Antibody-displaying extracellular vesicles for targeted cancer therapy.

Extracellular vesicles (EVs) function as natural delivery vectors and mediators of biological signals across tissues. Here, by leveraging these functionalities, we show that EVs decorated with an antibody-binding moiety specific for the fragment crystallizable (Fc) domain can be used as a modular delivery system for targeted cancer therapy. The Fc-EVs can be decorated with different types of immunoglobulin G antibody and thus be targeted to virtually any tissue of interest. Following optimization of the engineered EVs by screening Fc-binding and EV-sorting moieties, we show the targeting of EVs to cancer cells displaying the human epidermal receptor 2 or the programmed-death ligand 1, as well as lower tumour burden and extended survival of mice with subcutaneous melanoma tumours when systemically injected with EVs displaying an antibody for the programmed-death ligand 1 and loaded with the chemotherapeutic doxorubicin. EVs with Fc-binding domains may be adapted to display other Fc-fused proteins, bispecific antibodies and antibody-drug conjugates.

Tumor cell derived osteopontin and prostaglandin E2 synergistically promote the expansion of myeloid derived suppressor cells during the tumor immune escape phase.

The immune escape stage in cancer immunoediting is a pivotal feature, transitioning immune-controlled tumor dormancy to progression, and augmenting invasion and metastasis. Tumors employ diverse mechanisms for immune escape, with generating immunosuppressive cells from skewed hematopoiesis being a crucial mechanism. This led us to suggest that tumor cells with immune escape properties produce factors that induce dysregulations in hematopoiesis. In support of this suggestion, this study found that mice bearing advanced-stage tumors exhibited dysregulated hematopoiesis characterized by the development of splenomegaly, anemia, extramedullary hematopoiesis, production of immunosuppressive mediators, and expanded medullary myelopoiesis. Further ex vivo studies exhibited that conditioned medium derived from EL4lu2 cells could mediate the expansion of myeloid derived suppressor cells (MDSCs) in bone marrow cell cultures. The protein array profiling results revealed the presence of elevated levels of osteopontin (OPN), prostaglandin E2 (PGE2) and interleukin 17 (IL-17) in the culture medium derived from EL4luc2 cells. Accordingly, substantial levels of these factors were also detected in the sera of mice bearing EL4luc2 tumors. Among these factors, only PGE2 alone could increase the number of MDSCs in the BM cell cultures. This effect of PGE2 was significantly potentiated by the presence of OPN but not IL-17. Finally, in vitro treatment of EL4luc2 cells with pioglitazone, a modulator of OPN and cyclooxygenase 2 (COX-2) resulted in a significant reduction in cell proliferation in EL4luc2 cells. Our findings highlight the significant role played by tumor cell-derived OPN and PGE2 in fostering the expansion of medullary MDSCs and in promoting tumor cell proliferation. Furthermore, these intertwined cancer processes could be key targets for pioglitazone intervention.

Safety and Efficacy of Autologous Melanocyte/Keratinocyte Transplantation in Patients with Refractory Stable Vitiligo.

BACKGROUND: Vitiligo is a common depigmentation skin disease associated with significant psychosocial morbidity and profound effect on the quality of life. The treatment of vitiligo is still a major challenge in the field of dermatology. Currently, topical steroids, calcineurin inhibitors, ultraviolet phototherapy, surgery, and cultured and non-cultured epidermal melanocyte transplantation are used for the treatment of vitiligo. However, the effectiveness of these treatment modalities is limited by the lack of response, long-term treatment periods, high cost, and inevitable adverse effects. OBJECTIVES: In this study, we aimed to evaluate the efficacy of intraepidermal injection of autologous non-cultured melanocytes and keratinocytes as an alternative therapy for the refractory and stable (RS) vitiligo. METHODS: The treatment procedure was performed on thirty-nine RS vitiligo patients. The autologous skin grafts obtained from the buttock area and epidermis were separated from dermis using dispase. Single-cell autologous melanocytes and keratinocytes were prepared from the epidermis by trypsin/ethylene diamine tetra acetic acid and injected at the concentration of 100-400 × 103 cells/cm2, intra-epidermally to the selected vitiligo lesions. Vitiligo re-pigmentation was monitored employing photography. Photographs were taken prior to and 2, 4, and 6 months after the cell transplantation. Improvement of the skin depigmentation was classified as follows: <25% as minimal response, 26-50% as moderate response, 51-75% as good response, and finally 76-100% as excellent response. RESULTS: Cell infusion appeared to be safe as none of the patients exhibited any adverse effects. At the end of the sixth month follow-up period, of the treated patients, 12.8% demonstrated an excellent response, 36% exhibited a good response, and 51.2% showed a moderate to minimal response to the administered therapy. Obtained significant p value for Wilcoxon test over the checkpoints at 2nd, 4th, and 6th month (p = 0.03, 0.04, and 0.039, respectively) post-cell transplantation confirmed notable growing trend in the re-pigmentation. CONCLUSION: Our findings provide a strong support for the therapeutic efficacy of autologous non-cultured melanocytes and keratinocytes in patients with RS vitiligo.

COL-3-Induced Molecular and Ultrastructural Alterations in K562 Cells.

Tetracycline-3 (4-dedimethylamino sancycline, COL-3) is a non-antibiotic tetracycline derivative. COL-3 exerts potent anti-metalloproteinase activity and its antitumor effects have been reported both in vitro and in vivo. In this study, we investigated the mechanisms of COL-3-induced cytotoxicity in a chronic myeloid leukemia cell line, K562, characterized by the BCR-ABL fusion protein. COL-3 induced K562 cell death in a concentration-dependent manner with an IC50 of 10.8 µg/mL and exhibited features of both apoptosis and necrosis. However, flow cytometry analysis revealed that necrotic cells dominated over the early and late apoptotic cells upon treatment with COL-3. Transmission electron microscopy analysis in combination with Western blotting (WB) analysis revealed early mitochondrial swelling accompanied by the early release of cytochrome c and truncated apoptosis inducing factor (tAIF). In addition, ultrastructural changes were detected in the endoplasmic reticulum (ER). COL-3 affected the levels of glucose-regulated protein-94 (GRP94) and resulted in m-calpain activation. DNA double strand breaks as a signature for DNA damage was also confirmed using an antibody against γH2AX. WB analyses did not demonstrate caspase activation, while Bcl-xL protein remained unaffected. In conclusion, COL-3-induced cell death involves DNA damage as well as mitochondrial and ER perturbation with features of paraptosis and programmed necrosis.

Advanced therapeutic modalities in hepatocellular carcinoma: Novel insights.

Hepatocellular carcinoma (HCC), the most common type of liver cancer, is usually a latent and asymptomatic malignancy caused by different aetiologies, which is a result of various aberrant molecular heterogeneity and often diagnosed at advanced stages. The incidence and prevalence have significantly increased because of sedentary lifestyle, diabetes, chronic infection with hepatotropic viruses and exposure to aflatoxins. Due to advanced intra- or extrahepatic metastasis, recurrence is very common even after radical resection. In this paper, we highlighted novel therapeutic modalities, such as molecular-targeted therapies, targeted radionuclide therapies and epigenetic modification-based therapies. These topics are trending headlines and their combination with cell-based immunotherapies, and gene therapy has provided promising prospects for the future of HCC treatment. Moreover, a comprehensive overview of current and advanced therapeutic approaches is discussed and the advantages and limitations of each strategy are described. Finally, very recent and approved novel combined therapies and their promising results in HCC treatment have been introduced.

The kidney injury caused by the onset of acute graft-versus-host disease is associated with down-regulation of αKlotho.

Acute graft-versus-host disease (aGVHD) and kidney injury are the major complications after allogeneic hematopoietic stem cell transplantation (HSCT). Although the underlying mechanisms for the development of these complications are not yet fully understood, it has been proposed that emergence of aGVHD contributes to the development of kidney injury after HSCT. We have shown previously that aGVHD targets the kidney in a biphasic manner: at the onset, inflammatory genes are up-regulated, while when aGVHD becomes established, donor lymphocytes infiltrate the kidney. Here, we characterize renal manifestations at the onset of aGVHD. Mice receiving allogeneic bone marrow and spleen cells displayed symptoms of aGVHD and elevated serum levels of tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) within 4 days. There was concurrent kidney injury with the following characteristics: (1) elevated expression of the kidney injury biomarker, neutrophil gelatinase-associated lipocalin (NGAL), (2) accumulation of hetero-lysosomes in proximal tubule epithelial cells, and (3) reductions in αKlotho mRNA and protein and increased serum levels of fibroblast growth factor 23 (Fgf23), phosphate and urea. This situation resembled acute renal injury caused by bacterial lipopolysaccharide. We conclude that the onset of aGVHD is associated with kidney injury involving down-regulation of αKlotho, a sight that may inspire novel therapeutic approaches.

Simultaneous formulation of influenza vaccine and chitosan nanoparticles within CpG oligodesoxi nucleotides leads to dose-sparing and protects against lethal challenge in the mouse model.

Lack of efficient delivery systems for transporting antigenic molecules to the cytosol of antigen-presenting cells presents a major obstacle for antigen uptake by immune cells. To this end, influenza whole inactivated virus vaccines were formulated with chitosan nanoparticles and CpG oligonucleotide as a biodegradable delivery system and a Th1-specific adjuvant, respectively. Intradermal injections of a single high dose and low dose of formulated candidate vaccines were carried out. Thirty days after injection, cell proliferation assay (MTT), IFN-gamma and IL-4 ELISpot assays were conducted. Sera samples were collected 21 days after immunization to measure IgG1 and IgG2a levels. In addition, the mice challenged with mouse-adopted virus were monitored for weight loss. The results show a significant stimulation of both humoral and cellular immunities; also, weight gain and a decrease in mortality in the mice receiving both dosages of inactivated influenza virus vaccines with CpG and Chitosan coating were observed. Based on the results, it can be concluded that formulation of inactivated influenza virus with CpG and its delivery by chitosan as low-dose can return the same results as with high-dose balanced between cellular and humeral immune responses. This formulation could potentially lead to a significant saving in vaccine production.

miR-1266-5p and miR-185-5p Promote Cell Apoptosis in Human Prostate Cancer Cell Lines

Objective: Small non-coding RNA molecules are dysregulated in prostate cancer (PCa). In our previous study, downregulation of miR-1266 and miR-185 was demonstrated in PCa tissues and cell lines. The aim of the present study was to investigate whether miR-1266 and miR-185 are involved in the regulation of B-cell lymphoma (BCL) 2 and BCL2L1, respectively, and whether transfection of PCa cell lines with miR-1266 and miR-185 mimics can alter tumorigenic phenotypes. Methods: In order to investigate the regulation of BCL2 and BCL2L1 mRNA levels by miR-1266 and miR-185, respectively, a luciferase reporter assay was used. Real-time PCR was also used to analyze changes in the levels of BCL2 and BCL2L1 mRNAs in PCa cell lines following transfection with synthetic miR-1266 and miR-185. Cell apoptosis was determined by Annexin V protein expression analysis via flow cytometry. In addition to the MTT assay, a cell proliferation assay was performed. Result: A luciferase assay confirmed that the BCL2 and BCL2L1 genes may be targeted by miR-1266 and miR-185, respectively, through binding to their 3'UTR regions. Transfection of PC3 and DU145 cells with miR-1266 and miR-185 induced apoptosis and reduced proliferation, which also revealed an inverse correlation with BCL2 and BCL2L1 gene expression in the treated cells. Conclusion: Our data suggests that miR-1266 and miR-185 may be novel candidates for further research in PCa treatment through the anti-apoptotic pathway.

Downregulation of miR-1266-5P, miR-185-5P and miR-30c-2 in prostatic cancer tissue and cell lines.

Over the latest decade, the role of microRNAs (miRNAs/miRs) has received more attention. miRNAs are small non-coding RNAs that may serve a role as oncogenes or tumor suppressor genes. Certain miRNAs regulate the apoptosis pathway by influencing pro- or anti-apoptotic genes. We hypothesized that increases in the expression of B cell lymphoma 2 (BCL2) and BCL2-like 1 (BCL2L1) genes, which have been reported in various types of cancer tissues, may be due to the downregulation of certain miRNAs. The present study aimed to identify miRNAs that target BCL2 and BCL2L1 anti-apoptotic genes in prostate cancer (PCa) clinical tissue samples. Certain candidate miRNAs were selected bioinformatically and their expression in PCa samples was analyzed and compared with that in benign prostatic hyperplasia (BPH) tissue samples. The candidate miRNAs that targeted BCL2 and BCL2L1 genes were searched in online databases (miRWalk, microRNA.org, miRDB and TargetScan). A total of 12 miRNAs that target the 3'-untranslated region of the aforementioned genes and/or for which downregulation of their expression has previously been reported in cancer tissues. A total of 30 tumor tissue samples from patients with PCa and 30 samples tissues from patients with BPH were obtained and were subjected to reverse transcription-quantitative polymerase chain reaction for expression analysis of 12 candidate miRNAs, and the BCL2 and BCL2L1 genes. Additionally, expression of 3 finally selected miRNAs and genes was evaluated in prostate cancer PC3 and DU145 cell lines and human umbilical vein endothelial cells. Among 12 miRNA candidates, the expression of miR-1266, miR-185 and miR-30c-2 was markedly downregulated in PCa tumor tissues and cell lines. Furthermore, downregulation of these miRNAs was associated with upregulation of the BCL2 and BCL2L1 genes. An inverse association between three miRNAs (miR-1266, miR-185 and miR-30c-2) and two anti-apoptotic genes (BCL2 and BCL2L1) may be considered for interventional miRNA therapy of PCa.

Mathematical modeling of tumor-induced immunosuppression by myeloid-derived suppressor cells: Implications for therapeutic targeting strategies.

Myeloid-derived suppressor cells (MDSCs) belong to immature myeloid cells that are generated and accumulated during the tumor development. MDSCs strongly suppress the anti-tumor immunity and provide conditions for tumor progression and metastasis. In this study, we present a mathematical model based on ordinary differential equations (ODE) to describe tumor-induced immunosuppression caused by MDSCs. The model consists of four equations and incorporates tumor cells, cytotoxic T cells (CTLs), natural killer (NK) cells and MDSCs. We also provide simulation models that evaluate or predict the effects of anti-MDSC drugs (e.g., l-arginine and 5-Fluorouracil (5-FU)) on the tumor growth and the restoration of anti-tumor immunity. The simulated results obtained using our model were in good agreement with the corresponding experimental findings on the expansion of splenic MDSCs, immunosuppressive effects of these cells at the tumor site and effectiveness of l-arginine and 5-FU on the re-establishment of antitumor immunity. Regarding this latter issue, our predictive simulation results demonstrated that intermittent therapy with low-dose 5-FU alone could eradicate the tumors irrespective of their origins and types. Furthermore, at the time of tumor eradication, the number of CTLs prevailed over that of cancer cells and the number of splenic MDSCs returned to the normal levels. Finally, our predictive simulation results also showed that the addition of l-arginine supplementation to the intermittent 5-FU therapy reduced the time of the tumor eradication and the number of iterations for 5-FU treatment. Thus, the present mathematical model provides important implications for designing new therapeutic strategies that aim to restore antitumor immunity by targeting MDSCs.

Flavin-containing monooxygenase 3 (FMO3) role in busulphan metabolic pathway.

Busulphan (Bu) is an alkylating agent used in the conditioning regimen prior to hematopoietic stem cell transplantation (HSCT). Bu is extensively metabolized in the liver via conjugations with glutathione to form the intermediate metabolite (sulfonium ion) which subsequently is degraded to tetrahydrothiophene (THT). THT was reported to be oxidized forming THT-1-oxide that is further oxidized to sulfolane and finally 3-hydroxysulfolane. However, the underlying mechanisms for the formation of these metabolites remain poorly understood. In the present study, we performed in vitro and in vivo investigations to elucidate the involvement of flavin-containing monooxygenase-3 (FMO3) and cytochrome P450 enzymes (CYPs) in Bu metabolic pathway. Rapid clearance of THT was observed when incubated with human liver microsomes. Furthermore, among different recombinant microsomal enzymes, the highest intrinsic clearance for THT was obtained via FMO3 followed by several CYPs including 2B6, 2C8, 2C9, 2C19, 2E1 and 3A4. In Bu- or THT-treated mice, inhibition of FMO3 by phenylthiourea significantly suppressed the clearance of both Bu and THT. Moreover, the simultaneous administration of a high dose of THT (200µmol/kg) to Bu-treated mice reduced the clearance of Bu. Consistently, in patients undergoing HSCT, repeated administration of Bu resulted in a significant up-regulation of FMO3 and glutathione-S-transfrase -1 (GSTA1) genes. Finally, in a Bu-treated patient, additional treatment with voriconazole (an antimycotic drug known as an FMO3-substrate) significantly altered the Bu clearance. In conclusion, we demonstrate for the first time that FMO3 along with CYPs contribute a major part in busulphan metabolic pathway and certainly can affect its kinetics. The present results have high clinical impact. Furthermore, these findings might be important for reducing the treatment-related toxicity of Bu, through avoiding interaction with other concomitant used drugs during conditioning and hence improving the clinical outcomes of HSCT.

Rapid and Robust Quantification of p-Xyleneselenocyanate in Plasma via Derivatization.

p-Xyleneselenocyanate (p-XSC) is one of the most investigated selenium compounds in cancer-prevention and -therapy. Despite the potent anticancer property, there is still no proper method to perform the quantitative analysis of p-XSC in plasma. In this investigation, we aimed at developing a method based on liquid chromatography-mass spectrometry (LC-MS) for the measurement of p-XSC in plasma. Direct deproteinization was first used to extract parent p-XSC from plasma, but failed to achieve high recovery rate (<2%) due to formation of selenium-sulfur bond between p-XSC and plasma protein. To overcome this problem, we modified the extraction method to three steps: (1) break the selenium-sulfur bond by tris(2-carboxyethyl)phosphine; (2) stabilize the newly formed intermediate selenol by N-ethylmaleimide; (3) deproteinization. This three-step method efficiently recovered bound p-XSC by more than 75%. In in vivo study, p-XSC was injected intravenously into mice and plasma was collected for LC-MS analysis. Consistently, p-XSC was undetectable in its parent form, whereas the bound form was readily quantified, employing the modified extraction method. In summary, we describe a novel, robust, and sensitive method for quantification of p-XSC in plasma. The present method will enable pharmacokinetic and pharmacodynamic studies of p-XSC in both clinical and preclinical settings.

Differential effects of low-dose fludarabine or 5-fluorouracil on the tumor growth and myeloid derived immunosuppression status of tumor-bearing mice.

Myeloid-derived suppressor cells (MDSCs) accumulate in tumor-bearing hosts and play a key role in the suppression of the innate and adaptive immunity. Chemotherapeutic strategies have been developed to deplete or deactivate MDSCs in different tumor models. The pyrimidine analog, 5-fluorouracil (5-FU) is found to reduce the tumor size by depleting MDSCs. Here, we asked whether the purine analog, fludarabine (Flu), could exert similar effects. Employing a lymphoma model, we demonstrated that in mice with advanced tumors (where MDSC-induced suppression was present), treatment with a single low-dose Flu (25, 50, 100mg/kg) elevated the numbers of splenic MDSCs and serum arginase activity, and simultaneously, increased the tumor growth (only the highest dose). On the other hand, in mice with palpable tumors (where the MDSC-induced suppression was in progress), treatment with Flu had no significant effects on the tumor growth or the number of splenic MDSCs. In contrast to Flu, treatment with low-dose 5-FU, irrespective of tumor stage, caused tumor regression which coincided with significant reductions in the numbers of splenic MDSCs and blood neutrophils, but increases in the ratios of splenic CD4+ T and CD8+ T cells to suppressive MDSCs. Finally, in healthy mice (where MDSC-induced immuosuppression did not exist), 5-FU, but not Flu induced significant decreases in the number of myeloid cells in the bone marrow, naturally occurring splenic MDSCs and thymocytes. In conclusion, Flu exacerbates MDSC-induced immunosuppression in a tumor stage-dependent manner, whereas 5-FU alleviates the suppressive effects of MDSC at all stages of tumor development.

Biodistribution of biodegradable polymeric nano-carriers loaded with busulphan and designed for multimodal imaging.

BACKGROUND: Multifunctional nanocarriers for controlled drug delivery, imaging of disease development and follow-up of treatment efficacy are promising novel tools for disease diagnosis and treatment. In the current investigation, we present a multifunctional theranostic nanocarrier system for anticancer drug delivery and molecular imaging. Superparamagnetic iron oxide nanoparticles (SPIONs) as an MRI contrast agent and busulphan as a model for lipophilic antineoplastic drugs were encapsulated into poly (ethylene glycol)-co-poly (caprolactone) (PEG-PCL) micelles via the emulsion-evaporation method, and PEG-PCL was labelled with VivoTag 680XL fluorochrome for in vivo fluorescence imaging. RESULTS: Busulphan entrapment efficiency was 83% while the drug release showed a sustained pattern over 10 h. SPION loaded-PEG-PCL micelles showed contrast enhancement in T 2 *-weighted MRI with high r 2* relaxivity. In vitro cellular uptake of PEG-PCL micelles labeled with fluorescein in J774A cells was found to be time-dependent. The maximum uptake was observed after 24 h of incubation. The biodistribution of PEG-PCL micelles functionalized with VivoTag 680XL was investigated in Balb/c mice over 48 h using in vivo fluorescence imaging. The results of real-time live imaging were then confirmed by ex vivo organ imaging and histological examination. Generally, PEG-PCL micelles were highly distributed into the lungs during the first 4 h post intravenous administration, then redistributed and accumulated in liver and spleen until 48 h post administration. No pathological impairment was found in the major organs studied. CONCLUSIONS: Thus, with loaded contrast agent and conjugated fluorochrome, PEG-PCL micelles as biodegradable and biocompatible nanocarriers are efficient multimodal imaging agents, offering high drug loading capacity, and sustained drug release. These might offer high treatment efficacy and real-time tracking of the drug delivery system in vivo, which is crucial for designing of an efficient drug delivery system.

Evaluation of interleukin 12 and CD56+ lymphocyte cells in pediatric hematopoietic stem cell transplantation for early diagnosis of acute graft versus host disease.

The present study tried to explain CD56+ lymphocyte cells activities and possible prognostic role of these cells in Graft-Versus-Host-Disease (GVHD). The role of IL-12 activation and function is of interest in this study. Peripheral blood samples of 51 Hematopoietic Stem Cell Transplantation (HSCT) recipients collected at before (day -8) and after (days 7 and 14). PBMC were collected by Ficoll separation and analyzed by Flow Cytometry using triple antibody (CD45-PerCP, CD56-FITC, and CD69-PE staining and control antibody. Levels of the cytokine IL-12 in the patient's serum were evaluated by ELISA. Percentage of CD56+ lymphocytes (CD56+bright) cells was significantly increased at day 14 in patients with acute GVHD and percentage of lymphocytes expressing CD69 was significantly increased at days 7 and 14 posts HSCT in patients with acute GVHD in comparison to those in non-GVHD patients. Baseline serum IL-12 levels (pre-HSCT, day -8) were significantly higher in those HSCT recipients who did not develop GVHD. This study showed that post-transplant CD56+ lymphocytes and pre-transplant serum levels of IL-12 play significant roles in the induction of and protection against GVHD, respectively. The increase in the percentage of CD69+ cells indicates the activation of lymphocyte in acute GVHD group.

Suppressive effects of low-dose 5-fluorouracil, busulfan or treosulfan on the expansion of circulatory neutrophils and myeloid derived immunosuppressor cells in tumor-bearing mice.

As reported previously, large numbers of neutrophils appear in the circulation during tumor development. However, the relationship between these cells and myeloid-derived suppressor cells (MDSCs), as well as their susceptibility to myelosuppressive drugs have not been yet investigated. Here, we employed a lymphoma model to characterize tumor-associated circulating neutrophils, including their sensitivity to 5-fluorouracil (5-FU), busulfan (Bu) or treosulfan (Treo). Tumor-bearing mice exhibited pronounced elevations in the numbers of splenic MDSCs and circulating neutrophils, MDSCs, and granulocytic-MDSCs (G-MDSCs). Although these cells were not affected by 5-FU at a dose of 17mg/kg, 50 and 100mg/kg were equally effective in causing dramatic tumor regression, normalizing neutrophil number, and significantly reducing the numbers of splenic MDSCs, B cells, and bone marrow myeloid cells. Treatment with Bu (10, 30 or 60mg/kg) only reduced the number of circulating neutrophils, with no effects on these other parameters. At a concentration of 500mg/kg, Treo was ineffective, whereas, doses of 1500 and 3000mg/kg comparably reduced the tumor size as well as the numbers of circulatory neutrophils and bone marrow myeloid cells. Finally, in comparison to 5-FU alone, a combined treatment with low-dose 5-FU and Treo resulted in a more pronounced reduction in the numbers of circulatory neutrophils and bone marrow myeloid cells, together with longer survival time. We conclude that tumor-associated circulatory neutrophils represent blood MDSCs/G-MDSCs that are highly sensitive to 5-FU and Treo, but not Bu. Moreover, the efficacy of 5-FU can be potentiated by Treo.

DNA damage, lysosomal degradation and Bcl-xL deamidation in doxycycline- and minocycline-induced cell death in the K562 leukemic cell line.

We investigated mechanisms of cytotoxicity induced by doxycycline (doxy) and minocycline (mino) in the chronic myeloid leukemia K562 cell line. Doxy and mino induced cell death in exposure-dependent manner. While annexin V/propidium iodide staining was consistent with apoptosis, the morphological changes in Giemsa staining were more equivocal. A pancaspase inhibitor Z-VAD-FMK partially reverted cell death morphology, but concurrently completely prevented PARP cleavage. Mitochondrial involvement was detected as dissipation of mitochondrial membrane potential and cytochrome C release. DNA double strand breaks detected with γH2AX antibody and caspase-2 activation were found early after the treatment start, but caspase-3 activation was a late event. Decrement of Bcl-xL protein levels and electrophoretic shift of Bcl-xL molecule were induced by both drugs. Phosphorylation of Bcl-xL at serine 62 was ruled out. Similarly, Bcr/Abl tyrosine kinase levels were decreased. Lysosomal inhibitor chloroquine restored Bcl-xL and Bcr/Abl protein levels and inhibited caspase-3 activation. Thus, the cytotoxicity of doxy and mino in K562 cells is mediated by DNA damage, Bcl-xL deamidation and lysosomal degradation with activation of mitochondrial pathway of apoptosis.

Immunotoxicological effects of streptozotocin and alloxan: in vitro and in vivo studies.

Streptozotocin (STZ) and alloxan (ALX), widely used to induce diabetes in experimental animals, have different structures and mechanisms of action. We investigated those effects of these drugs on the immune system that might influence engraftment efficiency and graft survival in transplantation models, and their cytotoxicity on hematopoietic cell lines. We used the minimum dose to induce diabetes in a mouse, i.e. 180 mg/kg i.v. STZ and 75 mg/kg i.v. ALX. Both groups exhibited significant decrease in body weight during 4 days post-treatment as compared to controls. We found that blood glucose in ALX-injected mice increased faster than in STZ-injected mice. The total number of recovered splenocytes was lower in STZ-injected animals than in ALX-injected animals. The survival periods of rat islet grafts in recipient mice were longer and more diverse in STZ-injected recipients (7-24 days) compared to ALX-injected recipients (6-7 days). The in vitro study showed that ALX was less cytotoxic in cell lines with IC50 values of 2809, 3679 and >4000 µg/ml for HL60, K562 and C1498 cells respectively. STZ was more toxic, especially in HL60 cells, with IC50 values of 11.7, 904 and 1024 µg/ml for HL60, K562 and C1498 cells respectively. Furthermore, in response to concanavalin A (Con-A), splenocytes from STZ-injected mice produced higher amounts of interferon-gamma (IFN-γ) than those from ALX-injected mice. In conclusion, STZ was more cytotoxic than ALX in vitro and in vivo. STZ caused lymphocytopenia, which may result in longer graft survival in STZ-treated animals than in ALX-treated animals.

Complement activation is involved in the hepatic injury caused by high-dose exposure of mice to perfluorooctanoic acid.

High-dose exposure of mice to perfluorooctanoate (PFOA) induces both hepatotoxicity and immunotoxicity. Here, we characterized the effects of 10-day dietary treatment with PFOA (0.002-0.02%, w/w) on the liver and complement system of male C57BL/6 mice. At all four doses, this compound caused hepatomegaly and reduced the serum level of triglycerides (an indicator for activation of the peroxisome proliferator-activated receptor-alpha (PPARα)). At the highest dose (0.02%, w/w), this hepatomegaly was associated with the hepatic injury, as reflected in increased activity of alanine aminotranferase (ALAT) in the serum, severe hepatocyte hypertrophy and hepatocellular necrosis. PFOA-induced hepatic injury was associated with in vivo activation of the complement system as indicated by (i) significant attenuation of the serum activities of both the classical and alternative pathways; (ii) a marked reduction in the serum level of the complement factor C3; and (iii) deposition of the complement factor C3 fragment (C3a) in the hepatic parenchyma. PFOA did not activate the alternative pathway of complement in vitro. At doses lower than 0.02%, PFOA induced hepatocyte hypertrophy without causing liver injury or activating complement. These results reveal substantial involvement of activation of complement in the pathogenesis of PFOA-induced hepatotoxicity.