Fengqin Miao

Preparation of folic acid-conjugated, doxorubicin-loaded, magnetic bovine serum albumin nanospheres and their antitumor effects in vitro and in vivo

Background This study aimed to generate targeted folic acid-conjugated, doxorubicin-loaded, magnetic iron oxide bovine serum albumin nanospheres (FA-DOX-BSA MNPs) that lower the side effects and improve the therapeutic effect of antitumor drugs when combined with hyperthermia and targeting therapy. A new nanodrug using magnetic nanospheres for heating and addition of the folate receptor with cancer cell specificity was prepared. The characteristics of these nanospheres and their antitumor effects in nasopharyngeal carcinoma were explored. Methods FA-DOX-BSA MNPs comprising encapsulated magnetic iron oxide nanoparticles were prepared using a desolvation cross-linking method. Activated folic acid (N-hydroxysuccinimide ester of folic acid) was conjugated to the surface of albumin nanospheres via amino groups. Results Folic acid was successfully expressed on the surface of the nanospheres. Electron microscopy revealed that the FA-DOX-BSA MNPs were nearly spherical and uniform in size, with an average diameter of 180 nm. The nanomaterial could deliver doxorubicin at clinically relevant doses with an entrapment efficiency of 80%. An increasing temperature test revealed that incorporation of magnetic iron oxide into nanospheres could achieve a satisfactory heat treatment temperature at a significantly lower dose when placed in a high-frequency alternating magnetic field. FA-DOX-BSA MNPs showed greater inhibition of tumors than in the absence of folic acid in vitro and in vivo. Compared with chemotherapy alone, hyperthermia combined with chemotherapy was more effective against tumor cells. Conclusion Folic acid-conjugated bovine serum albumin nanospheres composed of mixed doxorubicin and magnetic iron oxide cores can enable controlled and targeted delivery of anticancer drugs and may offer a promising alternative to targeted doxorubicin therapy for nasopharyngeal carcinoma.

KIR and HLA Loci Are Associated with Hepatocellular Carcinoma Development in Patients with Hepatitis B Virus Infection: A Case-Control Study

Background Natural killer (NK) cells activation has been reported to contribute to inflammation and liver injury during hepatitis B virus (HBV) infection both in transgenic mice and in patients. However, the role of NK cells in the process of HBV-associated hepatocellular carcinoma (HCC) development has not been addressed. Killer cell immunoglobulin-like receptors (KIRs) are involved in regulating NK cell activation through recognition of specific human leukocyte antigen (HLA) class I allotypes. Methodology/Principal Findings To investigate whether KIR and HLA genes could influence the risk of HBV-associated HCC development, 144 HBV-infected patients with HCC and 189 well-matched HBV infectors with chronic hepatitis or cirrhosis as non-HCC controls were enrolled in this study. The presence of 12 loci of KIR was detected individually. HLA-A, -B, -C loci were genotyped with high-resolution. HLA-C group 1 homozygote (OR = 2.02; p = 0.005), HLA-Bw4-80I (OR = 2.67; p = 2.0E-04) and combination of full-length form and 22 bp-deleted form of KIR2DS4 (KIR2DS4/1D) (OR = 1.89; p = 0.017) were found associated with HCC incidence. When the combined effects of these three genetic factors were evaluated, more risk factors were observed correlating with higher odds ratios for HCC incidence (P trend = 7.4E-05). Because all the risk factors we found have been reported to result in high NK cell functional potential by previous studies, our observations suggest that NK cell activation may contribute to HBV-associated HCC development. Conclusions/Significance In conclusion, this study has identified significant associations that suggest an important role for NK cells in HCC incidence in HBV-infected patients. Our study is useful for HCC surveillance and has implications for novel personalized therapy strategy development aiming at HCC prevention in HBV-infected patients.

Enhanced antitumor effects of the BRBP1 compound peptide BRBP1-TAT-KLA on human brain metastatic breast cancer.

Novel molecularly targeted agents that block the development and metastasis of human brain metastatic breast cancer hold great promise for their translational value. In this study, we constructed a novel targeting composite peptide BRBP1-TAT-KLA comprising of three elements: a brain metastatic breast carcinoma cell (231-BR)-binding peptide BRBP1, a cell penetrating peptide TAT, and a proapoptotic peptide KLA. This composite peptide efficiently internalized in 231-BR cells and consequently induced mitochondrial damage and cellular apoptosis. Exposure of 231-BR cells to BRBP1-TAT-KLA significantly decreased cell viability and increased apoptosis compared with the cells treated with the control peptides. In vivo relevance of these findings was further corroborated in the 231-BR tumor-bearing mice that demonstrated significantly delayed tumor development and metastasis following administration of BRBP1-TAT-KLA compared with those treated with TAT-KLA alone. Interestingly, BRBP1-TAT-KLA inhibited the formation of both large and micro-metastases, while TAT-KLA alone failed to significantly reduce micro-metastases in the breast cancer brain metastasis mice. BRBP1-TAT-KLA selectively homed to the tumors in vivo where it induced cellular apoptosis without significant toxicity on non-tumor tissues. Our findings therefore demonstrated the enhanced antitumor effects of the BRBP1 compound peptide BRBP1-TAT-KLA, providing insights toward development of a potential therapeutic strategy for brain metastatic breast cancer.

The spatio-temporal expression of MHC class I molecules during human hippocampal formation development

In the immune system, the major histocompatibility complex (MHC) class I molecules mediate both the innate and adaptive immune responses in vertebrates. There has been a dogma that the central nervous system (CNS) is immune privileged and healthy neurons do not express MHC class I molecules. However, recent studies have indicated that the expression and non-immunobiologic roles of MHC class I in mammalian CNS. But data referring to humans are scarce. In this study we report the expression and cellular localization of MHC class I in the human fetal, early postnatal and adult hippocampal formation. The expression of MHC class I was very low in the hippocampus at 20 (gestational weeks) GW and slowly increased at 27-33 GW. The gradually increased expression in the somata of some granular cells in dentate gyrus (DG) was observed at 30-33 GW. Whereas, a rapid increase in MHC class I molecules expression was found in the subiculum and it reached high levels at 31-33 GW and maintained at postnatal 55 days. No expression of MHC class I was found in hippocampal formation in adult. MHC class I heavy chain and β2 microglobulin (β2M) showed similar expression in some cells of the hippocampal formation at 30-33 GW. Moreover, MHC class I molecules were mainly expressed in neurons and most MHC class I-expressing neurons were glutamatergic. The temporal and spatial patterns of MHC class I expression appeared to follow gradients of pyramidal neurons maturation in the subiculum at prenatal stages and suggested that MHC class I molecules are likely to regulate neuron maturation. This article is part of a Special Issue entitled Priority to Publish.

Killer artificial antigen-presenting cells deplete alloantigen-specific T cells in a murine model of alloskin transplantation

FasL-expressing killer antigen-presenting cells (KAPCs) have the ability to delete antigen-specific T cells and, therefore, could potentially be used for the treatment of allograft rejection and autoimmunity; however, their cellular nature markedly limits their clinical use. Novel bead-based killer artificial antigen-presenting cells (KaAPCs), which are generated by coupling major histocompatibility complex (MHC) class I antigens together with the apoptosis-inducing anti-Fas monoclonal antibody (mAb) onto magnetic beads, have recently attracted more attention. KaAPCs have a number of advantages over KAPCs and are able to deplete specific T cells in cocultures. However, it remains unknown whether bead-based KaAPCs can also induce apoptosis of alloreactive or autoreactive T cells and, consequently, generate hyporesponsiveness in vivo. In this study, H-2K(b)/peptide monomers and anti-Fas mAb have been covalently coupled to latex beads and administered intravenously into BALB/c mice (H-2K(d)) that had previously been grafted with skin squares from C57BL/6 mice (H-2K(b)). Alloskin graft survival was prolonged for 6 days. A 60% decrease of H-2K(b) antigen-alloreactive T cells was demonstrated by several measures 2 days after each injection of KaAPCs, but intact immune function, including antitumor activity, was maintained. These data provide the first in vivo evidence that bead-based KaAPCs can selectively deplete antigen-specific T cells without the loss of overall immune responsiveness and, therefore, highlight the therapeutic potential of this novel strategy for the treatment of allograft rejection and autoimmune disorders.

Inhibition of heat-shock protein 90 sensitizes liver cancer stem-like cells to magnetic hyperthermia and enhances anti-tumor effect on hepatocellular carcinoma-burdened nude mice

Purpose To explore the thermoresistance and expression of heat-shock protein 90 (HSP90) in magnetic hyperthermia-treated human liver cancer stem-like cells (LCSCs) and the effects of a heat-shock protein HSP90 inhibitor 17-allylamino-17-demethoxgeldanamycin (17-AAG) on hepatocellular carcinoma-burdened nude mice. Methods CD90+ LCSCs were isolated by magnetic-activated cell sorting from BEL-7404. Spheroid formation, proliferation, differentiation, drug resistance, and tumor formation assays were performed to identify stem cell characteristics. CD90-targeted thermosensitive magnetoliposomes (TMs)-encapsulated 17-AAG (CD90@17-AAG/TMs) was prepared by reverse-phase evaporation and its characteristics were studied. Heat tolerance in CD90+ LCSCs and the effect of CD90@17-AAG/TMs-mediated heat sensitivity were examined in vitro and in vivo. Results CD90+ LCSCs showed significant stem cell-like properties. The 17-AAG/TMs were successfully prepared and were spherical in shape with an average size of 128.9±7.7 nm. When exposed to magnetic hyperthermia, HSP90 was up-regulated in CD90+ LCSCs. CD90@17-AAG/TMs inhibited the activity of HSP90 and increased the sensitivity of CD90+ LCSCs to magnetic hyperthermia. Conclusion The inhibition of HSP90 could sensitize CD90+ LCSCs to magnetic hyperthermia and enhance its anti-tumor effects in vitro and in vivo.

20(s)-ginsenoside Rg3-loaded magnetic human serum albumin nanospheres applied to HeLa cervical cancer cells in vitro

20(s)-ginsenoside Rg3 is extracted from traditional Chinese medicine, red ginseng. However, due to its poor aqueous solubility and low oral bioavailability, the use of 20(s)-Rg3 is limited. This study aimed to explore a method of preparing nano-sized 20(s)-ginsenoside Rg3 particle named 20(s)-ginsenoside Rg3-loaded magnetic human serum albumin nanospheres (20(s)-Rg3/HSAMNP) to change dosage form to improve its aqueous solubility and bioavailability. 20(s)-Rg3/HSAMNP were prepared by the desolvation-crosslinking method. The character of 20(s)-Rg3/HSAMNP was detected. An antiproliferative effect and cell apoptosis rates of 20(s)-Rg3/HSAMNP on human cervical cancer cells were determined by the MTT assay and flow cytometry, respectively. TEM analysis showed that 20(s)-Rg3/HSAMNP were approximately spherical and uniform in size. Thermodynamic testing showed that the corresponding magnetic fluid of a specific concentration rosed to a steady temperature of 42-65○C. Iron content was approximately 3 mg/mL. Drug encapsulation efficiency was approximately 70%. The potential of 20(s)-Rg3/HSAMNP combined with magnetic hyperthermia therapy to inhibit cell growth and induce apoptosis was much more prominent than that of the other groups. A new dosage form of 20(s)-Rg3 was prepared, which effectively induced apoptosis in HeLa cervical cancer cells in vitro when combined with hyperthermia.

Combination of Human Leukocyte Antigen and Killer Cell Immunoglobulin-Like Receptor Genetic Background Influences the Onset Age of Hepatocellular Carcinoma in Male Patients with Hepatitis B Virus Infection

To investigate whether killer cell immunoglobulin-like receptor (KIR) and human leukocyte antigen (HLA) genetic background could influence the onset age of hepatocellular carcinoma (HCC) in patients with hepatitis B virus (HBV) infection, one hundred and seventy-one males with HBV-related HCC were enrolled. The presence of 12 loci of KIR was detected individually. HLA-A, -B, and -C loci were genotyped with high resolution by a routine sequence-based typing method. The effect of each KIR locus, HLA ligand, and HLA-KIR combination was examined individually by Kaplan-Meier (KM) analysis. Multivariate Cox hazard regression model was also applied. We identified C1C1-KIR2DS2/2DL2 as an independent risk factor for earlier onset age of HCC (median onset age was 44 for C1C1-KIR2DS2/2DL2 positive patients compared to 50 for negative patients, P = 0.04 for KM analysis; HR = 1.70, P = 0.004 for multivariate Cox model). We conclude that KIR and HLA genetic background can influence the onset age of HCC in male patients with HBV infection. This study may be useful to improve the current HCC surveillance program in HBV-infected patients. Our findings also suggest an important role of natural killer cells (or other KIR-expressing cells) in the progress of HBV-related HCC development.

IRF-1 and p65 mediate upregulation of constitutive HLA-A antigen expression by hepatocellular carcinoma cells.

Malignant transformation of hepatocytes is frequently associated with upregulation of HLA-A expression. Currently there is no information available regarding the mechanisms underlying this phenotypic change. We investigated HLA-A expression in 165 paraffin embedded tissues and 21 fresh tissues from liver cancer patients. Utilizing truncated HLA-A promoter-reporter constructs and gel-shift assay we had identified the regulatory elements and transcription factors required for HLA-A upregulation. 54% of the paraffin embedded tissues showed increased HLA-A expression in their cancerous part. 43% of the fresh liver cancer tissues had increased HLA-A complex expression with the HLA-A heavy chain gene demonstrating the highest level of upregulation (62%). Enhanced HLA-A expression in the liver cell lines QGY7701 and BEL7402 was found to be mediated by binding of interferon regulatory factor 1 (IRF-1) to interferon stimulated response element, and of nuclear transcription factor p65 binding to enhancer A element in the HLA-A promoter of these cell lines. The in vivo relevance of these findings was indicated by the association of the enhanced expression of IRF-1 and accumulation of nuclear p65 with HLA-A upregulation in 8 of the 21 liver cancer lesions investigated. Our results indicated that HLA-A upregulation in liver cancer was mediated by both increased nuclear aggregation of transcription factor p65 and upregulation of transcription factor IRF-1.

GSI-I has a better effect in inhibiting hepatocellular carcinoma cell growth than GSI-IX, GSI-X, or GSI-XXI.

Current studies are ongoing to find new drugs for the treatment of hepatocellular carcinoma (HCC). The discovery of drugs depends on the identification of molecules that can play essential roles in the development of liver cancer, for example, Notch pathway molecules. &ggr;-Secretase inhibitors (GSIs) can inhibit the cleavage of intramembranous substrates of all Notch receptors and subsequently suppress Notch signaling. However, whether the inhibition of the Notch pathway can suppress or promote HCC growth is still under debate. In this study, we examined the expression of Notch pathway molecules in 20 pairs of HCC tissue with their normal couterparts and a panel of eight HCC cell lines. We also determined the effects of different types of GSI treatments on the cell growth of those HCC cell lines. Our results showed that the molecules of the Notch pathway were expressed in six of the eight HCC cell lines. Those six HCC cell lines were more sensitive to GSI-I treatment than the nonexpression ones. Among the four inhibitors, GSI-X and GSI-XXI exerted no effect on HCC cells growth at all. GSI-IX inhibited the growth of four HCC cell lines at 40 &mgr;mol/l. In contrast, most of these HCC cell lines were susceptible to a low concentration of GSI-I (1.2 &mgr;mol/l) treatment. The suppressive effect of GSI-I on cell growth was because of the inhibition of C-Myc, a Notch target gene. In addition, 80% (16/20) of the specimens showed either an increased expression of at least one Notch receptor or an augmented expression of Jagged1 compared with their normal counterparts. Our study reports for the first time that different kinds of GSIs can block the growth of several HCC cell lines. Our finding suggests that GSI-I is a potential chemical reagent and warrants additional testing in liver cancer therapeutics.