Colony Formation Rate Research Articles

MicroRNA-518-3p suppresses cell proliferation, invasiveness, and migration in colorectal cancer via targeting TRIP4.

MicroRNA (miR)-518-3p has been shown to function as a tumor suppressor. This study was conducted to investigate the effects of miR-518-3p in colorectal cancer (CRC). The miR-518-3p mimic, mimic negative control (NC), miR-518-3p inhibitor, inhibitor-NC, ShRNA-TRIP4, and ShRNA-NC vectors were transfected into SW480 cells using Lipofectamine 2000. Cell viability was detected using CCK-8. Colony formation, cell invasiveness, and cell migration were assessed by plate colony formation, Transwell assays, and wound healing assays, respectively. Relative mRNA and protein levels were detected using RT-qPCR and Western blot, respectively. The target gene thyroid hormone receptor interactor 4 (TRIP4) of miR-518-3p was identified and further verified using dual-luciferase reporter assay. Compared with normal tissues, levels of miR-518-3p were decreased and TRIP4 was significantly increased in the tissues from patients with CRC. Following transfection with a miR-518-3p mimic or ShRNA-TRIP4, cell viability decreased in a time-dependent manner, and colony formation rate, wound closure rate, and the number of invasive cells were much lower for the transfected cells than in the corresponding NC and control groups. miR-518-3p overexpression or silencing of TRIP4 significantly down-regulated the expression of MMP-2 and MMP-9. Knockdown of miR-518-3p had the opposite effects, and TRIP4 was identified as a target of miR-518-3p. The inhibitory effects of miR-518-3p on the progressions of CRC are associated with TRIP4.

MiR-30a inhibits androgen-independent growth of prostate cancer via targeting MYBL2, FOXD1, and SOX4.

Castrate-resistant prostate cancer (CRPC) is an aggressive and lethal disease. The pathogenesis of CRPC is not fully understood and novel therapeutic targets need to be identified to improve the patients' prognosis. MicroRNA-30a (miR-30a) has been demonstrated to be a tumor suppressor in many types of solid malignancies. However, its role in androgen-independent (AI) growth of prostate cancer (PCa) received limited attention as yet. The clinical association of miR-30a and its potential targets with AI growth was characterized by bioinformatics analyses. Regulation of cell proliferation and colony formation rates by miR-30a were tested using PCa cell models. Xenograft models were used to measure the regulation of prostate tumor growth by miR-30a. The real-time quantitative polymerase chain reaction was used to validate whether miR-30a and its targets regulate cell cycle control genes and androgen receptor (AR)-dependent transcription. Bioinformatics tools, Western blot, and luciferase reporter assays were utilized to identify miR-30a targets. Bioinformatic analysis showed that low expression of miR-30a is associated with castration resistance of PCa patients and poor outcomes. Transfection of miR-30a mimics inhibited the AI growth of PCa cells in vitro and in vivo. Upregulation of miR-30a in 22RV1 cells altered the expression of cell cycle control genes and AR-mediated transcription, while downregulation of miR-30a in LNCaP cells had the opposite effects to AR-mediated transcription. MYBL2, FOXD1, and SOX4 were identified as miR-30a targets. Downregulation of MYBL2, FOXD1, and SOX4 affected the expression of cell cycle control genes and AR-mediated transcription and suppressed the AI growth of 22RV1 cells. Our results suggest that miR-30a inhibits AI growth of PCa by targeting MYBL2, FOXD1, and SOX4. They provide novel insights into developing new treatment strategies for CRPC.

Effects and mechanism of rat epidermal stem cells treated with exogenous vascular endothelial growth factor on healing of deep partial-thickness burn wounds in rats

Effects and mechanism of rat epidermal stem cells treated with exogenous vascular endothelial growth factor on healing of deep partial-thickness burn wounds in rats

Overexpression of CD36 in mammary fibroblasts suppresses colony growth in breast cancer cell lines

Human breast tumors are not fully autonomous. They are dependent on nutrients and growth-promoting signals provided by the supporting stromal cells. Within the tumor microenvironment, one of the secreted macromolecules by tumor cells is activin A, where we show to downregulate CD36 in fibroblasts. Downregulation of CD36 in fibroblasts also increases the secretion of activin A by fibroblasts. We hypothesize that overexpression of CD36 in fibroblasts inhibits the formation of solid tumors in subtypes of breast cancer models. For the first time, we show that co-culturing organoid models of breast cancer cell lines of MDA-MB-231 (e.g., a triple-negative line) or MCF7 (e.g., a luminal-A line) with CD36+ fibroblasts inhibit the growth and normalizes basal and lateral polarities, respectively. In the long-term anchorage-independent growth assay, the rate of colony formation is also reduced for MDA-MB-231. These observations are consistent with the mechanism of tumor suppression involving the downregulation of pSMAD2/3 and YY1 expression levels. Our integrated analytical methods leverage and extend quantitative assays at cell- and colony-scales in both short- and long-term cultures using brightfield or immunofluorescent microscopy and robust image analysis. Conditioned media are profiled with the ELISA assay.

Radiosensitization effect of selenocystine on HeLa cells in vitro

AbstractObjectivesTo study the effect of selenocystine (Selenocystine, SeC) on the radiosensitivity of human cervical cancer Hela cells, and to provide a new target for radiosensitivity of cervical cancer.MethodsSeC were added to the cervical cancer Hela cells, and then irradiate with 200 cGy/min X‐rays. The radiosensitization effect of SeC on cervical cancer Hela cells were confirmed with the experiments of cell growth inhibition, clone formation, flow cytometry and Western blotting to detect apoptosis.ResultsSeC were added to the Hela cells at different doses of 0, 5, 10, and 20 μmol/L and incubating for 24 hours, the cell growth inhibition rates were: 0%, 22.68%, 41.71%, 74.19%, and the calculated IC50 was 14.83 μmol/L. The growth inhibition rate of Hela cells was dose‐dependent with the concentration of SeC. The results of the colony formation experiments showed that the cell colony formation rates of the single X‐ray irradiation doses of 0, 2, 4, 6, and 8 Gy were 100%, 77.56%, 46.89%, 28.67%, and 10.89%, respectively. After adding 5μmol/L SeC under the same irradiation dose, the colony formation rate was reduced to 70.98%, 59.45%, 30.67%, 14.29%, 3.72%; 5μmol/L SeC combined with radiation therapy can shift the survival curve to the left, and the D0 and Dq values were significantly smaller than those in the radiation group, P < 0.05. Hela cell apoptosis were detected by the flow cytometry showed that when the single X‐ray irradiation dose was 0, 2, 4, 8 Gy, the apoptosis scores were: 2.51 ± 0.06, 6.76 ± 0.03, 14.51 ± 0.15, 21.41 ± 0.08; When 5 μmol/L SeC was added and then combined with X‐ray irradiation, and the irradiation dose was 0, 2, 4, 8Gy, the apoptosis score of each group was: 10.89 ± 0.36, 15.18 ± 0.21, 31.19 ± 0.08, 45.53 ± 0.28, SeC combined with X‐ray had an interaction effect on Hela cells, and the apoptosis rate increased (F = 21.52, P < 0.01). When Hela cells were treated with SeC and X‐rays, the expression of PARP protein and phosphorylated p53 protein was significantly increased compared with the control group.ConclusionsSeC may have a radiosensitizing effect on Hela cells, and its mechanism may be related to promoting apoptosis. SeC will be expected to become a new sensitization target for cervical cancer radiotherapy.

Role of mitochondrial damage in cadmium-induced cell apoptosis and DNA damage of hepatocytes

To investigate the role of mitochondrial damage mediated by reactive oxidative species(ROS) in cadmium-induced cell apoptosis and DNA damage of L02 hepatocytes, so as to provide experimental basis for the subsequent study and protection of people exposed to Cd. The L02 hepatocytes were cultured in vitro treated with 0-90 μmol/L Cd for 24 h, and the methylthiazolyldiphenyltetrazoliumbromide assay was used to detect the cell viability. The colony formation assay, flow cytometry, comet assay, 2', 7'-dichlorofluorescein diacetate, MitoTracker Red CMXRos and 10-N-nonyl-acridine-orange, mitochondrial membrane potential detection kit(JC-1) and adenosine triphosphate(ATP) assay kits and Western Blot were used to investigate cell growth and proliferation, cell apoptosis, DNA damage, ROS levels, mitochondrial morphology, mitochondrial membrane potential, mitochondrial mass, ATP content and related proteins after the cells exposed to 0, 20, 40 μmol/L Cd for 24 h. The cells were pretreated with vitamin C before adding Cd exposure, and ROS levels, mitochondrial function, cell apoptosis, DNA damage and proteins were measured. The cell viability was significantly inhibited with the increase of Cd concentration and treatment time. The cells were treated with Cd for 24 h for further study according to the result of MTT assay. Compared with control group, the colony formation rate were 8. 23% and 6. 17% respectively in 20 and 40 μmol/L Cd treatment and the apoptosis rate were 15. 85% and 26. 26%, respectively. We also found that the B cell lymphoma/leukemia(Bcl-2) gene protein was significantly reduced, while the levels of Bcl-2 associated X protein(Bax) and cleaved cysteine aspastic acid-specific protease 3(cleaved-caspase-3) were increased in a dose-dependent manner. Cd treatment also induced DNA damage and accumulation of intracellular ROS, accompanied by a mitochondrial morphological change, significant decrease in Δψm, mitochondrial mass, ATP content, mitochondrial cytochrome C(cyt c) and an increase in cytoplasmic cyt c expression(P<0. 05). In addition, pretreatment with antioxidant vitamin C not only significantly increased cyt c, mitochondrial mass, ATP content and mitochondrial cyt c, but also reduced the expression of cytoplasmic cyt c(P<0. 05), cell apoptosis and DNA damage induced by Cd. Cd exposure could induce ROS accumulation in L02 hepatocytes, which can lead to mitochondrial damage, and ultimately lead to cell apoptosis and DNA damage.

Exosomal microRNA-144 from bone marrow-derived mesenchymal stem cells inhibits the progression of non-small cell lung cancer by targeting CCNE1 and CCNE2

BackgroundMesenchymal stem cells (MSCs) are pluripotent mesenchymal cells present in various adult tissues. MSCs secrete exosomes as regulators of the tumor niche, with involvement in tumorigenesis and metastasis. The regulatory role of microRNAs (miRs or miRNAs) in MSCs via targeting cyclin E1 (CCNE1) or cyclin E2 (CCNE2) has been extensively reported. Since exosomes are considered as protective and enriched sources of shuttle miRNAs, we hypothesized that exosomal transfer of miR-144 from bone marrow-derived MSCs (BMMSCs) would affect the development of non-small cell lung cancer (NSCLC) cells by targeting CCNE1 and CCNE2.MethodsWe first quantified the levels of miR-144, CCNE1, and CCNE2 in NSCLC tissues and cell lines and then undertook gain- and loss-of-function studies of miR-144, CCNE1, and CCNE2 to investigate their roles in the biological characteristics of NSCLC in vitro. NSCLC cells (A549) were exposed to exosomes derived from MSCs, and cell proliferation and colony formation rate were determined using in vitro assays. Finally, effects of BMMSC-derived exosomal miR-144 on tumor development were studied in vivo.ResultsIn NSCLC tissues and cell lines, miR-144 was expressed poorly and CCNE1 and CCNE2 were expressed highly. Artificially elevating miR-144 inhibited cell proliferation, colony formation, and the number of S phase-arrested cells in NSCLC by downregulating CCNE1 and CCNE2. Additionally, BMMSC-derived exosomal miR-144 led to restrained NSCLC cell proliferation and colony formation. These inhibitory effects of BMMSC-derived exosomes carrying miR-144 on NSCLC were confirmed by experiments in vivo.ConclusionCollectively, these findings revealed inhibitory effects of BMMSC-derived exosomal miR-144 on NSCLC progression, which were mediated by downregulation of CCNE1 and CCNE2.

Effect and mechanism of down-regulating REV7 expression on the radiosensitivity of human colon cancer cell HCT116

Objective To evaluate the effect and mechanism of down-regulating the expression of REV7 on the radiosensitivity of human colon cancer cell HCT116. Methods HCT116 cells were cultured and the expression of REV7 was down-regulated by RNA interference technique. HCT116 cells were divided into the blank group, negative control transfected with negative RNA oligo group and REV7 expression down-regulation transfected with REV7 RNA oligo group, respectively. The cell proliferation was determined by colony formation assay. The expression levels of the proteins of relevant genes were detected by Western blot. The level of cell apoptosis and non-homologous end joining was evaluated. Results The colony formation rate was significantly reduced in THE REV7 siRNA group after 6Gy irradiation (P 60% in the REV7 siRNA group. The expression levels of γH2AX and Caspase9 were significantly up-regulated, whereas those of KU80 and XRCC4 were remarkably down-regulated in the REV7 siRNA group (all P<0.05). Conclusions The radiosensitivity of human colon cancer cell HCT116 can be increased by down-regulating the expression of REV7. The underlying mechanism may be related to the lower incidence rate of non-homologous end joining. Key words: REV7 gene; HCT116 cell line; Radiosensitivity; Non-homologous end joining

Abstract P3-03-02: Integrative proteogenomic analyses identifies CPT1A and fatty acid oxidation as a potential therapeutic strategy in hormone receptor positive breast cancer

Abstract Background: Clinically, approximately two-thirds of the nearly 250,000 breast cancer cases diagnosed each year in the United States are hormone receptor positive (HR+), luminal breast tumors. Slower growing luminal breast tumors are often successfully treated using endocrine based therapies resulting in a relatively good prognosis for these patients. However more highly proliferative luminal tumors are often resistant, or become resistant, to current therapies leading to a worse outcome for these patients. As such, there is a critical need to identify genomic alterations and dysregulated signaling pathway in this subset of tumors that may represent novel therapeutic opportunities. Methods: In order to identify genetic events responsible for tumorigenesis and to identify potential drug-able genetic alterations and/or pathways associated with high levels of proliferation in HR+ tumors, we used an integrative genomics-based strategy to interrogate orthogonal genome-wide data from more than 2,500 patients from the TCGA, METABRIC, CPTAC studies. Data from a genome-wide RNAi screen was used to delineate essential from non-essential genes in HR+ breast cancer cell lines. Genetic and pharmacological-based in vitro and in vivo studies were used to demonstrate the impact identified candidate genes on cell viability, proliferation, colony formation, spheroid formation and tumor growth rate. Results: Integrative genomic analyses of human breast tumors identified DNA amplification as well as mRNA and protein over-expression of carnitine palmitoyltransferase 1A (CPT1A) in highly proliferative luminal tumors. Further in silico analyses demonstrated that high CPT1A expression corresponded with protein markers of proliferation in human tumors and is prognostic in HR+ tumors in both the TCGA and METABRIC cohorts. Since CPT1A is the rate limiting enzyme responsible for fatty acid import into the mitochondria during Fatty Acid β Oxidation (FAO), these data indicate that highly proliferative luminal tumors may utilize FAO as a prominent energy source. As such, we hypothesized that inhibiting CPT1A and/or FAO in this subset of tumors or cell lines may represent a potential therapeutic opportunity. Consistent with our hypothesis, analyses of data from a genome-wide RNAi screen in 27 breast cancer cell lines suggested that HR+ cells lines are dependent on CPT1A expression. Importantly, we experimentally demonstrated that in vitro genetic silencing of CPT1A or pharmacological inhibition of FAO in ER+ breast cancer cell lines results in reduced cell viability and increased cell death as well as decreased spheroid formation. Importantly, our data indicated that in vivo inhibition of CPT1A resulted in decreased tumor volume and growth rate in orthotopic xenograft tumor models. Conclusions: In this study, integrative proteogenomic analyses identified amplification and over-expression of CPT1A in highly proliferative ER+ breast tumors. In vitro and in vivo studies demonstrated that CPT1A is an essential oncogenic driver in HR+/luminal breast cancer and establish CTP1A/FAO inhibition as a putative therapeutic strategy for treatment of these tumors. Citation Format: Michael L Gatza, Vrushank Bhatt, Kimberly Parker, Guarav Mehta, Christen Khella, Yanxiang Guo, Nidhi Jariwala. Integrative proteogenomic analyses identifies CPT1A and fatty acid oxidation as a potential therapeutic strategy in hormone receptor positive breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-03-02.

Antimicrobial peptide CAMA-syn expressed in pulmonary epithelium by recombination adenovirus inhibited the growth of intracellular bacteria.

Intracellular bacteria, especially Mycobacterium tuberculosis, are important pathogenic microorganisms that endanger human health. Purified and synthesized cecropin A-magainin 2 (CAMA-syn) can exhibit a higher antibacterial activity and lower cytotoxicity. To enhance such antimicrobial potential, it would be desirable to deliver CAMA-syn expressed in lung epithelial cells by an adenovirus vector using gene therapy. A549 cells in vitro and lung epithelial cells in vivo were used to express CAMA-syn by transducing recombinant adenovirus Ad-SPC-CAMA/GFP, and the expression of CAMA-syn was determined by a reverse transcriptase-polymerase reaction (RT-PCR) and immunofluorescence. The antimicrobial activity in cells was investigated by colony-forming rate and growth curve. Forty Kunming mice of a Bacillus Calmette-Guerin (BCG) infection animal model were randomly divided into three groups: adenoviruses delivery of Ad-SPC-CAMA/GFP, Ad-CMV-CAMA/GFP and empty-virus Ad-CMV-GFP. The expression of CAMA-syn in mice was confirmed by RT-PCR and immunofluorescence. After tracheal injection of adenoviral vector for 3 days, lungs from the mouse model were extracted and homogenized for detection of colony-forming efficiency. CAMA-syn expressed in lung epithelial cells A549 conferred antimicrobial activity against a series of bacteria, including Salmonella abortusovis and BCG. The results obtained in vivo showed that the colony-forming rate of Ad-SPC-CAMA/GFP (74.54%) and Ad-CMV-CAMA/GFP (62.31%) transduced into mice was significantly lower than that of the control group. Lung epithelial-specific expression of antimicrobial peptide CAMA-syn mediated by adenovirus suppressed the growth of intracellular bacteria, providing a promising approach for the control of refractory intracellular infection.

Interference with SMO increases chemotherapy drug sensitivity of A2780/DDP cells by inhibiting the Hh/Gli signaling pathway.

Aberrant activation of the Hedgehog (Hh)/Gli pathway contributes to the tumorigenesis of several human cancers, including ovarian cancers. We investigated the function of SMO on cell growth, drug resistance, and invasive ability in A2780/DDP cells. Moreover, we also tested the levels of the downstream target genes of the Hh/Gli pathway in SMO short hairpin RNA (shRNA) lentivirus-infected A2780/DDP cells. Western blot analysis results revealed that the Hh/Gli pathway was activated in cisplatin-resistant A2780/DDP cells. After infection by SMO shRNA lentivirus, the colony formation rate and invasive rate of cisplatin-resistant A2780/DDP cells were decreased. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that upon transfection with SMO shRNA, cell growth was decreased and drug sensitivity to cisplatin was upregulated. Moreover, interference with SMO decreased the expression of MMP-2, MMP-9, VEGF, and Snail in cisplatin-resistant cells. Thus, the Hh/Gli signaling pathway was aberrantly activated in A2780/DDP cells. The colony formation rate and invasive rate were decreased in SMO shRNA lentivirus-infected A2780/DDP cells. All results showed that inhibiting Hh/Gli signaling may negatively regulate the proliferation, invasion, and metastasis of cisplatin-resistant A2780/DDP cells, as well as increase the sensitivity of A2780/DDP to the chemotherapeutic drug of cisplatin.

Expansion of Human Dental Pulp Cells In Vitro Under Different Cryopreservation Conditions.

To optimize the expansion of human dental pulp cells in vitro by exploring several cryopreservation methodologies. The intra-dental pulp tissues from healthy subjects were extracted and divided into three separate tissue segments, which were randomly divided into the three following groups; the fresh group, the 5% DMSO group, and the 10% DMSO group. In the fresh group, dental pulp cells were directly cultivated as primary cultures, whereas in the DMSO groups, the dental pulp cells were cultivated from cryopreserved pulp tissue segments one month later. The cell yield and the time it took for the cells to grow out of the pulp tissue and attach to the culture plate varied among the three groups; the 5% DMSO group was inferior to the fresh group but superior to the 10% DMSO group (p<0.05). Moreover, no differences were found at the 1st passage amongst the three groups regarding the following aspects (p>0.05); colony formation rate and cell survival rate. Furthermore, no differences were noted at the 3rd passage regarding the following aspects (p>0.05); proliferation ability, cell growth curve and surface marker expression of dental pulp cells. Five percent DMSO may be the most optimal condition for tissue storage to preserve stem cells in situ.

Knocking down LSD1 inhibits the stemness features of colorectal cancer stem cells.

As a top leading cause of cancer death in many countries, colorectal cancer (CRC) has drawn increasing attention to the study of the pathological mechanism. According to the “cancer stem cell hypothesis”, malignancies originate from a small fraction of cancer cells that show self-renewal properties to initiate and sustain tumor growth and tumor metastasis. Therefore, these cancer stem cells (CSC) probably play important roles in tumor recurrence, metastasis, and drug resistance. Previous research reported that lysine-specific histone demethylase 1 (LSD1) maintains cancer stemness through up-regulating stemness markers SOX2 and OCT4. CD133 is believed to be the most robust surface marker for CRC stem cells, however the regulatory effect of LSD1 on stemness of CD133+ CRC has never been reported. In this study, our objectives included: 1) to isolate pure CD133+ and CD133− cells from SW620 cell line; 2) to investigate the effect of LSD1 on the characteristics of CD133+ stem cancer cells by knocking down the target gene. Results suggested that the SW620 cell line had both CD133+ and CD133− subsets. The CD133+ subset exhibited more CSC-like characteristics compared with the CD133− subset with higher viability, colony formation rate, migration and invasion rate, resistance to anti-cancer drugs, and apoptosis in vitro. The CD133+ also induced faster tumor formation and larger tumors in vivo. In the LSD1-knockdown CD133+ cells, the CSC-like characteristics had been all weakened. We conclude that LSD1 was important for CSCs to maintain their “stemness” features, which could be a potential therapeutic target of CRC.

Structural maintenance of chromosomes 2 is identified as an oncogene in bladder cancer in vitro and in vivo.

Taurine upregulated gene 1 (TUG1) has been found to promote bladder cancer cell growth in our recent research. In this study, TUG1-depleted bladder cancer cells were used to identify potent players in bladder cancer. Human gene expression arrays were used for transcriptome profiling of TUG1-depleted bladder cancer cells. Cell proliferation was analyzed by MTT assay. Cell apoptosis and cell cycle were analyzed by flow cytometry. Colony formation assay was used to observe the changes of colony formation rates. Xenograft formation assay was performed in nude mice. Immunohistochemical staining was used to test the gene expression levels in tissues from bladder cancer patients. We found that deregulated genes were strongly enriched in cell cycle or pathways in cancer in TUG1-depleted bladder cancer cells. Structural maintenance of chromosomes 2 (SMC2) was inhibited after TUG1 knockdown. The depletion of TUG1 or SMC2 led to G2/M phase arrest in bladder cancer cells. SMC2 depletion inhibited bladder cancer cell proliferation, promoted apoptosis, decreased colony formation, and reduced tumor growth in xenograft nude mice. Overexpression of SMC2 restored the growth of TUG1-depleted cells. The expression levels of SMC2 were higher in human bladder cancer tissues than that in paired normal tissues. Our data suggest that SMC2 is an oncogene in bladder cancer and depletion of SMC2 might have potential therapeutical significance in bladder cancer.

MicroRNA-152 suppresses cell proliferation and tumor growth of bladder cancer by targeting KLF5 and MKK7

B ackground: In bladder cancer, miR-152 has been reported to be downregulated and accompanied by high DNA methylation. However, the molecular mechanisms underlying miR-152 in bladder cancer tumorigenesis remain unknown. M ethods: The expressions of miR-152 in human bladder cancer cell lines (J82, 5637, and T24) and a human bladder epithelial cell line (SV-HUC-1) were compared. The expression of miR-152 was observed after adding DNA methylation inhibitor 5-Aza-dC or after knocking down the major DNA methyltransferase DNMT1. The bladder cancer cell viability was determined using an MTT assay, and the colony formation rate was determined using a colony formation assay. The effects of miR-152 overexpression on cell proliferation and tumor growth were observed in vitro and in vivo. The interactions among miR-152, KLF5, and MKK7 were determined using a luciferase reporter assay. R esults: MiR-152 was downregulated in bladder cancer cell lines in comparison with normal ones. The expression of miR-152 was significantly promoted after adding 5-Aza-dC or after knocking down DNMT1. The overexpression of miR-152 suppressed cell proliferation and tumor growth both in vitro and in vivo. MiR-152 inhibited KLF5 and MKK7 expression by binding with 3’ untranslated regions of their mRNAs. Meanwhile, the overexpression of KLF5 induced MKK7 expression by increasing the transcriptional activity of MKK7. The simultaneous overexpression of miR-152 and KLF5 reversed the inhibition of cell proliferation caused by miR-152 overexpression alone. C onclusions: Our findings suggest that miR-152 acts as a tumor suppressor in bladder cancer, and the overexpression of miR-152 may be a potential strategy for treating bladder cancer. K eywords: cell proliferation, bladder cancer, DNA methylation, miR-152, transcription factor

Transformation Chlorophyll a of Spirulina platensis to Chlorin e6 Derivatives and Several Applications

BACKGROUND:Spirulina platensis contains a large amount of chlorophylls, chlorophyll a, that are starting materials to synthesize functionalized chlorins. Chlorin e6 (Ce6) as well as its derivatives are second generation sensitizers using in photodynamic therapy (PDT) of various cancers. In this study, we transfer chlorophyll a of S. platensis to Ce6 derivatives and determine their several applications.AIM:We aimed to evaluate the effects of Ce6 derivatives to treat cancer cells.METHODS:Ce6 trimethylester was created from methyl pheophorbide a2 in S. platensis provided by the Hidumi Company, Nghe An province, Viet Nam. Hela cells were incubated with Ce6 trimethylester and the irradiated with the diode laser dose of 1.2 J/cm2/min through the system of filters £ 650 nm. MTT assay and clonogenic assay were used to determine survival rate and cloning efficiency of cells. Antimicrobial effect of Ce6 trimethylester with halogen light were studied with Propionibacterium acnes VTCC 0218 and Staphylococcus aureus VTCC 0173.RESULTS:From dry biomass (700 g) of S. platensis, after extracting chlorophyll a and methanolysis, 4.2 g of methyl pheophorbide a was obtained. The reaction to give Ce6 trimethylester with 82% yield was performed with potassium hydroxide (KOH) in MeOH/THF/CHCl3. After irradiation with a 650 nm laser at 1.2 J, the cell viability in all samples decreased with Ce6 trimethylester treatment, the survival declining trend of Hela cells treated with Ce6 trimethylester were proportional when concentration of Ce6 trimethylester increased. The rate of colony formation was declined as the concentration of Ce6 trimethylester treated was increased. The growth of both S. aureus and P. acnes can be inactivated by Ce6 trimethylester PDT. The MIC99 value against P. acnes VTCC 0218 and S. aureus VTCC 0173 of Ce6 trimethylester with halogen light was 1.25 μg/ml.CONCLUSION:The Ce6 trimethylester from S. platensis cultivated in Viet Nam could be used as a potential photosentizer for photodynamic therapy for treatment of cancer and acne.

Effect of polyadenyl diphosphate ribose polymerase 1 inhibitor on the sensitivity of As2O3 in treating renal cancer cells in vitro study

Objective To explore the effect of inhibition of poly (ADP-ribose) polymerase-1 (PARP-1) (AG14361) on the sensitivity of cells to arsenic trioxide (As2O3) in treating renal cancer cells. Methods Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay, colony formation assay, single cell gel electrophoresis and micronucleus assay, flow cytometry were used to detect the cytotoxicity, genotoxicity and apoptosis of As2O3 combined with AG14361 on renal cancer cells. Results The colony formation rate [Combination group vs. As2O3 group: OS-RC-2, (43.23±3.12)% vs. (62.35±2.69)%; 786-O, (36.23±3.12)% vs. (58.31±2.42)%; HK-2, (56.32±3.46)% vs. (68.92±3.12)%], The difference was statistically significant (P<0.05); Single cell gel electrophoresis and micronucleus test showed that DNA and chromosome damage increased in the combination group compared with As2O3 group, and the difference was statistically significant (P<0.05). Flow cytometry shows, the combination group significantly enhanced the apoptotic rate of renal cancer cells compared with the As2O3 group [OS-RC-2, (59.90±86.75)% vs. (17.00±3.61)%; 786-O, (41.47±4.11)% vs. (15.17±1.99)%; HK-2, (30.43±4.92)% vs. (13.17±3.26)%]. Conclusion AG14361 has a sensitizing effect on the killing effect of As2O3-induced renal cancer cells, and its mechanism may be through inhibition of single-double-strand damage repair, which in turn causes chromosome damage. Key words: Renal cancer cells; As2O3; AG14361; Sensitizing

Influence of simvastatin on the biological behavior of nucleus pulposus-derived mesenchymal stem cells.

Objective(s):This research is to study the influences of different concentrations of simvastatin on the biological activities of nucleus pulposus-derived mesenchymal stem cells (NPMSC).Materials and Methods: NPMSC were cultured with different concentrations of simvastatin (0, 0.01, 0.1, and 1 μM) and assessed to determine the possible effects of simvastatin. The cell proliferation was assessed with CCK-8 assay. The flowcytometry and multilineage differentiation were also performed to identify the stem characterization of the cells. The mRNA expressions of aggrecan, collagen type II, glucose transporter 1 (GLUT-1), vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α) were determined by qRT-PCR.Results: The results demonstrated that the cells isolated from nucleus pulposus of healthy Sprague-Dawley (SD) rat met the criteria of MSC. NPMSC could form sunflower-like colonies and strongly expressed stem cell-related genes. In addition, NPMSC showed strong ability of chondrogenic, adipogenic and osteogenic differentiation. Simvastatin at certain range concentrations (0.01 μM-0.1 μM)) significantly promoted colony-forming rate and cell proliferation, and inhibited cell apoptosis. Simvastatin could promote expressions of aggrecan, collagen type II, HIF-1α, VEGF and GLUT-1, while 0.1 μmol/l concentration reached the maximum effect. Our study further demonstrated that HIF-1α-intermediated signaling pathway might participate in regulating the biological activities of NPMSC. Conclusion: Proper concentration of simvastatin can promote the biological behavior of NPMSC, and HIF-1α-intermediated signaling pathway might participate in the mechanism.

NF-YA overexpression protects from glutamine deprivation

The heterotrimeric transcription factor NF-Y binds to CCAAT boxes of genes of glutamine metabolism. We set out to study the role of the regulatory NF-YA subunit in this pathway. We produced U2OS and A549 clones stably overexpressing -OE- the two splicing isoforms of NF-YA. NF-YA OE cells show normal growth and colony formation rates, but they become resistant to cell death upon glutamine deprivation. Increased mRNA and protein expression of the key biosynthetic enzyme GLUL in U2OS entails increased production of endogenous glutamine upon deprivation. The use of GLUL inhibitors dampens the NF-YA-mediated effect. NF-YA OE prevents activation of the pro-apoptotic transcription factor CHOP/DDIT3. Elevated basal levels of SERCA1/2, coding for the molecular target of Thapsigargin, correlate with resistance of NF-YA OE cells to the drug. The work represents a proof-of-principle that elevated levels of NF-YA, as found in some tumor types, helps altering cancer metabolic pathways.

Overexpression of Shrm4 promotes proliferation and differentiation of neural stem cells through activation of GABA signaling pathway.

Shrm4 is a protein that is exclusively expressed in polarized tissues. The physiological function of Shrm4 in the brain was required to be elucidated. Thus, we aimed to explore how the Shrm4-mediated gamma-aminobutyric acid (GABA) pathway affected neural stem cells (NSCs). At first, the Nestin expression in cultured NSCs was identified. After determination of the interaction of Shrm4 and GABAB1, a series of in vitro experiment were performed to detect cell proliferation, the ability of cell colony formation, degree that NSCs differentiated into neurons, the apoptosis rate, and cell cycle. The levels of Shrm4, GABAB1, Bcl-2-associated protein x (Bax), B cell lymphoma 2 (Bcl-2), cleaved Caspase-3, microtubule-associated protein 2 (MAP-2) as well as suppressor of cytokine signaling 2 (SOCS2) were detected to further assess the role of Shrm4 and GABA pathway in NSCs. Initially, we found that Shrm4 could bind to GABAB1, and overexpression of Shrm4 or activation of GABAB1 increased the number of positive cells, and promoted cell viability, colony formation rate and differentiation of NSCs. After overexpression of Shrm4 or activation of GABAB1, cells in the G1 phase were decreased, while those in the S phase were increased with an inhibited cell apoptosis rate in the NSCs. Besides, the overexpression of Shrm4 or activation of GABAB1 upregulated the levels of Shrm4, GABAB1, Bcl-2, MAP-2 and SOCS2, while downregulated Bax and cleaved Caspase-3 in NSCs. Overall, overexpression of Shrm4 activated GABAB1 to stimulate the proliferation and differentiation of NSCs. Thus, Shrm4 might be considered as a novel target for promoting the proliferation and differentiation of NSCs.