolymerisation thereby disrupting the actin cytoskeleton which leads to significant reduction in the lamellipodia and filopodia formation on cell surfaces thus retarding cell migration. Considering the biodegradability, non-toxicity and effectivity of PEG₄₀₀₀-TQ-Nps against cancer cell migration, TQ-Nps may provide new insights into specific therapeutic approach for cancer treatment.     

Expression of bcl-2 oncoprotein in renal cell tumours

Expression of bcl-2 is associated with inhibition of apoptosis and extension of cell survival. The importance of apoptosis in relation to the development and progression of renal cell neoplasia remains undefined so far. In order to determine the expression of bcl-2 oncoprotein in normal and neoplastic renal cells, 37 renal tumours were investigated by immunolabelling, including 13 clear cell carcinomas, ten tubulopapillary carcinomas, four chromophobic renal cell carcinomas, and ten oncocytomas. Twenty-six samples of adjacent normal renal tissue served as controls. bcl-2 expression was correlated with cell proliferation activity as estimated by Ki67 antigen expression, and p53 protein expression in the tumour samples. The results demonstrate that in the normal kidney, positive bcl-2 immunostaining was present in glomerular parietal epithelial cells, in distal tubular cells, and in sparse proximal tubule cells. Renal cell tumours showed heterogeneous bcl-2 expression according to the tumour cell type. While the majority of carcinomas of clear cell type were usually negative or contained sparsely distributed positive cells, all tubulopapillary carcinomas were consistently positive for bcl-2. In oncocytomas and chromophobic carcinomas, there was a low percentage of bcl-2 immunoreactive tumour cells; some nuclear bcl-2 positivity was detected in one chromophobic tumour. These findings indicate variable bcl-2 oncoprotein expression in different types of renal cell tumours, with the highest level of expression in tubulopapillary carcinomas. No clear relationship was found between nuclear grade, cell proliferation activity, and level of bcl-2 expression. p53 protein was detected in only one tubulopapillary carcinoma.     

A Gene and Neural Stem Cell Therapy Platform Based on Neuronal Cell Type-Inducible Gene Overexpression

Purpose

Spinal cord injury (SCI) is associated with permanent neurological damage, and treatment thereof with a single modality often does not provide sufficient therapeutic outcomes. Therefore, a strategy that combines two or more techniques might show better therapeutic effects.

Materials and Methods

In this study, we designed a combined treatment strategy based on neural stem cells (NSCs) introduced via a neuronal cell type-inducible transgene expression system (NSE::) controlled by a neuron-specific enolase (NSE) promoter to maximize therapeutic efficiency and neuronal differentiation. The luciferase gene was chosen to confirm whether this combined system was working properly prior to using a therapeutic gene. The luciferase expression levels of NSCs introduced via the neuronal cell type-inducible luciferase expression system (NSE::Luci) or via a general luciferase expressing system (SV::Luci) were measured and compared in vitro and in vivo.

Results

NSCs introduced via the neuronal cell type-inducible luciferase expressing system (NSE::Luci-NSCs) showed a high level of luciferase expression, compared to NSCs introduced via a general luciferase expressing system (SV::Luci-NSCs). Interestingly, the luciferase expression level of NSE::Luci-NSCs increased greatly after differentiation into neurons.

Conclusion

We demonstrated that a neuronal cell type-inducible gene expression system is suitable for introducing NSCs in combined treatment strategies. We suggest that the proposed strategy may be a promising tool for the treatment of neurodegenerative disorders, including SCI.     

Protein conformational changes affect the sodium triple‐quantum MR signal

The aim of this study was to investigate possible sodium triple‐quantum (TQ) signal dependence on pH variation and protein unfolding which may happen in vivo. The model system, composed of bovine serum albumin (BSA), was investigated over a wide pH range of 0.70 to 13.05 and during urea‐induced unfolding. In both experimental series, the sodium and BSA concentration were kept constant so that TQ signal changes solely arose from an environmental change. The experiments were performed using unique potential to detect weak TQ signals by implementing a TQ time proportional phase increment pulse sequence. At a pH of 0.70, in which case the effect of the negatively charged groups was minimized, the minimum TQ percentage relative to single‐quantum of 1.34% ± 0.05% was found. An increase of the pH up to 13.05 resulted in an increase of the sodium TQ signal by 225%. Urea‐induced unfolding of BSA, without changes in pH, led to a smaller increase in the sodium TQ signal of up to 40%. The state of BSA unfolding was verified by fluorescence microscopy. Results of both experiments were well fitted by sigmoid functions. Both TQ signal increases were in agreement with an increase of the availability of negatively charged groups. The results point to vital contributions of the biochemical environment to the TQ MR signals. The sodium TQ signal in vivo could be a valuable biomarker of cell viability, and therefore possible effects of pH and protein unfolding need to be considered for a proper interpretation of changes in sodium TQ signals.     

Expression of high-affinity IL-4 receptors on human melanoma,ovarian and breast carcinoma cells

It has previously been shown that murine sarcoma cells express high-affinity IL-4 receptors (1L-4R) which are internalized after binding to the ligand (Puri et al., Cancer Res 1991; 51:3011-7). We have also reported that human renal cell carcinoma cells express high-affinity IL-4R. and IL-4 inhibits tumour growth in vitro (Obiri et al, J Clin Invest 1993; 91:88). In this study we investigated the expression and function of IL-4R on other human solid tumours. Human melanoma, ovarian carcinoma and breast carcinoma cell lines were assessed for the cell surface expression of IL-4R by radio-ligand receptor binding and for IL-4R gene expression by Northern blot analysis. Primary cultures of mesothelioma and neurofibrosarcoma cells were similarly investigated. Human melanoma, ovarian carcinoma and breast carcinoma cell lines expressed IL-4R on their cell surface with a dissociation constant (K_d) of 140-549 pM. These tumour lines expressed a single 4 kb species of mRNA for IL-4R. Similarly, primary cultures of mesothelioma and neurofibrosarcoma cells were positive for the IL-4R mRNA by Northern blot analysis. Fresh, non-cultured mesothelioma and neurofibrosarcoma tumour sections were also positive for the presence of IL-4R as determined by immunohistochemistry of frozen sections using anti-IL-4R antibody. In order to study possible functions of IL-4R, we evaluated the effects of 11.-4 on cell growth and its effect on MHC antigen expression in the presence or absence of interferon-gamma (IFN-γ)- In tissue culture, IL-4 reduced the growth of tumour cell lines and primary cell cultures studied. IL-4 had very title effect on MHC class I antigen expression on ovarian, breast and melanoma cell lines; however. MHC class I (HLA-DR) expression was enhanced on melanoma and breast carcinoma cells. IL-4 also enhanced the IFN-γ-induced class II expression on melanoma and breast carcinoma cells. Taken together, our observations indicate that IL-4R are expressed on a variety of human solid tumours and these receptors may be functional. IL-4 alone and in combination with IFN-γ may play a role in host immune response against cancers.     

MiR-125b promotes migration and invasion by targeting the vitamin D receptor in renal cell carcinoma

Purpose: To investigate the expression of miR-125b and vitamin D receptor (VDR) in renal cell carcinoma (RCC) and assess the biological function of miR-125b in RCC.Methods: We used quantitative real-time polymerase chain reaction (RT-PCR) to detect the expression of nucleic acids and western blotting to analyze the protein abundance in RCC cell lines. MiR-125b mimic and inhibitor were employed to investigate the function and behavior of miR-125b in RCC cell lines. The relationship between miR-125 and VDR was verified using luciferase assays.Results: Overexpression of miR-125b promoted migration and invasion and prevent cell apoptosis in ACHN cells. In contrast, miR-125b deficiency suppressed migration and invasion and induced cell apoptosis in 786-O cells. Luciferase assays indicated the interaction between miR-125b and VDR. In collected samples, miR-125b was significantly higher in RCC tissues and negatively correlated to VDR (r=-0.444, p=0.04).Conclusion: MiR-125b displays an oncogene profile in RCC, patients with high expression of miR-125b should be a more frequent follow-up. MiR-125B may be a potential therapeutic target for RCC.     

Quantitative and qualitative differences in growth, invasion and lung colonization of an anaplastic and a papillary human thyroid cancer cell linein vitro andin vivo

Invasion and metastasis remain major reasons for failure of anti-cancer therapy. Cell lines derived from human carcinomas are frequently used to investigate the molecular mechanisms that underlie invasion and metastasis. Unfortunately many of these cell lines do not retain the malignant characteristics of their parental tumors. We therefore conducted a series of experimentsin vivo andin vitro to identify which aspects of malignancy of a papillary (NPA'87) and an anaplastic (DR090-1) thyroid carcinoma were consistent with the pathology of the parental tumor types. We evaluated tumor growth, invasion and metastasis of DRO90-1 and NPA'87in vivo following inoculation of the tumor cells under the dermis, under the renal capsule and into the lateral tail vein of nude mice. This evaluationin vivo showed that the anaplastic carcinoma had a faster growth rate compared with the papillary carcinoma. Furthermore, the papillary carcinoma cells could destroy and infiltrate surrounding tissue but were not capable of extravasation and colonization of lung tissue. The anaplastic cells formed lung nodules following injection into the tail vein of nude mice. This lung colonizing capability of DR090-1 correlated with their capacity to secrete an active 62 kDa gelatinase and to migrate through reconstituted basement membranein vitro.     

RNAi-mediated silencing of the Skp-2 gene causes inhibition of growth and induction of apoptosis in human renal carcinoma cells

Renal cancer ranks one of the most frequent causes of cancer death in the world. S-phase kinase-associated protein 2 (SKP 2) is overexpressed in human tumors and has prognostic value in many cancers including renal cancer, indicating its potential as a therapeutic target. In this study, we investigated the therapeutic potential of Skp-2 in renal cancer using the technique of RNA silencing via short hairpin RNA (shRNA). Synthetic shRNA duplexes against Skp-2 were introduced to down-regulate the expression of Skp-2 in a highly malignant renal carcinoma cell line, ACHN. The results indicated that siRNA targeting of Skp-2 could lead to an efficient and specific inhibition of endogenous Skp-2 activity. Furthermore, we found that depletion of Skp-2 caused a dramatic cell cycle arrest, followed by massive apoptotic cell death, and eventually resulted in a significant decrease in growth, viability and tumor formation in renal cancer cell lines studied.     

Immunohistochemical demonstration of neuron-specific enolase in neoplasms of the CNS and other tissues

In normal conditions, neuron-specific enolase (NSE) is histochemically demonstrable only in neurons and cells of the amine precursor uptake and decarboxylation (APUD) system. This has been found not to be true for neoplastic cells. Several types of CNS tumors, including glioblastoma, astrocytoma, oligodendroglioma, ependymoma, medulloblastoma, pineocytoma , meningioma, and choroid plexus papilloma, focally stained positively for NSE. Reactive astrocytes were also frequently positive. In the peripheral nervous system, neuroblastoma, ganglioneuroma, and paraganglioma stained positively for NSE. A number of non-APUD tumors were focally positive. These included schwannoma, carcinoma and fibroadenoma of the breast, renal cell carcinoma, giant cell tumor of the tendon sheath, and chordoma. Caution should be exercised in relying on the immunohistochemical demonstration of NSE as a diagnostic marker in those tumors that do not belong to the APUD cell system. It seems of little value as evidence of differentiation in CNS tumors.     

Correlation between BOLD-MRI and HIF expression level in renal carcinoma

Occupying about 2%~3% of all malignant tumors, renal carcinoma is the most common primary cancer in kidney. The oxidative level of tumor cells is of vital role for optimizing treatment plan, evaluating efficacy and predicting prognosis. This study thus investigated the R2^* value in mouse renal carcinoma model and the correlation between tumor hypoxia and expression level of hypoxia inducible factor-1 (HIF-1). A total of 20 BALB/C nude mice (4~6 weeks old) were inoculated with human ACHN renal carcinoma cells to generate renal cancer model. After the tumor diameter reached 0.5 cm, all animals were examined by BOLD-MRI, both under normal inhalation (R2a^*) and carbogen treatment (R2b^*). The alternation of R2^* values (ΔR2^*=R2a^* - R2b^*) was calculated. Mice were then sacrificed for Immunohistochemical (IHC) staining targeting HIF-1α and HIF-2α. The positive score of HIF was then analyzed for its correlation with R2^* value. In 18 mice finished both experiments, Pearson correlation analysis revealed significant negative correlation between R2a^* and ΔR2^* (r=-0.48, P<0.05) and positive relationship between ΔR2^* and HIF-2α (r=0.38, P<0.05). HIF-1α level, however, did not correlated with tumor R^* values. The positive correlation between ΔR2^* and HIF-2α, but not HIF-1α, suggested potential role of combined BOLD-MRI technique and HIF-1α staining in clinical diagnosis of renal carcinoma. HIF-2α may work as biological marker for renal cancer.     

Sporadic hemangioblastoma of the kidney with PAX2 and focal CD10 expression: report of a case

In this study, we presented an additional case of renal hemangioblastoma, which demonstrates PAX2 and focal CD10 expression. Histologically, the tumor consisted of sheets of oval or polygonal cells and a prominent vascular network. The tumor cells varied in size, and possessed pale or eosinophilic cytoplasm that sometimes contained sharply delineated fine vacuoles. The tumor cell nuclei with inconspicuous nucleoli showed moderate nuclear atypia and pleomorphism. Focal areas of stromal hyalinization and sclerosis were detected. On account of its strong or moderate immunoreactivity for the a-inhibin, S100, NSE, and EGFR, the diagnosis of renal hemangioblastoma was established. For further evidence of VHL deficiency, the tumor was subjected to VHL sequence analysis of all three exons and fluorescence in situ hybridization (FISH) detection for chromosome 3p deletion. None of the VHL gene mutations and chromosome 3p deletion was detected in the tumor. Because of several shared morphological and immunophenotypic features, renal hemangioblastoma may be underrecognized and should be included in the differential diagnosis of primary renal tumors, in particular clear cell renal cell carcinoma. The unexpected positive staining of PAX2 and CD10 in renal hemangioblastoma should be particular concerned. Using a combination of immunoprofile may be helpful to the differential diagnosis of these renal tumors.     

Bilateral renal tumors; conventional clear cell carcinoma and contralateral t(6;11)/t(X;17)-like tumor Histomorphologic, immunohistochemical, ultrastructural and molecular genetic studies including the report of a novel mutation in the VHL gene

A 34-year-old pregnant woman with bilateral kidney tumors 9.5 and 2.5 cm in maximum diameter is presented. The larger tumor was clear renal cell carcinoma. The smaller contralateral tumor was focally HMB45 positive and had unusual histomorphology, including features resembling clear renal cell carcinoma with features of both t(6;11)- and t(X;17)/ASPL-TFE3 carcinomas. This tumor displayed a complex karyotype. A novel germ line mutation in the VHL gene (c.439A>G/p.I147V) was also identified in this patient.