RelB regulates the homeostatic proliferation but not the function of Tregs.

BACKGROUND: RelB, a member of the NF-κB family, plays a critical role in the development of T cells. However, the role of RelB in Foxp3+ regulatory T cells (Tregs) remains controversial. RESULTS: Using a bone marrow chimeric mouse model, we demonstrated that the expansion of Foxp3+ Tregs in vivo could be mediated by extrinsic mechanisms. RelB plays an important role in inhibiting the homeostatic proliferation of Tregs, but not their survival. Even with the heightened expansion, RelB-/- Treg cells displayed normal suppressive function in vitro. Among the expanded populations of Treg cells, most were nTreg cells; however, the population of iTregs did not increase. Mechanistically, RelB seems to regulate Treg proliferation independently of the signal transducer and activator of transcription 5 (STAT5) pathway. CONCLUSIONS: These data suggest that RelB regulates Treg proliferation independently of the STAT5 pathway, but does not alter the function of Tregs. Further studies are warranted to uncover such mechanisms.

miR-373 Inhibits Glioma Cell U251 Migration and Invasion by Down-Regulating CD44 and TGFBR2.

Glioblastoma multiforme (GBM) is the most malignant glioma, unveiling the underlying mechanisms of its aggressiveness could promote the discovery of potential targets for effective treatment. MicroRNAs (miRNAs) are important participants in both development and disease, its involvement in cancers has long been recognized. In this study, we investigated the role of miRNA-373 (miR-373) in GBM cell line U251, demonstrated that although miR-373 does not affect cell growth of U251, it inhibits migration and invasion of U251. Forced expression of miR-373 down-regulates the expressions CD44 and TGFBR2, while knockdown of CD44 and TGFBR2 presents the similar phenotype as miR-373 overexpression, suggesting that CD44 and TGFBR2 are functional targets of miR-373, down-regulation of CD44 and TGFBR2 by miR-373 are partly responsible for the migration, and invasion suppressive role of miR-373 in U251.

Th1-, Th2-, and Th17-associated cytokine expression in hypopharyngeal carcinoma and clinical significance.

Th0 cells differentiate into Th1 or Th2 depending on multiple transcription factors acting on specific time points to regulate gene expression. Th17 cells, a subset of IL-17-producing T cells distinct from Th1 or Th2 cells, have been described as key players in inflammation and autoimmune diseases as well as cancer development. In the present study, 53 patients with hypopharyngeal cancer were included. The expression levels of Th1-, Th2- and Th17-associated cytokines in hypopharyngeal cancer tissues and pericarcinoma tissues were detected. The relationship between Th1, Th2, or Th17 infiltration and metastasis was studied. Our results showed that the mRNA and protein expressions of Th1 cytokines were lower, while the expressions of Th2 and Th17 cytokines were higher in tumor tissues, and the intensity of expression was strengthened with clinical stage increasing. Cancer tissues had higher level expressions of Th2 and Th17 cytokines than that of pericarcinoma tissues. From the above data, we speculated that high expressions of Th2- and Th17-associated cytokines in hypopharyngeal carcinoma may contribute to cancer development and metastasis.

A population pharmacokinetic study of tacrolimus in healthy Chinese volunteers and liver transplant patients.

AIM: To develop a population pharmacokinetic (PopPK) model of tacrolimus in healthy Chinese volunteers and liver transplant recipients for investigating the difference between the populations, and for potential individualized medication. METHODS: A set of 1100 sparse trough concentration data points from 112 orthotopic liver transplant recipients, as well as 851 dense data points from 40 healthy volunteers receiving a single dose of tacrolimus (2 mg, p.o.) were collected. PopPK model of tacrolimus was constructed using the program NONMEM. Related covariates such as age, hepatic and renal functions that were potentially associated with tacrolimus disposition were evaluated. The final model was validated using bootstrapping and a visual predictive check. RESULTS: A two-compartment model of tacrolimus could best describe the data from the two populations. The final model including two covariates, population (liver transplant recipients or volunteers) and serum ALT (alanine aminotransferase) level, was verified and adequately described the pharmacokinetic characteristics of tacrolimus. The estimates of V2/F, Q/F and V3/F were 22.7 L, 76.3 L/h and 916 L, respectively. The estimated CL/F in the volunteers and liver transplant recipients was 32.8 and 18.4 L/h, respectively. Serum ALT level was inversely related to CL/F, whereas age did not influence CL/F. Thus, the elderly (≥65 years) and adult (<65 years) groups in the liver transplant recipients showed no significant difference in the clearance of tacrolimus. CONCLUSION: Compared with using the sparse data only, the integrating modeling technique combining sparse data from the patients and dense data from the healthy volunteers improved the PopPK analysis of tacrolimus.

A Simple LC/MS/MS Method for the Determination of Moxifloxacin N-Sulfate in Rat Plasma and Its Application in a Pharmacokinetic Study.

Moxifloxacin N-sulfate is one of the main metabolites of moxifloxacin in phase II metabolism mediated by sulfotransferases. In this study, a simple, rapid, and sensitive LC/MS/MS method with one-step protein precipitation with methanol was developed and validated to quantify the concentration of moxifloxacin N-sulfate in rat plasma. The chromatographic separation was accomplished by using an Agilent Extend C18 column (4.6×250 mm; 5 µm) with a mobile phase consisting of acetonitrile and distilled water (30:70, v/v) containing 5 mM ammonium formate (pH=8.82 adjusted by ammonia) at a flow rate of 1.0 mL/min. A triple quadrupole tandem mass spectrometer with electrospray ionization source was used as a detector operated by multiple reaction monitoring in the negative-ion mode with m/z 480.2/436.3. The calibration curve was linear ranging from 2 to 200 ng/mL. The intraday and interday precision values (RSD) were less than 7.10%, and the intraday and interday accuracy values (relative error) were within -0.40 to 4.99%. Stabilities of all QC samples were within general assay acceptability criteria according to the U.S. Food and Drug Administration guidelines. No considerable matrix effect was found. A pharmacokinetic study of moxifloxacin N-sulfate after a single oral dose of moxifloxacin in rats was carried out using this new method.

The role of MMP-2 and MMP-9 in the metastasis and development of hypopharyngeal carcinoma.

INTRODUCTION: The role of matrix metalloproteinase-2 and 9 in the metastasis and development of hypopharyngeal carcinoma has not been clarified. OBJECTIVES: To observe the relationship between matrix metalloproteinase-2, matrix metalloproteinase-9 and the metastasis, development of hypopharyngeal carcinoma. METHODS: This study included 42 hypopharyngeal cancer patients. The mRNA and protein expression levels of matrix metalloproteinase-2 and 9 in hypopharyngeal carcinoma and paracancerous tissues were detected by reverse transcription-polymerase chain reaction and Western blot. RESULTS: Reverse transcription-polymerase chain reaction detection showed that the mRNA of matrix metalloproteinase-2 and 9 was expressed in both cancer and pericarcinoma tissues, but was almost not expressed in polypoid control tissues. The expression intensity in the cancer tissue was significantly higher than that in the pericarcinoma tissue (matrix metalloproteinase-2: t ＝ 2.529, p = 0.015; matrix metalloproteinase-9: t ＝ 4.781, p ＜ 0.001). The mRNA expression in the cancer tissue was enhanced with the increase of the tumor clinical stage (matrix metalloproteinase-2: F = 4.003, p = 0.026; matrix metalloproteinase-9: F = 5.501, p = 0.008). Its expression intensity was associated with the metastasis of lymph nodes (N staging) and increased with the degree of lymphatic metastasis (matrix metalloproteinases-2: F = 8.965, p = 0.005; matrix metalloproteinase-9: F = 5.420, p = 0.025). There was no significant change in T staging of tumor. With the increase of tumor pathological stage, the mRNA expression of matrix metalloproteinase-2 and 9 was strengthened (matrix metalloproteinase-2: F = 3.884, p = 0.029; matrix metalloproteinase-9: F = 3.783, p = 0.032). The protein expression level of matrix metalloproteinase-2 and 9 was the same as that of mRNA. CONCLUSION: The expression of matrix metalloproteinase-2 and 9 in hypopharyngeal carcinoma was significantly higher than that in pericarcinoma tissue, and it was enhanced with the increase of clinical stage. The expression level was related to lymph node metastasis and tumor pathological stage. Thus, matrix metalloproteinase-2 and 9 may be involved in the occurrence, development, invasion and metastasis of hypopharyngeal carcinoma through a variety of mechanisms.

Regulation of the Migration of Distinct Dendritic Cell Subsets.

Dendritic cells (DCs), a class of antigen-presenting cells, are widely present in tissues and apparatuses of the body, and their ability to migrate is key for the initiation of immune activation and tolerogenic immune responses. The importance of DCs migration for their differentiation, phenotypic states, and immunologic functions has attracted widespread attention. In this review, we discussed and compared the chemokines, membrane molecules, and migration patterns of conventional DCs, plasmocytoid DCs, and recently proposed DC subgroups. We also review the promoters and inhibitors that affect DCs migration, including the hypoxia microenvironment, tumor microenvironment, inflammatory factors, and pathogenic microorganisms. Further understanding of the migration mechanisms and regulatory factors of DC subgroups provides new insights for the treatment of diseases, such as infection, tumors, and vaccine preparation.

Association Between Interleukin 35 Gene Single Nucleotide Polymorphisms and the Uveitis Immune Status in a Chinese Han Population.

Autoimmune uveitis is characterized by immune disorders of the eyes and the whole body and is often recurrent in young adults, but its pathogenesis is still unclear. IL-35 is an essential regulatory factor in many autoimmune diseases, which is produced by Breg cells and can induce Breg cells to regulate the immune response. The relationship between the expression and gene polymorphism of IL-35 and the immune status of patients with autoimmune uveitis has not been reported. The peripheral blood of the subjects was collected from patients with Behçet's Disease (BD) and those with Vogt-Koyanagi-Harada (VKH) syndrome. The percentage of immune cell subsets including B cells, DC, and T cells, and the expression of IL-35 in serum of these two kinds of disease were analyzed. And then, the associations between seven IL-35 single nucleotide polymorphism (SNP) sites and disease susceptibility, the immune status, the clinical characteristics, and the serum IL-35 levels were analyzed. Our results showed that the percentage of Breg cells was significantly decreased in the blood of patients with VKH syndrome compared to that of healthy controls. The levels of IL-35 in the serum of patients with VKH syndrome or BD patients were not changed significantly, compared to that of healthy controls. Furthermore, the associations between two subunits of IL-35 (IL-12p35 and EBI3) and BD or VKH patients were analyzed. We found that there was an association between the EBI3 rs428253 and the occurrence of BD. There was an association between the IL-12p35 rs2243131 and the low level of Breg cell of VKH patients. In addition, there were associations between the polymorphisms of EBI3 rs4740 and the occurrence of headache and tinnitus of VKH patients, respectively. And the genotype frequency of IL-12p35 rs2243115 was related to the concentration of serum IL-35 in patients with VKH syndrome. Thus, the specific SNP sites change of IL-35 were correlated to the immune disorders in uveitis. And they may also play a guiding role in the occurrence of clinical symptoms in patients with uveitis, especially for VKH syndrome.

Soluble CD83 Regulates Dendritic Cell-T Cell Immunological Synapse Formation by Disrupting Rab1a-Mediated F-Actin Rearrangement.

Dendritic cell-T cell (DC-T) contacts play an important role in T cell activation, clone generation, and development. Regulating the cytoskeletal protein rearrangement of DCs can modulate DC-T contact and affect T cell activation. However, inhibitory factors on cytoskeletal regulation in DCs remain poorly known. We showed that a soluble form of CD83 (sCD83) inhibited T cell activation by decreasing DC-T contact and synapse formation between DC and T cells. This negative effect of sCD83 on DCs was mediated by disruption of F-actin rearrangements, leading to alter expression and localization of major histocompatibility complex class II (MHC-II) and immunological synapse formation between DC and T cells. Furthermore, sCD83 was found to decrease GTP-binding activity of Rab1a, which further decreased colocalization and expression of LRRK2 and F-actin rearrangements in DCs, leading to the loss of MHC-II at DC-T synapses and reduced DC-T synapse formation. Further, sCD83-treated DCs alleviated symptoms of experimental autoimmune uveitis in mice and decreased the number of T cells in the eyes and lymph nodes of these animals. Our findings demonstrate a novel signaling pathway of sCD83 on regulating DC-T contact, which may be harnessed to develop new immunosuppressive therapeutics for autoimmune disease.

Corrigendum: MicroRNA-138-5p Suppresses Non-small Cell Lung Cancer Cells by Targeting PD-L1/PD-1 to Regulate Tumor Microenvironment.

[This corrects the article DOI: 10.3389/fcell.2020.00540.].

MicroRNA-138-5p Suppresses Non-small Cell Lung Cancer Cells by Targeting PD-L1/PD-1 to Regulate Tumor Microenvironment.

Non-small cell lung cancer (NSCLC) is still challenging for treatment owing to immune tolerance and evasion. MicroRNA-138 (miR-138) not only acts as a tumor suppressor to inhibit tumor cell proliferation and migration but also regulates immune response. The regulatory mechanism of miR-138 in NSCLC remains not very clear. Herein, we demonstrated that miR-138-5p treatment decreased the growth of tumor cells and increased the number of tumor-infiltrated DCs. miR-138-5p not only down-regulated the expression of cyclin D3 (CCND3), CCD20, Ki67, and MCM in A549/3LL cells, but also regulated the maturation of DCs in A549-bearing nude mice and the 3LL-bearing C57BL/6 mouse model, and DCs' capability to enhance T cells to kill tumor cells. Furthermore, miR-138-5p was found to target PD-L1 to down-regulate PD-L1 on tumor cells to reduce the expression of Ki67 and MCM in tumor cells and decrease the tolerance effect on DCs. miR-138-5p also directly down-regulates the expression of PD-L1 and PD-1 on DCs and T cells. Similar results were obtained from isolated human non-small cell lung cancer (NSCLC) cells and DCs. Thus, miR-138-5p inhibits tumor growth and activates the immune system by down-regulating PD-1/PD-L1 and it is a promising therapeutic target for NSCLC.

Anticancer Effects of Constituents of Herbs Targeting Osteosarcoma.

Osteosarcoma is a rare primary malignancy of bone that is prone to early metastasis. Resection surgery and chemotherapeutic regimens are current standard treatments for osteosarcoma. However, the long-term survival rate of patients with osteosarcoma is low due to a high risk of metastasis. Hence, a new approach is urgently needed to improve the treatment of osteosarcoma. Compared with chemotherapy, natural active constituents isolated from herbs exhibit less adverse effects and better anti-tumor effects. This study aimed to summarize the anticancer effects of constituents of herbs on the progression and metastasis of osteosarcoma cells. It showed that many constituents of herbs inhibited osteosarcoma by targeting proliferation, matrix metalloproteinases, integrin and cadherin, and angiogenesis. The findings might be beneficial for the development of new drugs and treatment strategies.

The Expression of SOCS and NF-Ï°B p65 in Hypopharyngeal Carcinoma.

BACKGROUND: Hypopharyngeal carcinoma is one of the most common types of head and neck tumors. Suppressers of cytokine signalling (SOCS) family members are key regulators of cytokine homeostasis, they play important roles in the process of cell proliferation, differentiation, maturation and apoptosis, and participate in the occurrence and development of tumor. The abnormal activation of NF-Ï°B is an important feature of the tumor. The aim of this study was to investigate the relationships among SOCS, NF-Ï°B p65 and hypopharyngeal carcinoma development.C. METHODS: We included 72 hypopharyngeal cancer patients and 9 swallow cyst patients. The patients were recruited at The Second Hospital of Shandong University (Jinan, China) between 2014 and 2016. The mRNA and protein expression levels of SOCS-1, SOCS-3 and NF-Ï°B p65 in hypopharyngeal carcinoma tissues, para-cancerous tissues and control tissues were detected by RT-PCR and Western blot analysis, respectively. RESULTS: Hypopharyngeal carcinoma tissues had lower level expression of SOCS-1 and SOCS-3 than pericarcinoma tissues, but there was no significant difference, while cancer tissues had significantly higher level expression of NF-Ï°B p65 than that of pericarcinoma tissues (0.412±0.266, 0.281±0.231, t=2.969, P=0.004). The early stage patients had striking higher level expression of SOCS-1 and SOCS-3 than that in advanced stages (F=16.202, P<0.001; F=52.295, P<0.001), while the expression of NF-Ï°B p65 in early stages had lower level than that in advanced stages (F=3.383, P=0.04). CONCLUSION: SOCS-1, SOCS-3 may be protective factors while NF-Ï°B p65 could be a harmful factor in hypopharyngeal carcinoma.

[Constitutive characteristics and change trend of gynecological malignant tumors in 8009 hospitalized patients in Guangxi Zhuang Autonomous Region].

OBJECTIVE: To investigate the constitutive characteristics and the change trend of gynecologic malignant tumors in hospitalized patients in Guangxi Zhuang Autonomous Region over the recent 20 years. METHODS: Clinical data of 8009 in-patients who suffered from gynecologic malignant tumors in 23 hospitals from 1985 to 2004 in Guangxi Zhuang Autonomous Region were analyzed, with respect to the tumor types and change trend. RESULTS: (1) The leading 4 types of malignant tumors were cervical cancers, ovarian cancers, endometrial cancers, and malignant trophoblastic tumors according to the constitutive ratios of the tumors. The constitutive ratio of cervical cancer patients rose year by year, from 17.48% during the 1985-1989 period to 49.25% during the 2000-2004 period (P < 0.01). While the constitutive ratio of malignant trophoblastic tumors dropped year by year. The changes of ovarian cancer, endometrial cancer, vulvar and vaginal carcinomas, and sarcoma of the uterus were not obvious (P > 0.05). (2) The occurring age of cervical cancers became younger obviously, from > or = 60 years old dropped to < 40 years old. (3) Cervical cancers were found mainly in urban residents in the former 10 years, the constitutive ratio being 67.1%; while in the latter 10 years it gradually shifted to rural residents, accounting for 52.6% of the total gynecological tumors. (4) Patients were usually at stages II, III, IV when they visited a doctor for their diseases. Especially for ovarian cancer, malignant trophoblastic tumors, sarcoma of the uterus, these patients were in the intermediate or advanced stage when they were diagnosed, mainly because of lack of obvious symptoms. The constitutive ratio of these advanced patients was over 60%. CONCLUSIONS: We should strengthen the screening program of cervical cancer, and pay more attention to prevention and control of other gynecological reproductive organ tumors at the same time. On the other hand, we should explore better tumor markers, new methods of diagnosis and treatment to improve early diagnosis and treatment of gynecologic malignant tumors.

[Expression and clinical significance of MICA in laryngeal carcinoma tissue and cells].

OBJECTIVE: To investigate the mRNA and protein expression of MICA in laryngeal squamous cell carcinoma tissue and the Hep-2 cells. METHOD: Reverse transcriptase-polymerase chain reaction (RT-PCR) and Western-blot were used to detect the expression of MICA mRNA and protein levels in the Hep-2 cells and laryngeal cancer tissues. RESULT: The MICA mRNA showed higher expression in Hep-2 cells by RT-PCR. Compared with the control, the mRNA expression of MICA was significantly enhanced in laryngeal cancer tissues (t = 11.878, P < 0.01). The intensity of MICA expression is not related to the clinical stage of cancer. MICA protein demonstrated higher level expression by Western blot. The intensity of MICA protein expression was decreased with increased clinical stage in laryngeal cancer tissues. CONCLUSION: The MICA mRNA showed stronger expression in Hep-2 cells and laryngeal cancer tissues. The intensity of its expression is not related to clinical stage of cancer. The MICA protein expression was strong in Hep-2 cells. The intensity of MICA protein expression was decreased with increased clinical stage in laryngeal cancer tissues. MICA may play an important role in laryngeal carcinoma process.

Meta-analysis of the differentially expressed microRNA profiles in nasopharyngeal carcinoma.

MicroRNAs(miRNAs), as non-coding molecules, were proved to be correlated with gene expression in naspharyngeal carcinoma (NPC) development. In this research, a comprehensive meta-analysis of eight independent miRNA expression studies in NPC was preformed by using robust rank aggregation method (RRA), which contained a total of 775 tumor and 227 non-cancerous samples. There were 7 significant dysregulated miRNAs identified including three increased (miR-483-5p, miR-29c-3p and miR-205-5p) and four decreased (miR-29b-3p, let-7d-5p, miR-100- 5p and let-7g-5p) miRNAs. Subsequently, the miRNA target prediction and pathway enrichment analysis were carried out to find out the biological and functional relevant genes involved in the meta-signature miRNA regulation. Finally, several signaling and cancer pathogenesis pathways were suggested to be more frequently associated with the progression of NPC. In this research the meta-signature miRNA identified may be used to develop a series of diagnostic and prognostic biomarkers for NPC that serve specificity for use in clinics.

Expression of Th1- Th2- and Th17-associated cytokines in laryngeal carcinoma.

T-helper (Th) 0 cell differentiation into Th1 or Th2 cells is dependent on a number of transcription factors that act at specific time points to regulate gene expression. Th17 cells, a subset of interleukin (IL)-17-producing T cells distinct from Th1 or Th2 cells, are considered to exhibit a critical function in inflammation and autoimmune diseases, as well as cancer development. In the present study, the expression of Th1-, Th2- and Th17-associated cytokines in laryngeal cancer and pericarcinoma tissues obtained from 57 laryngeal carcinoma patients was investigated. The association between Th1, Th2 and Th17 infiltration and tumor development was also evaluated. Reverse transcription-polymerase chain reaction and western blotting results revealed that the mRNA and protein expression of Th2 cytokines was lower, while the expression of Th1 and Th17 cytokines was higher in tumor tissues than in pericarcinoma tissues. Furthermore, the early stage cancer patients exhibited a higher level of interferon-γ, IL-2 and IL-17 mRNA expression than those at advanced stages. Cancer tissues exhibited higher Th17 cytokine expression than pericarcinoma tissues. By contrast, Th1 cytokine expression was increased in pericarcinoma tissues compared with cancer tissues. These results indicate that high expression of Th1- and Th17-associated cytokines in laryngeal carcinoma may contribute to suppression of cancer development and a relatively good prognosis.

[EFFECT OF FETAL BOVINE SERUM ON OSTEOGENIC GROWTH PEPTIDE PROMOTING BONE MARROW MESENCHYMAL STEM CELLS PROLIFERATION AND DIFFERENTIATION].

OBJECTIVE: To explore the effect of fetal bovine serum (FBS) of different concentrations in the culture medium on osteogenic growth peptide (OGP) promoting bone marrow mesenchymal stem cells (BMSCs) proliferation and differentiation. METHODS: BMSCs were separated from limb bones of 8 Sprague Dawley rats (5 weeks old) and purified by adherence method, and BMSCs at passage 3 were divided into 4 groups according to OGP concentration: OGP 1 x 10(-10) mol/L group, OGP 1 x 10(-9) mol/L group, OGP 1 x 10(-8) mol/L group, and control group without OGP; and 0, 2%, 5%, 8%, and 10% FBS concentration gradient was used in each group. The cell proliferation rate was detected by MTT method at 1, 3, 5, 7, 9, and 12 days after culture, and the activity of intracellular alkaline phosphatase (ALP) was determined by the method of p-nitrophenyl phosphate disodium at 9 days after culture. RESULTS: BMSCs showed adherent growth, rapid proliferation, long fiber vortex, and typical morphology. MTT analysis showed that cells could not sustain proliferation when FBS concentration was less than 5% in each group; when FBS concentration was above 8%, cells proliferated continually. Proliferation promoting effect of OGP 1 x 10(-8) mol/L and 1 x 10(-9) mol/L groups was significantly higher than that of the control group in all serum concentrations (P < 0.05); when FBS concentration was lower than 10%, the proliferation promoting effect of OGP 1 x 10(-8) mol/L group was significantly higher than that of the other 2 OGP groups (P < 0.05), but when FBS concentration was 10%, OGP 1 x 10(-8) mol/L group had no advantage of promoting proliferation. ALP test results showed that as the FBS concentration increased, ALP activity of all groups also significantly increased (P < 0.05). Under the condition of 5% FBS and 8% FBS, the ALP activity of each OGP group was significantly greater than that of the control group, and it was the highest in OGP 1 x 10(-8) mol/L group (P < 0.05). Under the condition of 10% FBS, the ALP activity of each OGP group was still greater than that of the control group (P < 0.05), but no significant difference was found between the OGP 1 x 10(-8) mol/L group and OGP 1 x 10(-9) mol/L group (P > 0.05). CONCLUSION: The concentration of 8% FBS is the best concentration of serum for OGP promoting the proliferation and differentiation of BMSCs, and the most suitable concentration of promoting the proliferation and differentiation of BMSCs is OGP 1 x 10(-8) mol/L.

An LC-MS/MS method for the quantitation of cabozantinib in rat plasma: application to a pharmacokinetic study.

A simple, rapid and sensitive high-performance liquid chromatography tandem mass-spectrometric method (LC-MS/MS) for the novel multiple tyrosine kinase inhibitor (TKI) cabozantinib was developed and validated using erlotinib as internal standard (IS). Plasma samples were pre-treated by liquid-liquid extraction with ethyl acetate. Separation was achieved on a reversed phase C18 column (50×2mm, 5µm) at ambient temperature using isocratic elution with acetonitrile-water (45:55, v/v) containing 5mM ammonium formate buffer (finally adjusted to apparent pH*=5.0 with formic acid) at a flow rate of 0.4mL/min. The analytes were monitored by a triple quadrupole tandem mass spectrometer with electrospray ionization source in the positive ion mode. Calibration curve was linear (r>0.99) in a concentration range of 0.5-1000ng/mL with the lower limit of quantification (LLOQ) of 0.5ng/mL. Intra- and inter-day accuracy and precision were validated by relative error values (RE) and relative standard deviation values (RSD), respectively, which were both lower than the limit of 15%. This method was successfully applied to a pharmacokinetic study of cabozantinib in rats.

Suppression effect of recombinant adenovirus vector containing hIL-24 on Hep-2 laryngeal carcinoma cells.

The melanoma differentiation-associated gene-7 [MDA-7; renamed interleukin (IL)-24] was isolated from human melanoma cells induced to terminally differentiate by treatment with interferon and mezerein. MDA-7/IL-24 functions as a multimodality anticancer agent, possessing proapoptotic, antiangiogenic and immunostimulatory properties. All these attributes make MDA-7/IL-24 an ideal candidate for cancer gene therapy. In the present study, the human MDA-7/IL-24 gene was transfected into the human laryngeal cancer Hep-2 cell line and human umbilical vein endothelial cells (HUVECs) with a replication-incompetent adenovirus vector. Reverse transcription polymerase chain reaction and western blot analysis confirmed that the Ad-hIL-24 was expressed in the two cells. The expression of the antiapoptotic gene, Bcl-2, was significantly decreased and the IL-24 receptor was markedly expressed in Hep-2 cells following infection with Ad-hIL-24, but not in HUVECs. In addition, the expression of the proapoptotic gene, Bax, was induced and the expression of caspase-3 was increased in the Hep-2 cells and HUVECs. Methyl thiazolyl tetrazolium assay indicated that Ad-hIL-24 may induce growth suppression in Hep-2 cells but not in HUVECs. In conclusion, Ad-hIL-24 selectively inhibits proliferation and induces apoptosis in Hep-2 cells. No visible damage was found in HUVECs. Therefore, the results of the current study indicated that Ad-hIL-24 may have a potent suppressive effect on human laryngeal carcinoma cell lines, but is safe for healthy cells.