Andrographolide sensitizes KRAS-mutant colorectal cancer cells to cetuximab by inhibiting the EGFR/AKT and PDGFRß/AKT signaling pathways.

BACKGROUND: Cetuximab, an inhibitor targeting EGFR, is widely applied in clinical management of colorectal cancer (CRC). Nevertheless, drug resistance induced by KRAS-mutations limits cetuximab's anti-cancer effectiveness. Furthermore, the persistent activation of EGFR-independent AKT is another significant factor in cetuximab resistance. Nevertheless, the mechanism that EGFR-independent AKT drives cetuximab resistance remains unclear. Thus, highlighting the need to optimize therapies to overcome cetuximab resistance and also to explore the underlying mechanism. PURPOSE: This work aimed to investigate whether and how andrographolide enhance the therapeutic efficacy of cetuximab in KRAS-mutant CRC cells by modulating AKT. METHODS: The viabilities of CRC cell lines were analyzed by CCK-8. The intracellular proteins phosphorylation levels were investigated by Human Phospho-kinase Antibody Array analysis. Knockdown and transfection of PDGFRß were used to evaluate the role of andrographolide on PDGFRß. The western blotting was used to investigate Wnt/ß-catenin pathways, PI3K/AKT, and EMT in KRAS-mutant CRC cells. The animal models including subcutaneous tumor and lung metastasis were performed to assess tumor response to therapy in vivo. RESULTS: Andrographolide was demonstrated to decrease the expression of PI3K and AKT through targeting PDGFRß and EGFR, and it enhanced cetuximab effect on KRAS-mutant CRC cells by this mechanism. Meanwhile, andrographolide helped cetuximab to inhibit Wnt/ß-catenin, CRC cell migration and reduced Vimentin expression, while increasing that of E-cadherin. Lastly, co-treatment with cetuximab and andrographolide reduced the growth of KRAS-mutant tumors and pulmonary metastases in vivo. CONCLUSIONS: Our findings suggest that andrographolide can overcome the KRAS-mutant CRC cells' resistance to cetuximab through inhibiting the EGFR/PI3K/AKT and PDGFRß /AKT signaling pathways. This research provided a possible theory that andrographolide sensitizes KRAS-mutant tumor to EGFR TKI.

Gut mucosal microbiota profiles linked to colorectal cancer recurrence.

BACKGROUND: Emerging evidence links gut microbiota to various human diseases including colorectal cancer (CRC) initiation and development. However, gut microbiota profiles associated with CRC recurrence and patient prognosis are not completely understood yet, especially in a Chinese cohort. AIM: To investigate the relationship between gut mucosal microbiota profiles and CRC recurrence and patient prognosis. METHODS: We obtained the composition and structure of gut microbiota collected from 75 patients diagnosed with CRC and 26 healthy controls. The patients were followed up by regular examination to determine whether tumors recurred. Triplet-paired samples from on-tumor, adjacent-tumor and off-tumor sites of patients diagnosed with/without CRC recurrence were analyzed to assess spatial-specific patterns of gut mucosal microbiota by 16S ribosomal RNA sequencing. Next, we carried out bioinformatic analyses, Kaplan-Meier survival analyses and Cox regression analyses to determine the relationship between gut mucosal microbiota profiles and CRC recurrence and patient prognosis. RESULTS: We observed spatial-specific patterns of gut mucosal microbiota profiles linked to CRC recurrence and patient prognosis. A total of 17 bacterial genera/families were identified as potential biomarkers for CRC recurrence and patient prognosis, including Anaerotruncus, Bacteroidales, Coriobacteriaceae, Dialister, Eubacterium, Fusobacterium, Filifactor, Gemella, Haemophilus, Mogibacteriazeae, Pyramidobacter, Parvimonas, Porphyromonadaceae, Slackia, Schwartzia, TG5 and Treponema. CONCLUSION: Our work suggests that intestinal microbiota can serve as biomarkers to predict the risk of CRC recurrence and patient death.

Concurrent chemoradiotherapy using gemcitabine and nedaplatin in recurrent or locally advanced head and neck squamous cell carcinoma.

BACKGROUND: Patients with recurrent or locally advanced head and neck squamous cell carcinoma (HNSCC) typically have limited treatment options and poor prognosis. AIM: To evaluate the efficacy and safety of two drugs with potent radio-sensitization properties including gemcitabine and nedaplatin as concurrent chemoradiotherapy regimens in treating HNSCC. METHODS: This single-arm prospective study enrolled patients with HNSCC to receive gemcitabine on days 1 and 8 and nedaplatin on days 1 to 3 for 21 days. Intensity-modulated radiation therapy with a conventional fraction was delivered 5 days per week. Objective response rate (ORR), disease control rate, and toxicity were observed as primary endpoints. Overall survival (OS) and progression free survival were recorded and analyzed as secondary endpoints. RESULTS: A total of 24 patients with HNSCC were enrolled. During the median 22.4-mo follow-up, both ORR and disease control rate were 100%. The one-year OS was 75%, and one-year progression-free survival (PFS) was 66.7% (median PFS was 15.1 mo). Recurrent HNSCC patients had a poorer prognosis than the treatment-naïve patients, and patients who achieved complete response had better survival than those in the PR group (all P < 0.05). The most common grade 1-4 (100%) or grade 3-4 toxicities (75%) were hematological, and the most common grade 3-4 non-hematological toxicity was mucositis in 17 (71%) patients. CONCLUSION: Gemcitabine plus nedaplatin with concurrent chemoradiotherapy is a therapeutic option for HNSCC with predictable tolerability. Considering the high adverse event rate, the optimized dose and schedule must be further explored.

A high-sensitivity electrochemical aptasensor of carcinoembryonic antigen based on graphene quantum dots-ionic liquid-nafion nanomatrix and DNAzyme-assisted signal amplification strategy.

In this work, we have developed an electrochemical aptasensor for high-sensitivity determination of carcinoembryonic antigen (CEA) based on lead ion (Pb2+)-dependent DNAzyme-assisted signal amplification and graphene quantum dot-ionic liquid-nafion (GQDs-IL-NF) composite film. We designed hairpin DNA containing CEA-specific aptamers and DNAzyme chains. In the presence of CEA, hairpin DNA recognized the target and performed a DNAzyme-assisted signal amplification reaction to yield a large number of single-stranded DNA. The GQDs-IL-NF composite film was immobilized on the glassy carbon electrode for the interaction with single-stranded DNA through noncovalent π-π stacking interaction. Therefore, the methylene blue-labeled substrate DNA (MB-substrate) was fixed on the electrode and exhibited an initial electrochemical signal. Under optimal conditions, the response current change was proportional to the concentration of CEA, demonstrating a wide linear range from 0.5fgmL-1 to 0.5ngmL-1, with a low detection limit of 0.34fgmL-1. Furthermore, the proposed aptasensor was successfully applied in determining CEA in serum samples, showing its superior prospects in clinical diagnosis.

Ultrasound­targeted microbubbles combined with a peptide nucleic acid binding nuclear localization signal mediate transfection of exogenous genes by improving cytoplasmic and nuclear import.

The development of an efficient delivery system is critical for the successful treatment of cardiovascular diseases using non­viral gene therapies. Cytoplasmic and nuclear membrane barriers reduce delivery efficiency by impeding the transfection of foreign genes. Thus, a gene delivery system capable of transporting exogenous genes may improve gene therapy. The present study used a novel strategy involving ultrasound­targeted microbubbles and peptide nucleic acid (PNA)­binding nuclear localization signals (NLS). Ultrasound­targeted microbubble destruction (UTMD) and PNA­binding NLS were used to improve the cytoplasmic and nuclear importation of the plasmid, respectively. Experiments were performed using antibody­targeted microbubbles (AT­MCB) that specifically recognize the SV40T antigen receptor expressed on the membranes of 293T cells, resulting in the localization of ultrasound microbubbles to 293T cell membranes. Furthermore, PNA containing NLS was inserted into the enhanced green fluorescent protein (EGFP)­N3 plasmid DNA (NLS­PNA­DNA), which increased nuclear localization. The nuclear import and gene expression efficiency of the AT­MCB with PNA­binding NLS were compared with AT­MCB alone or a PNA­binding NLS. The effect of the AT­MCB containing PNA­binding NLS on transfection was investigated. The ultrasound and AT­MCB delivery significantly enhanced the cytoplasmic intake of exogenous genes and maintained high cell viability. The nuclear import and gene expression of combined microbubble­ and PNA­transfected cells were significantly greater compared with cells that were transfected with AT­MCB or DNA with only PNA­binding NLS. The quantity of EGFP­N3 plasmids in the nuclei was increased by >5.0­fold compared with control microbubbles (CMCB) and NLS­free plasmids. The gene expression was ~1.7­fold greater compared with NLS­free plasmids and 1.3­fold greater compared with control microbubbles. In conclusion, UTMD combined with AT­MCB and a PNA­binding NLS plasmid significantly improved transfection efficiency by increasing cytoplasmic and nuclear DNA import. This method is a promising strategy for the noninvasive and effective delivery of target genes or drugs for the treatment of cardiovascular diseases.

Efficient gene therapy with a combination of ultrasound­targeted microbubble destruction and PEI/DNA/NLS complexes.

Current strategies of gene transfection are not efficient at achieving a notable therapeutic effect. The aim of the present study was to combine ultrasound­targeted microbubble destruction (UTMD) with a polyethylenimine/pEGFP­N3 plasmid/nuclear localization sequence (PEI/DNA/NLS) complex gene delivery system, and evaluate the transfection efficiency of enhanced green fluorescent protein (EGFP) gene delivery to 293T cells using this system. The formation of PEI/DNA/NLS complexes and the protective effects of PEI/NLS were verified by gel electrophoresis. Solutions consisting of the plasmid alone, PEI/DNA complexes, PEI/DNA/NLS complexes, UTMD+DNA, UTMD+PEI/DNA complexes, and UTMD+PEI/DNA/NLS complexes were transduced into 293T cells via ultrasound irradiation. The expression of GFP was observed using an inverted microscope and transfection efficiency was detected by flow cytometry following 24 h incubation in vitro. Cell activity was detected using a Cell Counting kit (CCK)­8 assay. Gel electrophoresis confirmed the formation of PEI/DNA/NLS complexes and demonstrated that PEI/NLS exhibited protective effects on plasmid integrity for a limited time. Inverted microscope observations revealed that a greater GFP signal was observed with the combined action of PEI/DNA/NLS complexes with UTMD, and flow cytometry analysis demonstrated the highest level of transfection efficiency in this group. In addition, the viability of the cells detected by CCK­8 and treated with PEI/DNA/NLS complexes with UTMD was >80%. In conclusion, the combination of UTMD and PEI/DNA/NLS complexes was highly effective for the efficient transfection of 293T cells without causing excessive cell damage. This method may provide a novel and effective gene transduction system to be applied in clinical treatments.

A macrocyclic 1,4-bis(4-pyridylethynyl)benzene showing unique aggregation-induced emission properties.

A box-like macrocycle based on 1,4-bis(4-pyridylethynyl)benzene was derived in high yield. The macrocyclic fluorogen shows unique aggregation-induced emission properties.

[Development of IgHV family specific nucleic acid vaccine against lymphoma by construction of fusion gene of immunoglobulin heavy chain variable region and cytokine].

OBJECTIVE: To construct the gene co-expression vector of immunoglobulin heavy chain variable region (IgHV) gene and GM-CSF or IL-2 as IgHV family specific nucleic acid vaccine to lymphoma, and study the immune response of the immunized mice against lymphoma. METHODS: Six clones with significantly different length in gene fragments were selected from a gene bank constructed from normal fetal umbilical cord blood. These gene fragments in the clones were sequenced. The sequences were translated into IgHV peptides. T cell epitopes in the IgHV were predicted by bioinformatics. Meanwhile 6 clones of IgHV1 constructed before were analyzed. The most typical clone of IgHV1 and IgHV3 containing most of the T cell epitopes were selected. The IgHV gene fragments whose complementary determining region 3 (CDR3) was cut out and composed mainly of framework region were cloned into eukaryotic expression vector. The gene fragments of framework region of IgHV linking with gene of GM-CSF or IL-2 were cloned into pcDNA3.0 to form fusion genes of IgHV (FR)-GM-CSF/IL-2. Then they were transected into COS cells, African green monkey renal cells, by Lipofectin and their transient expression product was detected by ELISA. Twenty male Balb/c mice were randomly divided into 5 groups of 4 mice to be injected with different vectors at the weeks 0, 2, and 4: with IgHV3 (FR)/pcDNA3.0, with IgHV3 (FR)-IL-2/pcDNA 3.0, with blank vector pcDNA3.0 as control, with IgHV1 (FR)/pcDNA3.0, and with IgHV1 (FR)-IL-2/pcDNA3.0. At the weeks 0, 2, 4, 6, and 8 serum was collected from the mice to undergo indirect immunofluorescence examination to detect the antibodies against Namalwa cells, lymphoma cells from Africa green monkey, and normal lymphocytes. ELISA was used to examine the serum interferon (IFN)-gamma. RESULTS: About 30 of T cell epitopes existed in each IgHV peptides predicted by bioinformatics. Among the bioinformatics prediction, about 90% of the T cell epitopes were in the framework region (FR) of IgHV, and the first 10% with higher prediction score were in FR. The CDR3 of two typical IgHV sequences were cut down and the remaining sequences, mainly composed of FR, were used to construct the IgHV (FR)/pcDNA3.0 expression vectors. The 3' end of IgHV1 (FR) and IgHV3 (FR) were linked to GM-CSF or IL-2 gene successfully. The expression of GM-CSF in the group transfected with IgHV (FR)-GM-CSF/pcDNA3.0 were 200 times higher than in the group transfected with control pcDNA3.0. The expression of IL-2 in the group transfected with IgHV (FR)-IL-2/pcDNA3.0 were 60 times higher than in the group transfected with control pcDNA3.0. The antibody against IgHV1 family lymphoma cell line Namalwa could be detected since the second week in the mice immunized with IgHV1 (FR)-IL-2/pcDNA3.0. The antibody could be detected since the fourth week in the mice immunized with IgHV1 (FR). The antibody against lymphocyte line of IgHV3 family could be detected since the second week in the mice immunized with IgHV3 (FR)-IL-2/pcDNA3.0. The antibody could be detected since the fourth week in the mice immunized with IgHV3 (FR)/pcDNA3.0. The contents of serum IFN-gamma the mice immunized with IgHV1 (FR)-IL-2/pcDNA3.0 and with IgHV3 (FR)-IL-2/pcDNA3.0 was significantly higher than those of the mice immunized with IgHV (FR)/pcDNA3.0 or pcDNA3.0 (all P < 0.01). CONCLUSION: The gene fragments of IgHV (FR) can be used to construct IgHV family specific nucleic acid vaccine that induces anti-lymphoma immune response in mice by muscle injection. The expressing vectors of IgHV (FR)-GM-CSF/IL-2 induces stronger immunoreaction.

[The variation of T-cell clonal repertoire in patients with systemic lupus erythematosus (SLE) following autologous peripheral blood hematopoietic stem cell transplantation].

OBJECTIVE: Analyze the variation of T cell receptor(beta) (TCRbeta) CDR3 gene expressing repertoire in systemic lupus erythematosus (SLE) patients before and after autologous peripheral blood hematopoietic stem cell transplantation (APBSCT), to search the pathogenesis of SLE and characteristics of T cell reconstitute immune after APBSCT. METHODS: Reverse transcriptase - polymerase chain reaction (RT-PCR) amplified 25 subfamily genes of TCRbeta prom peripheral blood lymphocytes (PBLs) of SLE patient with APBSCT and run denaturation polyacrylamide sequencing gel electrophoresis, set up a map of TCRbetaCDR3 gene expressing repertoire. The bands of TCRbeta gene repertoire in the electrophoresis gel can be used to analyze the variety of T cell clones which expanded and disappeared in cases of SLE. Cut the bands and sequencing. Compare sequences of TCRbetagenes in normal and abnormal T cell clones to understand the relationship between TCRbeta gene and autoimmune diseases. RESULTS: In the normal control group, TCRbeta gene repertoires show more than 10 bands in each of 25 TCRbetaV families, characterized as Gaussian distribution. TCRbeta gene repertoire in the 8 patients with SLE were abnormally. Four patients expressed TCRbetaV13.1 gene preferentially. Three patients had TCRbetaV8, TCRbetaV9 and TCRbetaV18 genes over expressed. These over expressive genes represented the oligoclonal expansion of T cell populations. After APBSCT the TCRbeta gene repertoires regained CDR3 polymorphism in 5 patients, some of them recovered to normal completely. The sequencing analyse show dense and strong band in map of TCRbeta gene repertoires were T cell monoclonal or oligoclonal expansion frequently. In a case of SLE an amino acid sequence of TCRbetaV8 CDR3 was same to a sequence of disease correlative T cell clone with ankylosing spondylitis that had been reported in a literature before. CONCLUSION: SLE patients have oligoclonal expansion of T cell that related with the pathogenesis of autoimmune disease. After APBSCT, T cell clones recover to normal distribution. T cell clones that related with disease have been suppressed during immunological reconstitution. It may be a main reason the SLE get remission after transplantation.

[CIK cells acquired multidrug resistance and maintained cytotoxic activity to tumor cells after mdr1 gene transfection].

OBJECTIVE: To investigate whether the cytokine-induced killer (CIK) cells could acquire multidrug resistance and maintain the original cytotoxic activity after multidrug resistance (mdr1) genes transfection. METHODS: CIK cells were generated from peripheral blood cultured with IFN-gamma, CD(3) monoclonal antibody, IL-2, IL-1 and transfected with a plasmid (pHamdr) containing human mdr1 gene via electroporation. RT-PCR method was used to assay mRNA expression of mdr1 gene in transfected CIK cells, flow cytometry with anti-P-gp monoclonal antibody to detect P-glycoprotein (P-gp) expression on CIK cells membrane, and MTT assay to compare both the multidrug resistance to doxorubicin and colchicines and cytotoxic activity to human mammary cancer cell line MCF7 between transfected and non-transfected CIK cells. RESULTS: mdr1 expression was detected in the transfected CIK cells. There was a strong expression of P-gp on the transfected CIK cells and the percentages of P-gp positive cells were 21% - 37% (average 27%). The IC(50) of transfected CIK cells to doxorubicin was 22.3 - 45.8 times and 6.7 - 11.35 times to colchicines of those of non-transfected CIK cells. The cytotoxic activity to MCF7 remained unchanged (P > 0.05). CONCLUSION: It demonstrated that CIK cells transfected with mdr1 gene via electroporation could express multidrug resistance successfully without changes of cytotoxic activity.