Accurately Controlled Tumor Temperature with Silica-Coated Gold Nanorods for Optimal Immune Checkpoint Blockade Therapy.

Photothermal therapy (PTT) at mild temperatures ranging from 44 to 45 °C holds tremendous promise as a strategy for inducing potent immunogenic cell death (ICD) within tumor tissues, which can reverse the immunosuppressive tumor microenvironment (ITM) into an immune-responsive milieu. However, accurately and precisely controlling the tumor temperature remains a formidable challenge. Here, we report the precision photothermal immunotherapy by using silica-coated gold nanorods (AuNR@SiO2), and investigating the optimal administration routes and treatment protocols, which enabled to achieve the sustained and controlled mild heating within the tumor tissues. First, the highest photothermal performance of AuNR@SiO2 with 20-nm silica shell thickness than 5 or 40 nm was confirmed in vitro and in vivo. Then, the optimal conditions for precision immunotherapy were further investigated to produce mild temperature (44 to 45 °C) accurately in tumor tissues. The optimal conditions with AuNR@SiO2 result in a distinct cell death with high early/late apoptosis and low necrosis, leading to very efficient ICD compared to lower or higher temperatures. In colon tumor-bearing mice, intratumorally injected AuNR@SiO2 efficiently promotes a mild temperature within the tumor tissues by local irradiation of near-infrared (NIR) laser. This mild PTT substantially increases the population of mature dendritic cells (DCs) and cytotoxic T cells (CTLs) within tumor tissues, ultimately reversing the ITM into an immune-responsive milieu. Furthermore, we found that the combination mild PTT with AuNR@SiO2 and anti-PD-L1 therapy could lead to the 100% complete regression of primary tumors and immunological memory to prevent tumor recurrence. Collectively, this study demonstrates that AuNR@SiO2 with a robust methodology capable of continuously inducing mild temperature accurately within the ITM holds promise as an approach to achieve the precision photothermal immunotherapy.

Precise control over the silica shell thickness and finding the optimal thickness for the peak heat diffusion property of AuNR@SiO2.

Silica-coated gold nanorods (AuNRs) exhibit significantly enhanced photothermal effects and photoacoustic (PA) signal intensities, which is beneficial for various nanophotonic applications in materials science. However, the silica shell thickness for optimum enhancement is not fully understood and is even controversial depending on the physical state of the silica shell. This is because of the lack of systematic investigations of the nanoscale silica shell thickness and the photothermal effect. This study provides a robust synthetic method to control the silica shell thickness at the nanoscale and the physical state-dependent heat diffusion property. The selected base and solvent system enabled the production of silica-coated AuNRs (AuNR@SiO2) with silica shell thicknesses of 5, 10, 15, 20, 25, 30, 35, and 40 nm. AuNRs with a 20 nm silica shell showed the highest photothermal effect with a 1.45-times higher photothermal efficiency than that of AuNRs without a silica shell. The low density of the silica shell on the AuNRs showed a low photothermal effect and photostability. It was found that the disruption of cetyltrimethyl ammonium bromide (CTAB) layers on the AuNRs was responsible for the low photostability of the AuNRs. The simulation study for the heat diffusion property showed facilitated heat diffusion in the presence of a 20 nm silica shell. In a cell-based study, AuNRs with a 20 nm silica shell showed the most sensitive photothermal effect for cell death. The results of this robust study can provide conclusive conditions for the optimal silica shell thickness to obtain the highest photothermal effect, which will be useful for the future design of nanomaterials in various fields of application.

Surface decontamination of protective duplex oxide layers on stainless steel waste using deep eutectic solvents.

The decontamination capabilities of deep eutectic solvents (DESs) formed from choline chloride (ChCl) and p-toluenesulfonic acid monohydrate (PtsA), ChCl:PtsA, under different conditions (hydrated, heated, and agitated) were tested with simulant oxidized stainless steel 304 specimens. Although the leaching rates were satisfactory under all conditions, hydrated and stirred ChCl:PtsA at 60 °C showed the fastest leaching rate of 0.1647 mg/min. Oxidized specimens with an average mass gain of 1.2 ± 1 mg were leached, and their masses were reduced by 558 ± 22 mg after 26 h. These results were understood by improved physical properties of ChCl:PtsA upon hydration. Metal oxide solubility of CoO and NiO increased with water, and those of Cr2O3 and Fe3O4 decreased with hydration. Importantly, the use of choline chloride-based DESs in decontamination applications may significantly reduce the cost of decontamination because these DESs can be mass-produced and their components are both easily obtainable and economical. Also, DESs are biodegradable and eco-friendly. The different speciation of Co and Ni, which bond with Cl-, compared with Fe and Cr, which bond with H2O, illustrated the potential for a metal recovery for secondary liquid waste reduction.

Development of 8 ton/day gasification process to generate electricity using a gas engine for solid refuse fuel.

An 8 ton/day solid refuse fuel gasification process with air oxidant was operated under various conditions to generate electricity. Solid refuse fuels with fluff type feedstock were fabricated from municipal solid wastes. The tested experimental conditions included feedstock charging rate into the gasifier, equivalence ratio, and oxygen enrichment; varying these conditions resulted in different gasification characteristics, such as cold gas efficiency and carbon conversion ratio. Optimum conditions were a charging rate of 50 to 60% by volume (504.71 to 605.65 kg/Sm2) of feedstock in the gasifier, equivalence ratio of 0.21 to 0.33, and no oxygen enrichment. Average cold gas efficiency and carbon conversion ratio were 71.30% and 72.07%, respectively, at optimum conditions. Pollutants such as dust, tar, and gases, were analyzed at the outlet of the cleaning facility and gasifier, and their low concentrations in the producer gas were sufficient to allow for operation of the gas engine. The gasification process exhibited stable operation over 288 h, which included the facility check period. The average gasifier temperature was 825 °C, 17.14% by volume of producer gas was syngas, and gas engine power generation was 235 kWh during this period; power consumption of the entire system was 68 kWh. These results demonstrate that the gasification process for converting solid waste to energy can be operated at a commercial scale.

Near infrared dye-conjugated oxidative stress amplifying polymer micelles for dual imaging and synergistic anticancer phototherapy.

The recent advances in nanotechnology have led to the development of smart nanomaterials that combine diagnostic and therapeutic functions and provide synergistic anticancer effects through the combination of different treatment modalities. Here, we report a promising theranostic nanoconstruct that can translate into multiple functionalities: fluorescence/photoacoustic imaging, acid-triggered generation of ROS (reactive oxygen species), heat and singlet oxygen production under near infrared (NIR) laser irradiation, and coupling oxidative anticancer therapy to dual imaging-guided photothermal/photodynamic therapy. An NIR dye-conjugated hydroxyl radical generating biodegradable polymer (HRGP-IR) is employed as a theranostic nanoplatform. HRGP-IR could self-assemble to form micelles and elevate oxidative stress by generating hydrogen peroxide and hydroxyl radical. Under the NIR (808 nm) laser irradiation, HRGP-IR micelles also generate heat and singlet oxygen to induce cancer cell death. In mouse xenograft models, HRGP-IR micelles accumulated in tumors preferentially and the tumor could be detected by dual imaging. Effective tumor ablation was achieved by HRGP-IR micelles (5 mg/kg) combined with NIR laser irradiation, demonstrating the synergistic anticancer effects of oxidative stress with photothermal heating. Given their dual imaging capability, anticancer phototherapy and highly potent synergistic anticancer activity with NIR laser irradiation, HRGP-IR micelles hold great potential as a nanotheranostic agent for cancer treatment.

Nano-Fenton Reactors as a New Class of Oxidative Stress Amplifying Anticancer Therapeutic Agents.

Cancer cells, compared to normal cells, are under oxidative stress associated with an elevated level of reactive oxygen species (ROS) and are more vulnerable to oxidative stress induced by ROS generating agents. Thus, manipulation of the ROS level provides a logical approach to kill cancer cells preferentially, without significant toxicity to normal cells, and great efforts have been dedicated to the development of strategies to induce cytotoxic oxidative stress for cancer treatment. Fenton reaction is an important biological reaction in which irons convert hydrogen peroxide (H2O2) to highly toxic hydroxyl radicals that escalate ROS stress. Here, we report Fenton reaction-performing polymer (PolyCAFe) micelles as a new class of ROS-manipulating anticancer therapeutic agents. Amphiphilic PolyCAFe incorporates H2O2-generating benzoyloxycinnamaldehyde and iron-containing compounds in its backbone and self-assembles to form micelles that serve as Nano-Fenton reactors to generate cytotoxic hydroxyl radicals, killing cancer cells preferentially. When intravenously injected, PolyCAFe micelles could accumulate in tumors preferentially to remarkably suppress tumor growth, without toxicity to normal tissues. This study demonstrates the tremendous translatable potential of Nano-Fenton reactors as a new class of anticancer drugs.

EGFR negates the proliferative effect of oncogenic HER2 in MDA-MB-231 cells.

Members of the EGFR family are potent mediators of normal cell growth and development. HER2 possesses an active tyrosine kinase domain, but no direct ligand has been identified. To investigate the differential effect of HER2 in breast cell lines, HER2 was overexpressed in MCF-10A, MCF7 and MDA-MB-231 cells. HER2 overexpression promoted proliferation, survival and migration in MCF-10A and MCF-7 cells. No significant differences were seen in proliferation, survival or migration between MDA-MB-231 vec and HER2 cells. The activity of downstream HER2 proteins increased in MCF-10A HER2 and MCF-7 HER2 cells but not in MDA-MB-231 HER2 cells. Exogenously expressed HER2 failed to associate with EGFR or HER3 in MDA-MB-231 cells, while overexpression of HER2 enhanced HER family dimerization in MCF-10A and MCF-7 cells.

Amplification of oxidative stress by a dual stimuli-responsive hybrid drug enhances cancer cell death.

Cancer cells, compared with normal cells, are under oxidative stress associated with the increased generation of reactive oxygen species (ROS) including H2O2 and are also susceptible to further ROS insults. Cancer cells adapt to oxidative stress by upregulating antioxidant systems such as glutathione to counteract the damaging effects of ROS. Therefore, the elevation of oxidative stress preferentially in cancer cells by depleting glutathione or generating ROS is a logical therapeutic strategy for the development of anticancer drugs. Here we report a dual stimuli-responsive hybrid anticancer drug QCA, which can be activated by H2O2 and acidic pH to release glutathione-scavenging quinone methide and ROS-generating cinnamaldehyde, respectively, in cancer cells. Quinone methide and cinnamaldehyde act in a synergistic manner to amplify oxidative stress, leading to preferential killing of cancer cells in vitro and in vivo. We therefore anticipate that QCA has promising potential as an anticancer therapeutic agent.

Dual pH-sensitive oxidative stress generating micellar nanoparticles as a novel anticancer therapeutic agent.

Cancer cells are under oxidative stress due to a large production of reactive oxygen species (ROS), which involve in cell proliferation and cancer promotion and progression. On the other hand, ROS promotes cell death, depending on the rate of ROS production and the activity of antioxidant systems. Recently, "oxidation therapy" has arisen as a promising anticancer strategy, which can be achieved by inducing the generation of cytotoxic level of ROS or inhibiting the antioxidant systems in tumor cells. Here, we report oxidative stress amplifying nanoplatforms as novel anticancer therapeutics, which are able not only to suppress antioxidant but also to generate ROS simultaneously in acidic tumor microenvironments. The oxidative stress amplifying nanoplatforms are composed of dual pH-sensitive PBCAE copolymer, polymeric prodrug of BCA (benzoyloxycinnamaldehyde) and heme oxygenase-1 (HO-1) inhibiting zinc protoporphyrin (ZnPP). PBCAE was designed to incorporate ROS-generating BCA in its backbone via acid-cleavable acetal linkages and self-assemble to form micelles that encapsulate ZnPP. In vitro proof-of-concept studies revealed that ZnPP encapsulated in PBCAE micelles suppressed HO-1 to make cancer cells more vulnerable to BCA-induced ROS, leading to enhanced apoptotic cell death. In addition, ZnPP-loaded PBCAE micelles significantly suppressed the tumor growth in human cancer xenograft mouse models. We believe that oxidative stress amplifying micellar nanoparticles have a great potential as novel redox anticancer therapeutics.

S100A4 negatively regulates ß-catenin by inducing the Egr-1-PTEN-Akt-GSK3ß degradation pathway.

S100A4, also known as the mts1 gene, has been reported as an invasive and metastatic marker for many types of cancers. S100A4 interacts with various target genes that affect tumor cell metastasis; however, little is known about cellular signaling pathways elicited by S100A4. In the current study, we demonstrate an inhibitory effect of S100A4 on ß-catenin signaling in breast cancer cells. By overexpressing S100A4 in MCF-7, MDA-MB-231 and MDA-MB-453 breast cancer cells, we observed the down-regulation of ß-catenin expression and ß-catenin-dependent TCF/LEF transcriptional activities. The activity of GSK3ß, which phosphorylates ß-catenin and induces proteasomal degradation of ß-catenin, was increased in S100A4-overexpressing cell lines. Blocking Glycogen Synthase Kinase (GSK3ß) activity by lithium chloride or Dvl gene overexpression restored ß-catenin expression. We also found that increased GSK3ß activity was due to decrease in Akt activity resulting from Egr-1-induced phosphatase and tensin homolog (PTEN) expression. S100A4 induced Egr-1 nuclear localization by increasing the association between Egr-1 and importin-7 and this effect was reduced in S100A4 mutants that harbored a defect in nuclear localization signals. Collectively, we verify herein that S100A4 may act as a tumor suppressor in breast cancers by down-regulating the central signaling axis for tumor cell survival.

Regulation of cell proliferation and migration by keratin19-induced nuclear import of early growth response-1 in breast cancer cells.

PURPOSE: Keratin19 (KRT19) is the smallest known type I intermediate filament and is used as a marker for reverse transcriptase PCR-mediated detection of disseminated tumors. In this study, we investigated the functional analysis of KRT19 in human breast cancer. EXPERIMENTAL DESIGN: Using a short hairpin RNA system, we silenced KRT19 in breast cancer cells. KRT19 silencing was verified by Western blot analysis and immunocytochemistry. We further examined the effect of KRT19 silencing on breast cancer cells by cell proliferation, migration, invasion, colony formation assay, cell-cycle analysis, immunocytochemistry, immunohistochemistry, and mouse xenograft assay. RESULTS: Silencing of KRT19 resulted in increased cell proliferation, migration, invasion, and survival. These effects were mediated by upregulation of Akt signaling as a result of reduced PTEN mRNA expression. Silencing of KRT19 decreased the nuclear import of early growth response-1 (Egr1), a transcriptional factor for PTEN transcription, through reduced association between Egr1 and importin-7. We also confirmed that silencing of KRT19 increased tumor formation in a xenograft model. CONCLUSIONS: KRT19 is a potential tumor suppressor that negatively regulates Akt signaling through modulation of Egr1 nuclear localization.

HER2 stabilizes survivin while concomitantly down-regulating survivin gene transcription by suppressing Notch cleavage.

In breast cancer, the HER2 (human epidermal growth factor receptor 2) receptor tyrosine kinase is associated with extremely poor prognosis and survival. Notch signalling has a key role in cell-fate decisions, especially in cancer-initiating cells. The Notch intracellular domain produced by Notch cleavage is translocated to the nucleus where it activates transcription of target genes. To determine the combinatory effect of HER2 and Notch signalling in breast cancer, we investigated the effect of HER2 on Notch-induced cellular phenomena. We found the down-regulation of Notch-dependent transcriptional activity by HER2 overexpression. Also, the HER2/ERK (extracellular-signal-regulated kinase) signal pathway down-regulated the activity of γ-secretase. When we examined the protein level of Notch target genes in HER2-overexpressing cells, we observed that the level of survivin, downstream of Notch, increased in HER2 cells. We found that activation of ERK resulted in a decrease in XAF1 [XIAP (X-linked inhibitor of apoptosis)-associated factor 1] which reduced the formation of the XIAP-XAF1 E3 ligase complex to ubiquitinate survivin. In addition, Thr(34) of survivin was shown to be the most important residue in determining survivin stability upon phosphorylation after HER2/Akt/CDK1 (cyclin-dependent kinase 1)-cyclin B1 signalling. The results of the present study show the combinatorial effects of HER2 and Notch during breast oncogenesis.

Protein kinase B/Akt1 inhibits autophagy by down-regulating UVRAG expression.

Autophagy, or autophagocytosis, is a selective intracellular degradative process involving the cell's own lysosomal apparatus. An essential component in cell development, homeostasis, repair and resistance to stress, autophagy may result in either cell death or survival. The targeted region of the cell is sequestered within a membrane structure, the autophagosome, for regulation of the catabolic process. A key factor in both autophagosome formation and autophagosome maturation is a protein encoded by the ultraviolet irradiation resistance-associated gene (UVRAG). Conversely, the serine/threonine-specific protein kinase B (PKB, also known as Akt), which regulates survival in various cancers, inhibits autophagy through mTOR activation. We found that Akt1 may also directly inhibit autophagy by down-regulating UVRAG both in a 293T transient transfection system and breast cancer cells stably expressing Akt1. The UVRAG with mutations at putative Akt1-phosphorylation sites were still inhibited by Akt1, and dominant-negative Akt1 also inhibited UVRAG expression, suggesting that Akt1 down-regulates UVRAG by a kinase activity-independent mechanism. We showed that Akt1 overexpression in MDA-MB-231 breast cancer cells down-regulated UVRAG transcription. Cells over-expressing Akt1 were more resistant than control cells to ultraviolet light-induced autophagy and exhibited the associated reduction in cell viability. Levels of the autophagosome indicator protein LC3B-II and mRFP-GFP-LC3 were reduced in cells that over-expressing Akt1. Inhibiting Akt1 by siRNA or reintroducing UVRAG gene rescued the level of LC3B-II in UV-irradiation. Altogether, these data suggest that Akt1 may inhibit autophagy by decreasing UVRAG expression, which also sensitizes cancer cells to UV irradiation.

Down-regulation of estrogen receptor α (ERα) transcriptional activity by p27 is mediated by inhibition of ERα nuclear localization and modulation of the ERα transcriptional complex.

To investigate the role of p27 on estrogen receptor (ER)α-mediated transcription, we generated MCF-7 cells with knocked down p27 via retroviral delivery of p27 shRNA. Suppression of p27 expression in MCF-7 cells resulted in up-regulation of ERα-mediated transcription by estradiol compared to the levels in control MCF-7 cells. Accordingly, transient transfection studies in 293T cells revealed that overexpression of p27 reduced ERα-mediated transcription. The effect of p27 on ERα transcriptional activity was independent of cell cycle arrest by p27, as cell cycle arrest induced by serum starvation did not significantly affect ERα-mediated transcription. Further, we observed that p27 inhibited nuclear localization of ERα, and that p27 was associated with ERα in the cytoplasm. We also investigated the role of p27 in the modulation of ERα transcriptional activity in the nucleus. We found that p27 negatively modulated ERα transcriptional activity by inhibiting association of cyclin D1 with ERα and recruiting BRCA1 to ERα transcriptional complex. Taken together, these data suggest that p27 inhibits ERα transcriptional activity by two independent mechanisms, namely, physical nuclear exclusion of ERα, and modulation of the ERα transcriptional complex.

CD24 regulates cell proliferation and transforming growth factor ß-induced epithelial to mesenchymal transition through modulation of integrin ß1 stability.

To determine the role of CD24 in breast cancer cells, we knocked down CD24 in MCF-7 human breast cancer cells by retroviral delivery of shRNA. MCF-7 cells with knocked down CD24 (MCF-7 hCD24 shRNA) exhibited decreased cell proliferation and cell adhesion as compared to control MCF-7 mCD24 shRNA cells. Decreased proliferation of MCF-7 hCD24 shRNA cells resulted from the inhibition of cell cycle progression from G1 to S phase. The specific inhibition of MEK/ERK signaling by CD24 ablation might be responsible for the inhibition of cell proliferation. Phosphorylation of Src/FAK and TGF-ß1-mediated epithelial to mesenchymal transition was also down-regulated in MCF-7 hCD24 shRNA cells. Reduced Src/FAK activity was caused by a decrease in integrin ß1 bound with CD24 and subsequent destabilization of integrin ß1. Our results suggest that down-regulation of Raf/MEK/ERK signaling via Src/FAK may be dependent on integrin ß1 function and that this mechanism is largely responsible for the CD24 ablation-induced decreases in cell proliferation and epithelial to mesenchymal transition.

Induction of apoptotic cell death by Pharbitis nil extract in HER2-overexpressing MCF-7 cells.

AIM OF THE STUDY: We performed this study to investigate the anti-cancer activity of Pharbitis nil (PN) ethanol extract which has been used for herbal medicinal treatment against diseases in East Asia. MATERIALS AND METHODS: We analyzed the effects of PN extract on proliferation of breast cancer cell lines, MCF-7 control vector (vec) and MCF-7 human epidermal growth factor receptor 2 (HER2) cells engineered to overexpress oncogenic HER2 via retroviral infection. We performed the proliferation assay to measure the growth rate of the cells. FACS analysis was used to analyze the cell cycle. Western blot analysis was used to investigate the effect of PN on the level and activation of intracellular molecules. RESULTS: We found that PN extract inhibited the proliferation of both MCF-7 vec and MCF-7 HER2 cells. This growth inhibition was accompanied with the increase of sub G0/G1 apoptotic fractions. When we check the efficiency of PN on the level of intracellular signaling molecules, we found that PN extract induced the inhibition of phosphorylation of HER2 and its downstream effectors, Akt and extracellular signal-regulated kinases (ERK). Active forms of both Akt and ERK were gradually decreased in PN-treated MCF-7 vec and MCF-7 HER2 cells suggesting that the growth suppressive activity of PN is related to signaling pathway. The level of cyclin D also diminished in PN-treated both cells suggesting that PN may inhibit the growth of MCF-7 vec and MCF-7 HER2 cells by perturbing cell cycle progression. It should be noted that PN decreased the growth rate of both MCF-7 vec and MCF-7 HER2 cells without changing the level and activation of p53. CONCLUSION: PN extract suppressed the proliferation rate of HER-2 overexpressing MCF-7 breast cancer cells inducing apoptotic cell death in vitro. Our data demonstrates that PN extracts contain useful anti-tumor activity especially against HER2 overexpressing breast cancer.

Akt isoform-specific inhibition of MDA-MB-231 cell proliferation.

To dissect the isoform-specific roles of Akt in breast cancer cells, constitutively active Akt isoforms were introduced into MDA-MB-231 cells. Both Akt1 and Akt2 efficiently inhibited the growth of MDA-MB-231 cells. Overexpression of Akt1 down-regulated ERK activity inhibiting Ser 259 phosphorylation of c-Raf and subsequent downstream signaling. Akt2 overexpression up-regulated the cell cycle inhibitor p27. Cycloheximide decay assays showed that Akt2 increased the stability and nuclear localization of p27, thus inhibiting the cyclin E/CDK2 complex. These results suggest that the inhibition of cell proliferation by Akt1 and Akt2 is mediated by isoform-specific mechanisms.

Danshen (Salvia miltiorrhiza) extract inhibits proliferation of breast cancer cells via modulation of Akt activity and p27 level.

Danshen is widely used in traditional Chinese medicine, often in combination with other herbs. To check the effect of Danshen on the proliferation of breast cancer cells, Danshen extract was used to treat MCF-7 and MCF-7 HER2 cells, the latter of which overexpresses HER2. HER2 is a receptor tyrosine kinase, and is involved in signal transduction pathways leading to tumor cell proliferation. MTT and cell proliferation assays revealed that Danshen strongly inhibited the proliferation of both MCF-7 vec cells and MCF-7 HER2 cells. Flow cytometry analyses indicated that Danshen induced cell cycle delay in the G1 phase. HER2 expression was shown to confer resistance to Danshen-induced inhibition of proliferation and cell cycle delay, suggesting that HER2 is responsible for the resistance to Danshen. Danshen treatment induced the down-regulation of Akt phosphorylation and an increase in p27 in MCF-7 vec and MCF-7 HER2 cells. Nevertheless, MCF-7 HER2 cells were more resistant to the Danshen-induced inhibition of Akt phosphorylation and p27 up-regulation.

Effect of p27 on motility of MDA-MB-231 breast cancer cells.

p27 is a member of the Kip family of cyclin-dependent kinase inhibitors and its overexpression results in cell cycle arrest at G1 and/or apoptosis. In addition to its role as a regulator of cell cycle progression, p27 can also participate in cell motility, especially when it is mislocalized in the cytosol. To further elucidate the role of p27 in the motility of MDA-MB-231 breast cancer cells, we performed p27 knockdown in MDA-MB-231 cells by RNA interference. Infection of MDA-MB-231 cells with retroviruses harboring p27 short hairpin RNA (shRNA) designed from human p27 cDNA resulted in efficient inhibition of p27 expression, while p27 shRNA designed from mouse p27 cDNA did not affect p27 expression in MDA-MB-231 cells. MDA-MB-231 cells infected with human p27 shRNA (MDA-MB-231 hp27shRNA) showed increased proliferation compared to control MDA-MB-231 cells and MDA-MB-231 cells infected with mouse p27shRNA (MDA-MB-231 mp27shRNA). Wound healing assays revealed that migration of MDA-MB-231 hpshRNA cells was markedly impaired compared to MDA-MB-231 mpshRNA cells, especially when cycloheximide was added to block protein synthesis. Immunostaining of p27 in MDA-MB-231 cells showed that p27 predominantly localized in the nuclei. These results suggest that both nuclear and cytosolic p27 can promote cancer cell motility.

A case of persistent anemia in a renal transplant recipient: association with parvovirus B19 infection.

We report an unexplained anemia that persisted for 4 months in a renal transplant patient who was receiving immunosuppression therapy that included prednisolone, tacrolimus and azathioprine. A bone marrow biopsy demonstrated pure erythroid hypoplasia and occasional giant pronormoblasts with intranuclear inclusions, characteristic of a parvovirus B19 infection. Both the serum and bone marrow cells were positive by parvovirus B19 DNA PCR. The anemia resolved 6 weeks after the administration of intravenous immunoglobulin (IVIG). Four months later, anemia redeveloped and IVIG was infused again. Hemoglobin levels were, however, still subnormal after 1 month of treatment and tacrolimus was then switched to cyclosporin A, resulting in a clear improvement. A parvovirus B19 infection should be included in the differential diagnosis of renal transplant recipients who present with anemia associated with a low reticulocyte count. Tacrolimus may possibly impair the clearance of a parvovirus B19 infection.