CRISPR/Cas9-mediated Knockout of KIFC1 Inhibits Proliferation and Induces Apoptosis of Cervical Cancer Cells / 肿瘤防治研究

Objective To investigate the functions of the KIFC1 gene in tumor cells and its effect on the proliferation of cervical cancer cells. Methods We designed sgRNAs targeting the KIFC1 gene and constructed a recombinant plasmid based on the pSpCas9 (BB)-2A-GFP vector, which was co-transfected into HeLa cells. We screened monoclonal knockout cell lines through flow cytometry sorting, limited dilution inoculation of cells, and sequencing. RT-qPCR, Western blot, and immunofluorescence were used to detect the transcription and protein expression levels of KIFC1 in knockout cells. Cell phenotypes such as nucleus and microtubule cytoskeleton were observed using phase-contrast microscopy and fluorescence confocal microscopy. Cell proliferation, cell cycle, and apoptosis were analyzed by growth curve plotting, EdU labeling, and acridine orange staining. Results The deletion of the KIFC1 gene resulted in the abnormal phenotypes of HeLa cells, with increased numbers of multinuclei, micronucleus, and disordered microtubules. The cell cycle was disrupted, accompanied with a significant increase in the ratio of late apoptotic cells and a decrease in cell proliferation (all P < 0.05). Conclusion KIFC1 gene deletion affects the assembly of microtubules and cell division in HeLa cells, leading to abnormal nuclear morphology, chromatin elimination, cell cycle arrest, and increased cell apoptosis.

Expression and prognostic significance of cell division cycle associated protein 5 in pancreatic cancer tissues / 肿瘤研究与临床

Objective:To analyze the expression of cell division cycle associated protein 5 (CDCA5) in pancreatic cancer tissues and its correlation with prognosis based on the bioinformatics.Methods:The RNA sequencing data (HTSeq-FPKM) and corresponding clinical information of 168 pancreatic cancer samples from January to December 2021 were downloaded from the Cancer Genome Atlas (TCGA) database, and the data of 179 pancreatic patients from January to December 2021 were downloaded from the GEPIA2 database, and 171 normal pancreatic tissues from TCGA and GTEx databases were simultaneously integrated. The relative expression level of CDCA5 mRNA in pancreatic cancer patients in GEPIA2 database and its relationship with overall survival (OS) and disease-free survival (DFS) were explored. Combined with the clinical data of the patients, univariate and multivariate Cox regression model analysis was used to analyze the factors influencing the OS of pancreatic cancer patients. Gene set enrichment analysis (GSEA) was performed to investigate the possibly involved signal pathways of CDCA5 in pancreatic cancer.Results:In the GEPIA2 database, the relative expression level of CDCA5 mRNA in pancreatic cancer tissues was higher than that in normal pancreatic tissues, and the difference was statistically significant ( P < 0.05). The pancreatic cancer patients were divided into the high CDCA5 mRNA expression group (89 cases) and the low CDCA5 mRNA expression group (89 cases) according to the median of relative expression level of CDCA5 mRNA (the case equal to the median value was not subgrouped). Survival analysis showed that patients with high CDCA5 mRNA expression had shorter OS ( P = 0.024) and DFS ( P = 0.025) compared with those with low CDCA5 mRNA expression. Multivariate Cox analysis showed that in TCGA database, N staging ( HR = 2.15, 95% CI 1.24-3.72, P = 0.006) and CDCA5 expression ( HR = 1.71, 95% CI 1.23-2.38, P = 0.001) were independent influencing factors of OS for pancreatic cancer patients. The results of GSEA enrichment analysis indicated that high CDCA5 mRNA expression was enriched in 13 biological pathways [all P < 0.05, false discovery rate (FDR) < 0.005] including cell cycle, DNA replication, homologous recombination, pyrimidine metabolism, mismatch repair, pentose phosphate pathway, glycolysis gluconeogenesis and p53. The expression of CDCA5 mRNA was positively correlated with the expressions of HK2, PKM, PGK1, ALDOA, EN01 and LDHA (all P < 0.05). Conclusions:CDCA5 is highly expressed in pancreatic cancer tissues and is associated with poor prognosis of patients, and it can be used as a prognostic marker for pancreatic cancer.

Linkage of blood cell division cycle 42 with T helper cells, and their correlation with anxiety, depression, and cognitive impairment in stroke patients

Cell division cycle 42 (CDC42) regulates T helper (Th) cell differentiation and is related to psychological disorders. This study aimed to assess the correlation between blood CDC42 and Th cells, and their association with mental issues in stroke patients. Peripheral blood samples were obtained from 264 stroke patients and 50 controls. Then, serum CDC42 was measured by enzyme-linked immunosorbent assay, and Th1, Th2, and Th17 cells were detected by flow cytometry. Hospital Anxiety and Depression Scale (HADS) and Mini Mental State Examination (MMSE) were applied to patients. CDC42 was decreased (P<0.001), Th1 (P=0.013) and Th17 (P<0.001) cells were elevated, while Th2 cells (P=0.108) showed no difference in stroke patients compared to controls. In addition, CDC42 was negatively associated to Th1 (P=0.013) and Th17 (P<0.001) cells in stroke patients but were not associated with Th2 cells (P=0.223). Interestingly, CDC42 was negatively associated with HADS-anxiety (P<0.001) and HADS-depression scores (P=0.034) and positively associated with MMSE score (P<0.001) in stroke patients. Lower CDC42 was associated to lower occurrence of anxiety (P=0.002), depression (P=0.001), and cognitive impairment (P=0.036) in stroke patients. Furthermore, increased Th17 cells were positively correlated with HADS-anxiety and HADS-depression scores and inversely correlated with MMSE score, which were also associated with higher occurrence of anxiety, depression, and cognitive impairment in stroke patients (all P<0.05). Blood CDC42 and Th17 cells were correlated, and both of them were linked to the risk of anxiety, depression, and cognitive impairment. However, the findings need further large-scale validation, and the implicated mechanism needs more investigation.

miR-185 inhibits the proliferation and migration of osteosarcoma MG63 cells via regulating CDC42 gene expression / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To analyze the expression of miR-185 and cell division cyclin 42 (CDC42) in osteosarcoma tissues and cells, and to preliminarily explore whether miR-185 affects the proliferation and migration of osteosarcoma MG63 cells by regulating CDC42. Methods: The cancer tissues and para-cancerous tissues of 28 patients with osteosarcoma that pathologically confirmed in the Fourth People's Hospital of Hengshui City from January 2020 to January 2021 were collected for this study. Immunohistochemistry was used to detect the expression of CDC42 in osteosarcoma tissues, and qPCR was used to detect the expression of miR-185 in osteosarcoma tissues. Dual-luciferase reporter gene experiment was applied to verify the targeting relationship between CDC42 and miR-185. According to different transfectants, MG63 cells were divided into miR-185 mimic group, miR-NC group, miR-185 inhibitor group, NC-inhibitor group, CDC42 group (transfected with CDC42 over-expression vector), and negative control (NC) group. The effects of miR-185 and CDC42 expression on the migration, proliferation and cell cycle of MG63 cells were detected by scratch healing assay, CCK-8 method and FCM, respectively. A nude mouse xenograft model was constructed by inoculating osteosarcoma MG63 cells. Immunohistochemistry, qPCR and WB methods were used to detect the effects of over-expression or knock-down of miR-185 on the expression of Ki67 and CDC42 in transplanted tumor tissues. Results: Compared with para-cancerous tissues, the expression of miR-185 in osteosarcoma tissues was significantly decreased, while the expression of CDC42 was significantly increased (all P<0.01). CDC42 was verified to be a target gene of miR-185. Compared with the control group, the migration and proliferation of MG63 cells in the miR-185 mimic group were inhibited (all P<0.01), while the migration and proliferation of MG63 cells in the CDC42 group were increased and the cell cycle was arrested in the S phase (all P<0.01). Compared with the miR-185 group, the migration and proliferation abilities of MG63 cells in the miR-185+CDC42 group were promoted, and the proportion of cells in S phase was increased (all P<0.01). Compared with the control group, the expression of Ki67 and CDC42 in the transplanted tumor tissues of miR-185 mimic group was significantly decreased (all P<0.01), while the opposite results were observed in miR-185 inhibitor group (all P<0.01). Conclusion: miR-185 is lowly expressed while CDC42 is highly expressed in osteosarcoma tissues. miR-185 can inhibit the proliferation and migration of osteosarcoma MG63 cells by negatively regulating the expression of CDC42.

Correlation study of abdominal aortic calcification and serum cell division cycle 42 in maintenance hemodialysis patients / 中国综合临床

Objective:To explore the correlation between abdominal aortic calcification and serum cell division cycle 42 (CDC-42) in maintenance hemodialysis (MHD) patients, and to explore the influencing factors of them.Methods:A cross-sectional study was conducted in the Blood Purification Center of Qingdao Municipal Hospital,112 patients who underwent MHD for more than 6 months from October 2019 to March 2021 were selected. The abdominal aortic calcification score (ACCs) was calculated by reference to the abdominal lateral X flat tablets. According to AACS, 50 cases were divided into no and mild calcification group (0≤AACS<5 points) and 62 cases were divided into moderate and severe calcification group (AACS≥5 points). The level of serum CDC-42 was detected by enzyme linked immunosorbent assay (ELISA). Taking the median serum CDC-42 level as the boundary, 56 cases were divided into low CDC-42 group and high CDC-42 group. Spearman correlation analysis was used to analyze the correlation between indicators. The risk factors of elevated CDC-42 and abdominal aortic calcification in MHD patients were explored by multivariate logistic regression analysis, and the variables were included by entry method.Results:In 112 patients, 91 cases (81.25%, 91/112) had abdominal aortic calcification, and the median serum CDC-42 level was 466.56 (335.56,623.57) ng/L. CDC-42, AACs, age, dialysis age, diabetic nephropathy, glycosylated hemoglobin, alkaline phosphatase, parathormone and calcium in the no and mild calcification groups were 347.77 (291.20, 419.53) ng/L, 1.00 (0.00, 3.00) points, (57.18±6.25) years, 31.50 (15.00, 49.25) months, 34.00%(17/50), (6.63±0.97)%, 116.22 (87.32, 152.13) U/L, 258.57 (143.40, 433.31) ng/L, (2.18±0.26) mmol/L, and in the moderate to severe calcification group were 602.69 (489.61, 762.73) ng/L, 10.00 (7.00, 16.25) points, (60.81±7.12) years, 49.00 (18.00, 67.00) months, 53.23%(33/62), (7.07±1.20)%, 144.34 (99.71, 201.76) U/L, 336.57 (230.63, 506.00) ng/L,(2.28±0.26) mmol/L, with statistically significant differences between the two groups(The statistical values were 6.99, 9.11, 2.83, 2.45, 4.14, 2.08, 2.04, 2.16 and 1.99, respectively, all P<0.05). CDC-42, AACs, glycosylated hemoglobin and parathormone in the low CDC-42 group were 336.50 (295.10, 395.25) ng/L, 2.00 (0.00, 4.00) points, (6.62±1.06) %, 250.60 (140.20, 462.02) ng/L,and in the high CDC-42 group were 622.92 (558.11, 836.65) ng/L, 10.00 (6.25, 15.75) points, (7.13±1.13) %, 347.21 (240.40,501.20) ng/L, with statistically significant differences between the two groups (The statistical values are 6.51, 5.21, 2.43 and 2.54, respectively,all P<0.05). Abdominal aortic calcification has positive correlations with CDC-42 ( r s=0.704, P<0.001), age ( r s=0.308, P=0.001), dialysis years ( r s=0.198, P=0.036), glycosylated hemoglobin ( r s=0.358, P<0.001), alkaline phosphatase ( r s=0.187, P=0.048), parathormone ( r s=0.437, P<0.001), serum calciu m( r s=0.323, P=0.001) and serum phospho-rus ( r s=0.251, P=0.007), and negative correlation with serum albumin( r s=-0.276, P=0.003). This study has confirmed that high serum CDC-42 ( OR=1.010, 95%CI:1.004-1.016, P=0.001) and senior dialysis age ( OR=1.033, 95%CI:1.006-1.061, P=0.018) were independent risk factors for moderate to severe abdominal aortic calcification.Serum CDC-42 levels has positive correlation with AACs ( r s=0.704, P<0.001), age ( r s=0.240, P=0.011), dialysis age ( r s=0.191, P=0.044), glycosylated hemoglobin ( r s=0.350, P<0.001), parathormone ( r s=0.380, P<0.001) and serum calcium ( r s=0.235, P=0.013). This study learned that,high AACs ( OR=1.185, 95%CI:1.037-1.354, P=0.013) and high parathormone ( OR=1.005, 95%CI:1.001-1.009, P=0.009) were independent risk factors for high CDC-42. The area under the receiver operating characteristic curve (ROC-AUC) of serum CDC-42 in predicting moderate and severe abdominal aortic calcification in MHD patients was 0.885. When the cut-off point was 466.56 ng/L, the predictive sensitivity and specificity were 79% and 86% respectively. Conclusion:The degree of abdominal aortic calcification in MHD patients was positively correlated with the level of serum CDC-42. High serum CDC-42 and high dialysis age were independent risk factors for abdominal aortic calcification in MHD patients. High AACS and high parathyroid hormone were independent risk factors for the increase of serum CDC-42 in MHD patients .

Effects of hepatitis C virus NS3/4A protein on proliferation, apoptosis and division of human hepatocarcinoma SMMC-7721 cells / 肿瘤研究与临床

Objective:To investigate the effects of hepatitis C virus (HCV) NS3/4A protein on proliferation, apoptosis and division of human hepatocarcinoma SMMC-7721 cells.Methods:The recombinant plasmid pLV-Green-NS3/4A and pLV-puro-NS3/4A were produced by homologous recombination method, and then the cells were divided into pLV-Green-NS3/4A or pLV-puro-NS3/4A transfection group and their corresponding control group (transfected with empty vector). The expression of recombinant protein was detected by immunofluorescence staining and Western blotting. Methyl thiazol tetrazolium (MTT) method was used to detect cell proliferation, DAPI staining was used to detect cell apoptosis, and immunofluorescence was used to analyze cell devision.Results:The growth state of SMMC-7721 cells in the pLV-Green-NS3/4A transfection group was worse than that in the corresponding empty vector control group. Compared with the corresponding empty vector control group, the proliferation activity of SMMC-7721 cells in the pLV-Green-NS3/4A transfection group on the 3rd and 4th day was inhibited (both P < 0.05). The apoptosis rates of cells in the pLV-Green-NS3/4A transfection group and the corresponding empty vector control group were (20.3±3.5)% and (5.3±1.5)%, and the difference was statistically significant ( t = -8.57, P < 0.001). Compared with the corresponding empty vector control group, the cells in the pLV-puro-NS3/4A transfection group had more multipolar spindles, and the multipolar division rates of the two groups were (2.33±0.58)% and (6.01±0.99)%, and the difference was statistically significant ( t = -5.50, P = 0.005). Conclusions:HCV NS3/4A can inhibit the proliferation, promote the apoptosis and multipolar division of human hepatocarcinoma SMMC-7721 cells.

The disruption of protein-protein interactions with co-chaperones and client substrates as a strategy towards Hsp90 inhibition

The 90-kiloDalton (kD) heat shock protein (Hsp90) is a ubiquitous, ATP-dependent molecular chaperone whose primary function is to ensure the proper folding of several hundred client protein substrates. Because many of these clients are overexpressed or become mutated during cancer progression, Hsp90 inhibition has been pursued as a potential strategy for cancer as one can target multiple oncoproteins and signaling pathways simultaneously. The first discovered Hsp90 inhibitors, geldanamycin and radicicol, function by competitively binding to Hsp90's N-terminal binding site and inhibiting its ATPase activity. However, most of these N-terminal inhibitors exhibited detrimental activities during clinical evaluation due to induction of the pro-survival heat shock response as well as poor selectivity amongst the four isoforms. Consequently, alternative approaches to Hsp90 inhibition have been pursued and include C-terminal inhibition, isoform-selective inhibition, and the disruption of Hsp90 protein-protein interactions. Since the Hsp90 protein folding cycle requires the assembly of Hsp90 into a large heteroprotein complex, along with various co-chaperones and immunophilins, the development of small molecules that prevent assembly of the complex offers an alternative method of Hsp90 inhibition.

7SK truncation at 128-179 nt suppresses embryonic stem cell proliferation / 南方医科大学学报

OBJECTIVE@#To explore the role of small nuclear noncoding RNA 7SK in embryonic stem cell (ESCs) proliferation and the value of 7SK as a target for early diagnosis and treatment for primordial dwarfism (PD).@*METHODS@#ESC line R1 was transfected with the CRISPR/Cas9 system, and sequencing of the PCR product and glycerol gradient analysis were performed to identify novel 7SK deletion mutations. A lentivirus system was used to knock down cyclin-dependent kinase 9 (CDK9) in clones with 7SK deletion mutations, and the effect of CDK9 knockdown on the protein level of cell division cycle 6 (CDC6) was analyzed with Western blotting.@*RESULTS@#We identified a novel deletion mutation of 7SK at 128-179 nt in the ESCs, which resulted in deficiency of cell proliferation. 7SK truncation at 128-179 nt significantly reduced the protein expressions of La-related protein 7 (LARP7) and CDC6.@*CONCLUSIONS@#7SK truncation at 128-179 nt can significantly impair proliferation of ESCs by downregulating CDC6. 7SK is a key regulator of proliferation and mediates the growth of ESCs through a mechanism dependent on CDK9 activity, suggesting the value of 7SK truncation at 128-179 nt as a potential target for early diagnosis and treatment of PD.

Relationship between CDCA8 and tumor progression as well as that between CDCA8 and stemness maintenance of stem cells / 国际肿瘤学杂志

Cell division cycle associated 8 (CDCA8) is involved in the accurate regulation of cell division cycle. The expression change of CDCA8 can affect the progression of many kinds of tumors as well as the prognosis of patients. In recent years, studies have suggested that CDCA8 may play an important role in maintaining the stemness of cancer stem cells. Exploring the mechanism of CDCA8 affecting tumor is of great significance for the diagnosis and treatment of tumors.

Identification of potential genetic markers in prognosis of oral squamous cell carcinoma patients by bioinformatic analysis / 口腔疾病防治

Objective@#To analyze the differentially expressed genes of patients with oral squamous cell carcinoma (OSCC) from paracarcinoma through biological information analysis to preliminarily identify OSCC-associated genes. @*Methods@#GSE23558, GSE37991 and GSE30784 were downloaded from the Gene Expression Omnibus (GEO), which is the mRNA expression profile dataset. The differentially expressed genes (DEGs) were identified based on the gene ontology and the Kyoto Encyclopedia of Genes and Genomes. Then, the protein-protein interaction (PPI) network was constructed using the STRING online tool, and Cytoscape was used to filter the critical genes. Furthermore, key genes involved in the survival of patients with OSCC were analyzed using Kaplan-Meier analysis. The expression of hub genes was validated based on GEPIA（http://gepia.cancer-pku.cn/）. @*Results @#A total of 212 DEGs were screened, and further analysis revealed 16 core genes, among which the core genes associated with prognosis included aurora kinase A (AURKA), aurora kinase B (AURKB), apoptosis inhibiting factor 5 (BIRC5), cell division cycle 6 (CDC6), E2F transcription factor 7 (E2F7), ubiquitin-like with PHD and ring finger domains 1 (UHRF1). These key genes were highly expressed in patients with oral squamous cell carcinoma, and the survival time of patients was short; the difference was statistically significant (P < 0.05).@*Conclusion @# AURKA, AURKB, BIRC5, CDC6, E2F7 and UHRF1 may be useful as potential biomarkers for OSCC prognosis prediction.

Formins: the key regulators of plant cell morphology and development / 生物工程学报

Formins are widely distributed in eukaryotes such as fungi, plants and animals. They play crucial roles in regulating the polymerization of actin, coordinating the synergistic interactions between actin and microtubules, and determining cell growth and morphology. Unlike formins from fungi and animals, plant formins have been evolved into two plant-specific types. Generally, type Ⅱ formins are believed to regulate the polarized growth of cells, and type Ⅰ formins may regulate the cell expansion and division processes. Recent studies on the function of plant formins suggest it is inappropriate to classify the function of formins purely based on their structures. This review summarizes the domain organization of formins and their corresponding functions, as well as the underpinning mechanisms. Furthermore, the unsolved or unexplored issues along with future perspectives on plant formins are proposed and discussed.

Effects of cell division protein-encoding genes knockout on solvent formation and cell morphology in Clostridium acetobutylicum / 生物工程学报

Clostridium acetobutylicum is an important strain for bio-butanol formation. In recent years, gene-editing technology is widely used for developing the hyper-butanol-production strains. In this study, three genes (cac1251, cac2118 and cac2125) encoding cell division proteins (RodA, DivIVA and DivIB) in C. acetobutylicum were knocked out. The cac2118-knockout strain had changed its cell morphology to spherical-shape during the solventogenesis, and obtained a higher butanol yield of 0.19 g/g, increasing by 5.5%, compared with the wild type strain. The glucose utilization and butanol production of cac1251-knockout strain decreased by 33.9% and 56.3%, compared the with wild type strain, reaching to 47.3 g/L and 5.6 g/L. The cac1251-knockout strain and cac2125-knockout strain exhibited poor cell growth with cell optical density decreased by 40.4% and 38.3%, respectively, compared with that of the wild type strain. The results indicate that cell division protein DivIVA made the differences in the regulation of cell morphology and size. Cell division proteins RodA and DivIB played significant roles in the regulation of cell division, and affected cell growth, as well as solventogenesis metabolism.

Screen of differentially expressed genes in liver cancer xenograft tissues after CDC25Agene silence / 中国肿瘤生物治疗杂志

@#Objective: To explore the genes that may be regulated by cell division cycle 25A (CDC25A) with gene chip technology, and to elucidate and verify that CDC25A has a regulatory effect on the expression of liver cancer related genes. Methods: CDC25A expression in human liver cancer HepG2 cells was silenced by siRNA interference technology and a nude mouse xenograft model of liver cancer was successfully constructed in our previous research. Affymetrix human gene expression profiling microarray was used to further screen differentially expressed genes (DEGs) after silencing CDC25A in liver cancer xenografts, and GO analysis and KEGG analysis were performed. Some of the DEGs were verified by qPCR. Results: The chip screened 188 DEGs in liver cancer xenograft tissues after CDC25A silence, including 78 up-regulated genes and 110 down-regulated genes. These DEGs mainly involved in cell proliferation, apoptosis, protein complex binding, extracellular space, etc., and associated with the changes in pathways such as focal adhesions and extracellular matrix (ECM) receptor interactions. qPCR showed that the expression of HIPK2 mRNA was up-regulated and the mRNA expressions of (microfibrillar-associated protein 5(MFAP5) and cyclin D1 (CCND1) were down-regulated, which were consistent with the results of microarray detection. Conclusion: Using human gene expression profiling chip, the DEGs in liver cancer xenograft tissues in nude mice after silencing CDC25Awere successfully screened, providing effective clues for exploring the effect of CDC25Aon the growth of liver cancer.

Overlapping Cervical Cell Image Segmentation Based on Bottleneck Detection and Watershed Algorithm / 中国医疗器械杂志

This study proposes an image segmentation method based on bottleneck detection and watershed algorithm to solve the problem of overlapping cervical cell image. First, we use polygon approximation to get all feature points on the cell contour and then use bottleneck detection and ellipse fitting to obtain the correct split point pairs. Therefore, the approximate range of the overlapping region was determined. The watershed algorithm was used to obtain the internal boundary information for the gradient image of the region. Finally, the segmentation results of the overlapped cells were obtained by superimposing with the outer contour. The experimental results show that this algorithm can segment the contour of a single cell from the overlapping cervical cell images with good accuracy and integrity. The segmentation result is close to that of doctors' manual marking, and the segmentation result is better than other existing algorithms.

Cytotoxic and genotoxic potential of industrialized powdered milk for infants and young children

This study aimed to evaluate the cytotoxicity and genotoxicity and determine the LC50 concentration of powdered infant formulas widely marketed in South American countries. To this, milk samples, called as A, B, C and D, were analyzed in root meristem cells of Allium cepa, at concentrations of 0.075; 0.15 and 0.30 g mL-1, for 24 and 48 hours; and through cell viability in culture of normal line cells, via MTT test, for 24 hours, in the concentrations 0.018; 0.0375; 0.075 and 0.15 g mL-1. In A. cepa, all dairy products in the three concentrations caused significant inhibition of cell division in the meristems within the first 24 hours of exposure. In the in vitro evaluation, all milk formulas at 0.15 g mL-1, as well as milk A at a concentration of 0.037 g mL-1, C at 0.075 g mL-1 and D at 0.037 g mL-1, significantly reduced the cellular viability of the cell culture exposed to the foods studied, being potentially toxic. The milk A was considered the most toxic, with LC50 of 0.031 g mL-1, and B as the least toxic, with LC50 of 0.15 g mL-1. Therefore, the milk evaluated caused significant instability in cells of the test systems used and were characterized as cytotoxic.

Dna Topoisomerases: A Review On Their Functionalaspects In Cell Division

DNA is double helical macromolecule which carries all the genetic information and it is usually found enveloped inside a nucleus. The DNA helix relaxes and supercoils itself frequently in order to derive information from the genes during processes like transcription, condensation, replication and recombination, which require mutable or immutable alterations to cause the separation of the two DNA strands. Due to problems caused by the helical structure of DNA, these topoisomerase enzymes perform the required DNA uncoiling. Their role in cell cycle is also significant as their mutation leads to failure of anaphase separation (1, 2). In the present review, the important roles of DNA topoisomerases and their inevitable role in cell growth and cell cycle are discussed viz. how they function in cell proliferation and what are the results when different inhibitors are added to the cells, affecting cell cycle at various checkpoints .

MCC1019, a selective inhibitor of the Polo-box domain of Polo-like kinase 1 as novel, potent anticancer candidate

Polo-like kinase (PLK1) has been identified as a potential target for cancer treatment. Although a number of small molecules have been investigated as PLK1 inhibitors, many of which showed limited selectivity. PLK1 harbors a regulatory domain, the Polo box domain (PBD), which has a key regulatory function for kinase activity and substrate recognition. We report on 3-bromomethyl-benzofuran-2-carboxylic acid ethyl ester (designated: MCC1019) as selective PLK1 inhibitor targeting PLK1 PBD. Cytotoxicity and fluorescence polarization-based screening were applied to a library of 1162 drug-like compounds to identify potential inhibitors of PLK1 PBD. The activity of compound MC1019 against the PLK1 PBD was confirmed using fluorescence polarization and microscale thermophoresis. This compound exerted specificity towards PLK1 over PLK2 and PLK3. MCC1019 showed cytotoxic activity in a panel of different cancer cell lines. Mechanistic investigations in A549 lung adenocarcinoma cells revealed that MCC1019 induced cell growth inhibition through inactivation of AKT signaling pathway, it also induced prolonged mitotic arrest-a phenomenon known as mitotic catastrophe, which is followed by immediate cell death apoptosis and necroptosis. MCC1019 significantly inhibited tumor growth in a murine lung cancer model without affecting body weight or vital organ size, and reduced the growth of metastatic lesions in the lung. We propose MCC1019 as promising anti-cancer drug candidate.

Bioinformatic analysis of direct protein targets of aspirin against human breast cancer proliferation / 南方医科大学学报

OBJECTIVE@#To explore the molecular mechanism underlying the inhibitory effects of aspirin against human breast cancer cell proliferation through bioinformatics analysis.@*METHODS@#Drug Bank 5.1.3 was searched to identify direct protein targets (DPTs) of aspirin, and the protein-protein interaction (PPI) network of the DPTs was constructed online using STRING and the signaling pathways involved were identified. The genetic alterations of 6 DPTs associated with human breast cancer was analyzed and visualized by cBio Portal and OncoPrint, respectively. The transcriptomic data of breast cancer and normal tissues were downloaded from TCGA database, and the overexpressed genes were analyzed by DECenter. The intersection between the genes associated with the DPTs obtained by STRING analysis and the differentially over-expressed genes in TCGA was determined to confirm the candidate DPTs as a potential target of aspirin, and GO functional enrichment analysis was performed using Gene Ontology. The potential targets of aspirin against the proliferation of human breast cancer cells were verified by Western blotting.@*RESULTS@#Eleven DPTs of aspirin were identified. KEGG pathway enrichment indicated that 6 genes (EDNRA, IKBKB, NFKB2, NFKBIA, PTGS2 and TP53) were associated with the occurrence and development of cancer. A total of 10 220 differentially expressed genes were identified from the TCGA database, and among them 4 genes (, , , ) were found to be the potential targets for aspirin. These genes were involved mostly in the regulation of cell cycle and cell division. Western blotting showed that aspirin could down-regulate the expression levels of several pivotal proteins that regulated cell cycle and cell division, including , , and .@*CONCLUSIONS@#, , and may be potential targets for aspirin to inhibit the proliferation of human breast cancer cells, by affecting the progress of cell cycle and cell division.

Expression of polo-like kinase 1 in pre-implantation stage murine somatic cell nuclear transfer embryos

Somatic cell nuclear transfer (SCNT) has various applications in research, as well as in the medical field and animal husbandry. However, the efficiency of SCNT is low and the accurate mechanism of SCNT in murine embryo development is unreported. In general, the developmental rate of SCNT murine embryos is lower than in vivo counterparts. In previous studies, polo-like kinase 1 (Plk1) was reported to be a crucial element in cell division including centrosome maturation, cytokinesis, and spindle formation. In an initial series of experiments in this study, BI2536, a Plk1 inhibitor, was treated to in vivo-fertilized embryos and the embryos failed to develop beyond the 2-cell stage. This confirmed previous findings that Plk1 is crucial for the first mitotic division of murine embryos. Next, we investigated Plk1's localization and intensity by immunofluorescence analysis. In contrast to normally developed embryos, SCNT murine embryos that failed to develop exhibited two types of Plk1 expressions; a low Plk1 expression pattern and ectopic expression of Plk1. The results show that Plk1 has a critical role in SCNT murine embryos. In conclusion, this study demonstrated that the SCNT murine embryos fail to develop beyond the 2-cell stage, and the embryos show abnormal Plk1 expression patterns, which may one of the main causes of developmental failure of early SCNT murine embryos.

Distribution of adeG, adeB, adeE, adeY, abeM, and adeJ Efflux Pump Genes in Clinical Isolates of Acinetobacter Species from Korea

BACKGROUND: The aim of the present study was to determine the frequency of six efflux pump genes in Acinetobacter clinical isolates collected from South Korean hospitals. METHODS: In this study, we used a total of 339 Acinetobacter strains, comprising 279 Acinetobacter calcoaceticus–Acinetobacter baumannii (ACB) complex and 60 non-ACB complex strains. We performed specific PCR assays to detect adeG, adeB, adeE, adeY, abeM, and adeJ, transporter genes of the multidrug efflux pumps AdeFGH, AdeABC, AdeDE, AdeXYZ, AbeM, and AdeIJK, respectively. RESULTS: Frequencies of six efflux pump genes varied according to the species of Acinetobacter. Frequencies of adeE, abeM, and adeJ between A. baumannii group and A. nosocomialis group were found to be significantly different. Significant differences were found in the frequencies of adeB, adeE, adeY, and adeJ among the susceptible A. baumannii (SAB), multidrug-resistant A. baumannii (MDRAB), and extensively drug-resistant A. baumannii (XDRAB) groups within the 154 strains of A. baumannii. The frequencies of efflux pump genes in imipenem-susceptible and imipenem-nonsusceptible groups were significantly different for adeB, adeY, and adeJ. The frequencies of efflux pump genes in ciprofloxacin-susceptible and ciprofloxacin-nonsusceptible groups were significantly different for adeB and adeY. No significant difference was found in the frequency of efflux pump genes among groups sampled from different regions of Korea, across 86 strains of A. baumannii collected in 2012. CONCLUSIONS: The frequencies of six efflux pump genes obtained in this study demonstrate the fundamental epidemiological feature of efflux pump genes in Korean Acinetobacter clinical isolates.