Clinical efficacy analysis of mesenchymal stem cell therapy in patients with COVID-19: A systematic review.

BACKGROUND: Currently, ongoing trials of mesenchymal stem cells (MSC) therapies for coronavirus disease 2019 (COVID-19) have been reported. AIM: In this study, we investigated whether MSCs have therapeutic efficacy in novel COVID-19 patients. METHODS: Search terms included stem cell, MSC, umbilical cord blood, novel coronavirus, severe acute respiratory syndrome coronavirus-2 and COVID-19, applied to PubMed, the Cochrane Controlled Trials Register, EMBASE and Web of Science. RESULTS: A total of 13 eligible clinical trials met our inclusion criteria with a total of 548 patients. The analysis showed no significant decrease in C-reactive protein (CRP) levels after stem cell therapy (P = 0.11). A reduction of D-dimer levels was also not observed in patients after stem cell administration (P = 0.82). Furthermore, interleukin 6 (IL-6) demonstrated no decrease after stem cell therapy (P = 0.45). Finally, we investigated the overall survival (OS) rate after stem cell therapy in COVID-19 patients. There was a significant improvement in OS after stem cell therapy; the OS of enrolled patients who received stem cell therapy was 90.3%, whereas that of the control group was 79.8% (P = 0.02). CONCLUSION: Overall, our analysis suggests that while MSC therapy for COVID-19 patients does not significantly decrease inflammatory markers such as CRP, D-dimer and IL-6, OS is improved.

The incidence of cytokine release syndrome and neurotoxicity of CD19 chimeric antigen receptor-T cell therapy in the patient with acute lymphoblastic leukemia and lymphoma.

Our objective was to summarize the side effect of chimeric antigen receptor (CAR)-T cell therapy in patients with acute lymphocytic leukemia (ALL) and lymphoma. Two independent reviewers extracted relevant data. A total of 35 hematologic malignancy studies with CD19 CAR-T cell were included (1412 participants). Severe cytokine release syndrome (sCRS) proportion was experienced by 18.5% (95% confidence interval [CI], 0.128-0.259; P = 0.000) of 982 patients with the National Cancer Institute/Lee/common terminology criteria for adverse events grading system. The pooled neurotoxicity proportion was 21.7% (95% CI, 0.167-0.287; P = 0.000) of 747 patients with the same grading system. For all of the 25 clinical trials with the same grading system, subgroup analysis was performed. Based on the different disease type, a pooled prevalence of 35.7% was observed with event rate (ER) of 0.358 (95% CI, 0.289-0.434; P = 0.000) for ALL in 12 clinical trials. For lymphoma, a pooled prevalence of 13% was observed with ER of 0.073 (95% CI, 0.028-0.179; P = 0.000) in eight clinical trials. It was demonstrated that the patients who were older than 18 years of age have the lower sCRS incidence of 16.1% (95% CI, 0.110-0.250; P = 0.000) compared with 28.6% of the remaining population who were younger than 18 years of age (95% CI, 0.117-0.462: P = 0.023) in our analysis. Based on the different co-stimulatory domain, the sCRS of 16.5% was observed with ER of 0.175 (95% CI, 0.090-0.312; P = 0.000) for 4-1BB. The sCRS of 22.2% was observed with ER of 0.193 (95% CI, 0.107-0.322; P = 0.000) for CD28. For both the CD28 and 4-1BB, the sCRS of 17.3% was observed with ER of 0.170 (95% CI, 0.067-0.369; P = 0.003). Sub-analysis sCRS of the impact with cell dose and specific disease indication were also demonstrated. Limitations include heterogeneity of study populations, as well as high risk of bias of included studies. These results are helpful for physicians, patients and the other stakeholders to understand the adverse events and to further promote the improvement of CAR-T cell therapy in the future.

Assessment of the efficacy of passive cellular immunotherapy for glioma patients.

To evaluate the therapeutic efficacy of passive cellular immunotherapy for glioma, a total of 979 patients were assigned to the meta-analysis. PubMed and the Cochrane Central Register of Controlled Trials were searched initially from February 2018 and updated in April 2019. The overall survival (OS) rates and Karnofsky performance status (KPS) values of patients who underwent passive cellular immunotherapy were compared to those of patients who did not undergo immunotherapy. The proportion of survival rates was also evaluated in one group of clinical trials. Pooled analysis was performed with random- or fixed-effects models. Clinical trials of lymphokine-activated killer cells, cytotoxic T lymphocytes, autologous tumor-specific T lymphocytes, chimeric antigen receptor T cells, cytokine-induced killer cells, cytomegalovirus-specific T cells, and natural killer cell therapies were selected. Results showed that treatment of glioma with passive cellular immunotherapy was associated with a significantly improved 0.5-year OS (p = 0.003) as well as improved 1-, 1.5-, and 3-year OS (p ≤ 0.05). A meta-analysis of 206 patients in one group of clinical trials with 12-month follow-up showed that the overall pooled survival rate was 37.9% (p = 0.003). Analysis of KPS values demonstrated favorable results for the immunotherapy arm (p < 0.001). Thus, the present meta-analysis showed that passive cellular immunotherapy prolongs survival and improves quality of life for glioma patients, suggesting that it has some clinical benefits.

Advances in precise regulation of CRISPR/Cas9 gene editing technology.

Gene editing is a genetic engineering technology that can modify, delete, or insert a small piece of DNA at a specific point in the genome of cells and organisms. Gene editing technology holds great promises in the fields of disease treatment, gene function regulation, gene detection, drug research and development, and crop breeding. However, side effects, such as off-target editing, genotoxicity and other issues, have gradually emerged in the application. In the CRISPR (clustered regularly interspaced short palindromic repeats) system, the Cas9 nuclease can specifically recognize the target DNA by the base pairing of a guide RNA (gRNA) with the target DNA. Upon target recognition, the two DNA strands are cleaved by distinct domains of the Cas9 nuclease. Since both Cas9 nuclease and gRNA possess different characteristics in their own activities, recognition sites and binding ability to specific target, it is essential to precisely regulate the activity of Cas9 nuclease and gRNA in both time and space manners, thus preventing the risk of side effects and enhancing the precise regulation of the CRISPR/Cas9 gene editing technology. In this review, we summarize the advances in the precise control of gene editing, especially CRISPR/cas9 over several dimensions using fusion Cas9 proteins regulated by light, temperature and drugs, exploiting and screening anti-CRISPRs proteins, synthesizing and identifying small molecules- inhibitors, and developing other therapeutic agents, thereby providing a reference and research ideas for human disease treatment, crop and livestock improvement and prevention of biotechnology misuse.

The efficacy of anti-CD19 chimeric antigen receptor T cells for B-cell malignancies.

Immunotherapy with chimeric antigen receptor T (CAR-T) cells has proved remarkably effective in recently published clinical trials. In this meta-analysis, we performed a systematic review in terms of the clinical response treated with CAR-T cells in acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL) and lymphomas patients. Thirty-eight published clinical studies including 665 patients were eligible for response rate (RR) evaluation. The overall pooled RR of CD19-CAR-T cells was 72% (95% confidence interval: 62-77%). The various clinical parameters were analyzed. RR was 81% in ALL, 68% in lymphoma and 70% in CLL. RR in patients who received interleukin (IL)-2 was 70%, whereas in those who did not receive IL-2, it was 74%. RR was 75% with lymphodepletion and 56% without lymphodepletion. RR with autologous cells was 76% and 57% with allogeneic cells. In conclusion, this meta-analysis showed a high clinical RR of CD19-CAR-T cell-based immunotherapy in patients with refractory B-cell malignancies.

Validity of combination active specific immunotherapy for colorectal cancer: a meta-analysis of 2993 patients.

BACKGROUND AIMS: The aim of this study was to investigate whether active specific immunotherapy (ASI) is able to demonstrate therapeutic efficacy against colorectal cancer. METHODS: We conducted a systematic review of published papers from MEDLINE, the Cochrane Central Register of Controlled Trials, EMBASE, the Wanfang Database, the China Science and Technology Periodical Database and China Journal Net. Published data were extracted independently by two authors who used predefined database templates. The effects of ASI were compared with those of surgery alone, and a pooled analysis was performed with the use of the data from random- or fixed-effect models. RESULTS: Twelve trials matched our inclusion criteria (n = 2993, including 1842 control subjects). The overall analysis showed a significant survival benefit [1-, 2-, 3-, 4-, 5-, 6- and 7-year overall survival (OS), P < 0.05; 10-year OS, P < 0.001] in favor of ASI immunotherapy combined with surgery, but there was not an improvement in the 8- or 9-year OS (P > 0.05). The disease-free survival (DFS) rate was improved after the combination of ASI immunotherapy (2-, 3-, 5- and 10-year DFS, P < 0.05), but no significant improvement was noted for the 1-, 4-, 6-, 7-, 8- or 9-year DFS (P > 0.05). In addition, the disease-specific survival (DSS) was improved at some time points after the combination of ASI immunotherapy and surgery (2-, 3-, 4-, 5- and 6-year DSS, P < 0.05, but not the 1-, 7-, 8- or 9-year DSS, P > 0.05). An improved 2-, 3-, 4-, 5- and 6-year recurrence-free interval (RFI) (P < 0.05) was also observed in patients who received ASI therapy, but this was not observed for the 1-year RFI (P > 0.05). Furthermore, an analysis of the recurrence-free survival (RFS) showed that it was significantly increased in the ASI plus surgery group (1-, 2-, 3-, 4-, 5- and 6-year RFS, P < 0.001). The funnel plots showed that the analyses were relatively reliable and the publication bias was small. CONCLUSIONS: The combination of ASI immunotherapy and surgery was superior in prolonging the overall survival time and enhancing the recurrence-free survival rate compared with surgery alone.

Evaluation of tumour vaccine immunotherapy for the treatment of advanced non-small cell lung cancer: a systematic meta-analysis.

OBJECTIVES: Our meta-analysis performed a systematic evaluation on the therapeutic efficacy and safety of tumour vaccines for the treatment of advanced non-small cell lung cancer (NSCLC). DESIGN: Systematic review and meta-analysis of randomised controlled trials (RCT). DATA SOURCES: PubMed, the Cochrane Center Register of Controlled Trials, Science Direct and EMBASE were searched from January 1980 until January 2015. ELIGIBILITY CRITERIA FOR SELECTING STUDIES: RCT were included; the control arm had to receive either placebo or chemotherapy or no treatment. MAIN OUTCOME MEASURES: The quality of the data from individual papers was assessed for overall survival (OS), clinical response rate and side effects. RESULTS: Overall, 11 RCT of advanced NSCLC with a total of 3986 patients were conducted for meta-analysis. The results showed that the vaccine arm significantly extended primary endpoint median overall survival compared with control group (p<0.00001) (HR 0.760; 95% CI 0.644 to 0.896; p=0.001). Three subgroup patients with tumour vaccine at 1-year, 2-year and 3-year survival rates also gained significant benefits compared with their corresponding control group (p=0.0004, 0.03 and 0.19, respectively). Besides, a significant improvement in median time to progression (TTP), median progression-free survival (PFS) and a trend of improvement in objective response rate were observed after tumour vaccine treatment (p=0.001, 0.005 and 0.05, respectively; median PFS HR 0.842; 95% CI 0.744 to 0.954; p=0.007). A few severe adverse effects occurred in the tumour vaccine group, but fewer side effects were observed in the vaccine group compared with the control group (p<0.00001). CONCLUSIONS: Taken together, NSCLC tumour vaccines markedly prolong median OS (p<0.00001), median TTP (p=0.001) and median PFS (p=0.005), improve clinical response rate (p=0.05) and lessen adverse side effects (p<0.00001). Our meta-analysis suggests tumour vaccines improve the efficacy of the treatment, and also provide superiority in treatment of patients with advanced NSCLC among a variety of immunotherapy strategies.

Clinical efficacy of autologous stem cell transplantation for the treatment of patients with type 2 diabetes mellitus: a meta-analysis.

BACKGROUND AIMS: In this study, we investigate whether bone marrow mononuclear cells (BM-MNC) or peripheral blood mononuclear cells (PB-MNC) have therapeutic efficacy in type 2 diabetes (T2D). METHODS: Search terms included stem cell, bone marrow cell, peripheral blood cell, umbilical cord blood and T2D in MEDLINE, the Cochrane Controlled Trials Register, EMBASE, the Wanfang Database, the China Science and Technology Periodical Database and China Journal Net. RESULTS: Fifteen trials met our inclusion criteria (n = 497). One group included 266 cases with BM-MNC therapy and the other group contained 231 cases with PB-MNC treatment. Glycosylated hemoglobin was decreased after BM-MNC or PB-MNC therapy compared with that before (12 months: P < 0.001; 6 months: P < 0.001; 3 months: P < 0.05). Fasting plasma glucose was reduced in BM-MNC therapy group compared with control after 12-month follow-up (P < 0.001) and after BM-MNC therapy compared with that before (9 months: P < 0.001) but was not obvious in other stages. Meanwhile, the analysis showed that C-peptide level increased after BM-MNC and PB-MNC therapy compared with the control therapy (12 months: P < 0.001) and with that before therapy (6 months: P < 0.05). Insulin requirement reduction was also observed in patients receiving BM-MNC therapy (3, 6, 9 and 12 months: P < 0.05). CONCLUSIONS: To a certain extent, BM-MNC or PB-MNC therapy for T2D demonstrated superiority of glycemic control, increased insulin biosynthesis and elevated insulin secretion from existing ß-cells and might prevent islet cell loss.

Adoptive immunotherapy of cytokine-induced killer cell therapy in the treatment of non-small cell lung cancer.

AIM: The aim of this study was to systemically evaluate the therapeutic efficacy of cytokine-induced killer (CIK) cells for the treatment of non-small cell lung cancer. MATERIALS AND METHODS: A computerized search of randomized controlled trials for CIK cell-based therapy was performed. The overall survival, clinical response rate, immunological assessment and side effects were evaluated. RESULTS: Overall, 17 randomized controlled trials of non-small cell lung cancer (NSCLC) with a total of 1172 patients were included in the present analysis. Our study showed that the CIK cell therapy significantly improved the objective response rate and overall survival compared to the non-CIK cell-treated group. After CIK combined therapy, we observed substantially increased percentages of CD3+, CD4+, CD4+CD8+, CD3+CD56+ and NK cells, whereas significant decreases were noted in the percentage of CD8+ and regulatory T cell (Treg) subgroups. A significant increase in Ag-NORs was observed in the CIK-treated patient group (p = 0.00001), whereas carcinoembryonic antigen (CEA) was more likely to be reduced to a normal level after CIK treatment (p = 0.0008). Of the possible major side effects, only the incidence of fever in the CIK group was significantly higher compared to the group that received chemotherapy alone. CONCLUSION: The CIK cell combined therapy demonstrated significant superiority in the overall survival, clinical response rate, and T lymphocytes responses and did not present any evidence of major adverse events in patients with NSCLC.

Clinical efficacy of tumor antigen-pulsed DC treatment for high-grade glioma patients: evidence from a meta-analysis.

BACKGROUND: The effectiveness of immunotherapy for high-grade glioma (HGG) patients remains controversial. To evaluate the therapeutic efficacy of dendritic cells (DCs) alone in the treatment of HGG, we performed a systematic review and meta-analysis in terms of patient survival with relevant published clinical studies. MATERIALS AND METHODS: A total of 409 patients, including historical cohorts, nonrandomized and randomized controls with HGG, were selected for the meta-analysis. RESULTS: The treatment of HGG with DCs was associated with a significantly improved one-year survival (OS) (p<0.001) and 1.5-, 2-, 3-, 4-, and 5-year OS (p<0.001) compared with the non-DC group. A meta-analysis of the patient outcome data revealed that DC immunotherapy has a significant influence on progression-free survival (PFS) in HGG patients, who showed significantly improved 1-,1.5-, 2-, 3- and 4-year PFS (p<0.001). The analysis of Karnofsky performance status (KPS) demonstrated no favorable results for DC cell therapy arm (p = 0.23).The percentages of CD3+CD8+ and CD3+CD4+ T cells and CD16+ lymphocyte subset were not significantly increased in the DC group compared with the baseline levels observed before treatment (p>0.05), whereas CD56+ lymphocyte subset were significantly increased after DC treatment (p = 0.0001). Furthermore, the levels of IFN-γ in the peripheral blood of HGG patients, which reflect the immune function of the patients, were significantly increased after DC immunotherapy (p<0.001). CONCLUSIONS: Thus, our meta-analysis showed that DC immunotherapy markedly prolongs survival rates and progression-free time, enhances immune function, and improves the efficacy of the treatment of HGG patients.

Cytokine-induced killer cells in the treatment of patients with renal cell carcinoma: a pooled meta-analysis.

Cytokine-induced killer cells (CIKs) have been applied in multifarious cancer. Here, we address the connection between immune therapy and clinical responses by a systematic meta-analysis. A total of 385 patients (including 183 controls) were identified for renal cell cancer (RCC) in the seven selected trials. The estimated pooled complete response and partial response showed a significant improvement for patients receiving CIK immunotherapy compared with non-CIK therapy (p < 0.0001), which was up to 62% of clinical response. The overall analysis showed a significant survival benefit (1-year overall survival [OS]: p = 0.0002; 3-year OS: p < 0.0001) in favor of CIK-based therapy in RCC, thus a statistically significant effect of OS and clinical response was demonstrated in RCC patients.

Efficacy of autologous bone marrow mononuclear cell therapy in patients with peripheral arterial disease.

AIM: Peripheral arterial disease (PAD), particularly critical limb ischemia (CLI), is a severe cause of amputation and mortality. More than 50% of diabetic patients with CLI die within four to five years. The development of novel stem cell therapies may bring new hope to these patients. We aimed to assess the efficacy of autologous bone marrow cell therapy for treating CLI using a meta-analysis. METHODS: We searched the literature in PubMed, the Cochrane Central Registry of Controlled Trials, the Elsevier database and EBSCO for trials of autologous cell therapy in patients with severe PAD published before October 30, 2013. We chose objective clinical endpoints to assess the efficacy of therapy in the meta-analysis, including changes in the ankle-brachial index (ABI), transcutaneous oxygen tension (TcO2), pain scale (0-10 scale) and amputation-free survival (AFS). RESULTS: Thirty-one articles reporting clinical trials involving a total of 1,214 patients treated with bone marrow stem cell-based therapy were collected for the meta-analysis, in which the randomized controlled trials (RCTs) and other trials (non-RCTs) were classified into two groups. Regarding the efficacy of stem cell therapy, the ABI showed significant increases (P＜0.05) at 12 , 24 and 48 weeks after therapy in the non-RCT and RCT groups, but not after four to eight weeks in the non-RCT group. The TcO2 values also increased in the RCT group at four to eight weeks after therapy and 24 weeks after therapy (P＜0.001) and in the non-RCT group at four to eight weeks after therapy (P= 0.01), although no significant increases were observed in the RCT group at 12 weeks after therapy or the non-RCT group at 24 weeks after therapy. Meanwhile, pain was significantly reduced (P＜0.05) at four to eight weeks and 24 weeks after therapy in both the non-RCT and RCT groups, but not at four to eight weeks or 12 weeks after therapy in the RCT group. In addition, the long-term clinical trials demonstrated that the AFS rate improved after therapy with bone marrow stem cells (one-year AFS, P＜0.00001; three-year AFS, P=0.0003). CONCLUSIONS: The present results suggest that autologous bone marrow stem cells have an advantageous therapy effect in PAD patients who are not eligible for revascularization.

Adoptive cellular immunotherapy for the treatment of patients with breast cancer: a meta-analysis.

BACKGROUND: To evaluate the therapeutic efficacy of dendritic cells (DC) alone, cytokine-induced killer (CIK) cells alone and the combination of DC and CIK cells in the treatment of breast cancer, we performed a systemic review of the relevant published clinical studies, collectively referred to as DC-CIK cell therapy. METHODS: Six hundred thirty-three patients with breast cancer were assigned to cohorts, and a meta-analysis was conducted. RESULTS: The treatment of breast cancer with DC-CIK cells was associated with a significantly improved 1-year survival (P = 0.0001). The Karnofsky performance status scale of the patients treated with DC-CIK cells was significantly improved compared with that of the non-DC-CIK group (P < 0.0001). The percentage of T cells (CD3(+), CD4(+) and CD4(+)CD8(+)), CD16(+) monocytes, and CD3(+)CD56(+) natural killer T cells in the peripheral blood of cancer patients was significantly increased (P ≤ 0.05), whereas the percentage of CD4(+)CD25(+) regulatory T cells was not significantly decreased (P = 0.32) in the DC-CIK treatment group compared with the non-DC-CIK group. The levels of interleukin-2, interleukin-12, tumor necrosis factor-α, interferon-γ, and nucleolar organizer region protein in the peripheral blood of cancer patients, which reflect immune function, were significantly increased (P < 0.001) after DC-CIK cell treatment. Furthermore, after DC-CIK treatment, the average levels of the alpha-fetoprotein, cancer antigen embryonic antigen and carbohydrate antigen tumor markers were decreased (P < 0.00001). CONCLUSIONS: DC-CIK cell therapy markedly prolongs survival time, enhances immune function, and improves the efficacy of the treatment of breast cancer patients.

Combination of chemotherapy and immunotherapy for colon cancer in China: a meta-analysis.

AIM: To investigate whether autologous dendritic cell (DC)-cytokine-induced killer (CIK) cell therapy is able to improve the therapeutic efficacy of chemotherapy in colon cancer. METHODS: We conducted a systematic review of published papers from the sources of MEDLINE, the Cochrane Central Register of Controlled Trials, EMBASE, the Wanfang Database, the China Science and Technology Periodical Database and China Journal Net. Published data were extracted independently by two authors using predefined database templates. The quality of the data from individual papers was also assessed. The effects of chemotherapy were compared with those of chemotherapy in combination with DC-CIK immunotherapy. The pooled analysis was performed using the data from random or fixed-effect models. RESULTS: Seven trials matched our inclusion criteria (n = 533). The overall analysis showed significant survival benefit [one-year overall survival (OS), P < 0.0001; two-year OS, P = 0.009; three-year OS, P = 0.002] in favor of DC-CIK immunotherapy combined with chemotherapy. Disease-free survival (DFS) rate was improved after the combination of DC-CIK immunotherapy and chemotherapy (one-year DFS, P < 0.0001; two-year DFS, P = 0.002; three-year DFS, P = 0.02). An improved overall response rate (P = 0.009) was also observed in patients who received DC-CIK therapy. Furthermore, the analysis of T-lymphocyte subsets in peripheral blood indicated that the number of CD4⁺ T cells significantly increased in the DC-CIK plus chemotherapy group (P < 0.05). CONCLUSION: The combination of DC-CIK immunotherapy and chemotherapy was superior in prolonging the survival time and enhancing immunological responses.

Pluripotency-associated genes in human nasopharyngeal carcinoma CNE-2 cells are reactivated by a unique epigenetic sub-microenvironment.

BACKGROUND: There is increasing evidence that cancers contain their own stem-like cells, and particular attention has been paid to one subset of cancer-stem cells termed side population (SP). Stem cells under normal physical conditions are tightly controlled by their microenvironment, however, the regulatory role of the microenvironment surrounding cancer stem cells is not well characterized yet. In this study we found that the phenotype of SP can be "generated" by macrophage-like cells under conditioned culture. Furthermore the gene regulation pathway involved in cellular reprogramming process was investigated. METHODS: The selection and identification of SP in 50 CNE-2 single cell clones were performed by flow cytometry. The transwell assay and immunofluorescence staining were used to measure migration and cancer stem cell characters of non-SP single clone cells cultured with conditioned medium respectively. The subtraction suppression hybridization (SSH) technique and northern blotting analysis was applied to explore the pluripotency-associated genes under a unique epigenetic sub-microenvironment. RESULTS: Among 50 clones, only one did not possess SP subpopulation while others did. The non-SP cells induced by macrophage-like cells showed more aggressive characters, which increased cell migration compared with the control cells and showed some fraction of SP phenotype. These cells expressed distinguished level of pluripotency-associated genes such as ADP-ribosylation factor-like 6 interacting protein (ARMER), poly (rC) binding protein 1 (PCBP1) and pyruvate dehydrogenase E1-beta subunit (PDHB) when subjected to the environment. CONCLUSION: To our knowledge, this is the first study to demonstrate that non-SP single-clone cells can be induced to generate a SP phenotype when they are cultured with conditioned medium of macrophage-like cells, which is associated with the reactivation of pluripotency-associated genes.

Requirement of aurora-A kinase in astral microtubule polymerization and spindle microtubule flux.

Mitotic Aurora-A kinase was found to be required for formation of bipolar spindle, ensuring accurate chromosome segregation in mitosis. Recently, Aurora-A was shown to promote Ran-GTP-induced spindle formation and astral microtubule development. Here, by selective immunodepletion, we showed that Aurora-A was required for centrosome- but not Ran-GTP-induced astral microtubule formation in Xenopus egg extracts. Aurora-A enhanced microtubule polymerization in both centrosome- and Ran-GTP-induced aster assemblies: shortening the timing of aster assembly and increasing the aster size. Indeed, adding of Aurora-A protein alone induced microtubule clustering, which was abrogated by Aurora kinase inhibitory small molecule ZM447439. In addition, we showed that Aurora-A was indispensable for Ran-GTP-induced bipolar spindle formation. Inhibition of Aurora-A activity by adding of kinase inactive dominant mutant led to spindle collapse and formation of monopolar spindle whereas minus-end motor protein dynein/dynactin inhibitor p50/dynamitin rescued the bipolar structure. Lastly, we revealed that Aurora-A was necessary for microtubule poleward flux and this requirement depended on kinase activity. Thus, we showed that Aurora-A promoted microtubule polymerization and maintained microtubule flux in ensuring proper bipolar spindle assembly.

ZM 447439 inhibition of aurora kinase induces Hep2 cancer cell apoptosis in three-dimensional culture.

Mitotic Aurora kinases are essential for accurate chromosome segregation during cell division. Forced overexpression of Aurora kinase results in centrosome amplification and multipolar spindles, causing aneuploidy, a hallmark of cancer. ZM447439 (ZM), an Aurora selective ATP-competitive inhibitor, interferes with the spindle integrity checkpoint and chromosome segregation. Here, we showed that inhibition of Aurora kinase by ZM reduced histone H3 phosphorylation at Ser10 in Hep2 carcinoma cells. Multipolar spindles were induced in these ZM-treated G(2)/M-arrested cells with accumulation of 4N/8N DNA, similar to cells with genetically suppressed Aurora-B. Cells subsequently underwent apoptosis, as assessed by cleavage of critical apoptotic associated protein PARP. Hep2 cells formed a tumor-like cell mass in 3-dimensional matrix culture; inhibition of Aurora kinase by ZM either destructed the preformed cell mass or prevented its formation, by inducing apoptotic cell death as stained for cleaved caspase-3. Lastly, ZM inhibition of Aurora kinase was potently in association with decrease of Akt phosphorylation at Ser473 and its substrates GSK3alpha/beta phosphorylation at Ser21 and Ser9. Together, we demonstrated that Aurora kinase served as a potential molecular target of ZM for more selective therapeutic cancer treatment.

A male reproduction-related Kazal-type peptidase inhibitor gene in the prawn, Macrobrachium rosenbergii: molecular characterization and expression patterns.

Peptidase inhibitors in the male reproductive tract are well known in mammals, in which they play roles in protecting the tract epithelium against proteolytic damage or in regulating the fertilization process. By screening the subtracted cDNA clones enriched for male reproductive tract-specific transcripts, one clone encoding a putative protein that showed significant similarity to Kazal-type peptidase inhibitor (KPI) was obtained. This is the first report of an invertebrate in which a male reproductive tract-specific KPI gene has been identified and characterized. The gene contains a 405-bp open reading frame (ORF), a 72 bp 5' untranslated region (UTR), and a 259 bp 3' UTR. The conceptually translated protein consisted of a 21-amino-acid signal peptide and a 113-amino-acid mature polypeptide with two Kazal-type domains (named after the discoverer). Significant levels of the mRNA were observed only in the male reproductive tract, while mRNA expression was not detected in any other tissues tested. The transcription of the gene remained constant during maturation, although not in the postlarval stage. In situ hybridization demonstrated the presence of the mRNA in the secretory epithelial cells of vas deferens and terminal ampullae.

Molecular cloning and characterization of FGLamide allatostatin gene from the prawn, Macrobrachium rosenbergii.

Allatostatins are important regulatory neuropeptides that inhibit juvenile hormone (JH) biosynthesis by the corpora allata (CA) in insects. However, to date, the structure and expression of the gene encoding allatostatins have not been reported in any species other than insects. In this study, we used a combination of a semi-nested polymerase chain reaction (PCR) and screening of a central nervous system cDNA library of Macrobrachium rosenbergii to isolate and sequence a cDNA clone (2885 bp) encoding a 701 amino acid FGLamide allatostatin precursor polypeptide. This is the first reported allatostatin gene in crustacean. The deduced precursor was conceptually split into at least 35 FGLamide allatostatins at dibasic cleavage sites (Lys and Lys/Arg), far more than reported for any other known FGLamide allatostatin precursors from insects (13-14 allatostatins). Reverse transcription-polymerase chain reaction (RT-PCR) analysis demonstrated that the gene was expressed in the brain, gut, thoracic and abdominal ganglia, but not in the heart, muscle, ovary, gill, or hepatopancreas. Furthermore, developmentally-dependent expression of the gene was observed in the brain and thoracic ganglia of the prawn by using semi-quantitative RT-PCR analysis.

Identification of a novel male reproduction-related gene and its regulated expression patterns in the prawn, Macrobrachium rosenbergii.

To identify male-specific genes that could be involved in male development, we screened a subtracted male reproductive tract library and isolated a novel gene named Mar-Mrr (M. rosenbergii male reproduction-related gene). The Mar-Mrr cDNA sequence consists of 683 nucleotides with a 333 nucleotide open reading frame, encoding putative 110 amino acids (11.7473 kDa) precursor protein and a signal peptide consisting of 24 amino acids. Significant developmentally dependent accumulation of the mRNA was observed in the male reproductive tract, specifically in epithelial cells of vas deferens and terminal ampullae.