Myogenic Potential of Whole Bone Marrow Mesenchymal Stem Cells In Vitro and In Vivo for Usage in Urinary Incontinence

Urinary incontinence, defined as the complaint of any involuntary loss of urine, is a pathological condition, which affects 30% females and 15% males over 60, often following a progressive decrease of rhabdosphincter cells due to increasing age or secondary to damage to the pelvic floor musculature, connective tissue and/or nerves. Recently, stem cell therapy has been proposed as a source for cell replacement and for trophic support to the sphincter. To develop new therapeutic strategies for urinary incontinence, we studied the interaction between mesenchymal stem cells (MSCs) and muscle cells in vitro; thereafter, aiming at a clinical usage, we analyzed the supporting role of MSCs for muscle cells in vitro and in in vivo xenotransplantation. MSCs can express markers of the myogenic cell lineages and give rise, under specific cell culture conditions, to myotube-like structures. Nevertheless, we failed to obtain mixed myotubes both in vitro and in vivo. For in vivo transplantation, we tested a new protocol to collect human MSCs from whole bone marrow, to get larger numbers of cells. MSCs, when transplanted into the pelvic muscles close to the external urethral sphincter, survived for a long time in absence of immunosuppression, and migrated into the muscle among fibers, and towards neuromuscular endplates. Moreover, they showed low levels of cycling cells, and did not infiltrate blood vessels. We never observed formation of cell masses suggestive of tumorigenesis. Those which remained close to the injection site showed an immature phenotype, whereas those in the muscle had more elongated morphologies. Therefore, MSCs are safe and can be easily transplanted without risk of side effects in the pelvic muscles. Further studies are needed to elucidate their integration into muscle fibers, and to promote their muscular transdifferentiation either before or after transplantation.     

Cell Proliferation and Ageing In Mouse Colon

The descending colon of 4 month and 2 year old mice was exposed to 1250 rad X-rays. This killed most of the epithelial cells. the surviving cells formed new crypts and surface epithelium in animals of both ages. Not all the crypts were replaced. the irradiated area contained no more than 80% of the control number of crypts per section for at least 6 weeks after irradiation. In the young mice new crypts were much larger and the labelling index (LI) was much higher than in unirradiated animals during the first week after irradiation. In the old mice the overshoot in LI and crypt size began later and continued longer than in young animals. This may be because the control of cell proliferation was much less precise in old than in young mice. The irradiation was repeated, in an attempt to age prematurely the epithelial cells by increasing the number of divisions they underwent. the overshoot in LI and cells per crypt was smaller after a second dose than after the first in both young and old mice. There was almost no overshoot after a third dose was given to young mice. Increasing the number of divisions undergone by the surviving epithelial cells did not change the timing of repopulation in young mice compared to that found in old mice. Little evidence was found for the presence of a limited proliferative lifespan in colon epithelial cells.     

Quantitation of Satellite Cell Proliferation in Vivo Using Image Analysis

A nonisotopic, double fluorescence technique was developed to study myogenic satellite cell proliferation in posthatch turkey skeletal muscle. Labeled satellite cell nuclei were identified on enzymatically isolated myofiber segments using a mouse monoclonal antibody (anti-BrdU) followed by fluorescein-5-isothiocyanate (FITC) conjugated goat anti-mouse IgG secondary antibody. Myofiber nuclei (myonuclei + satellite cell nuclei) were counterstained with propidium iodide (PI). The myofiber segment length, myofiber segment diameter, and the number of PI and FITC labeled nuclei contained in each segment was determined using a Nikon fluorescence microscope, a SIT video camera and Image-1 software. Data collected by three different operators of the image analysis system revealed 5.0 ± 1.4 satellite cell nuclei per 1000 myofiber nuclei and 5284 ± 462 μm³ of cytoplasm surrounding each myofiber nucleus in the pectoralis thoracicus of 9-week-old tom turkeys. BrdU immunohistochemistry coupled with the new approach of PI staining of whole myofiber mounts is an effective combination to allow the use of an efficient semi-automated image analysis protocol.     

WT1 Promotes Cell Proliferation in Non-Small Cell Lung Cancer Cell Lines through Up-Regulating Cyclin D1 and p-pRb In Vitro and In Vivo

The Wilms’ tumor suppressor gene (WT1) has been identified as an oncogene in many malignant diseases such as leukaemia, breast cancer, mesothelioma and lung cancer. However, the role of WT1 in non-small-cell lung cancer (NSCLC) carcinogenesis remains unclear. In this study, we compared WT1 mRNA levels in NSCLC tissues with paired corresponding adjacent tissues and identified significantly higher expression in NSCLC specimens. Cell proliferation of three NSCLC cell lines positively correlated with WT1 expression; moreover, these associations were identified in both cell lines and a xenograft mouse model. Furthermore, we demonstrated that up-regulation of Cyclin D1 and the phosphorylated retinoblastoma protein (p-pRb) was mechanistically related to WT1 accelerating cells to S-phase. In conclusion, our findings demonstrated that WT1 is an oncogene and promotes NSCLC cell proliferation by up-regulating Cyclin D1 and p-pRb expression.     

Comparative Cardiac Toxicity of Anthracyclines In Vitro and In Vivo in the Mouse

Purpose

The antineoplastic efficacy of anthracyclines is limited by their cardiac toxicity. In this study, we evaluated the toxicity of doxorubicin, non-pegylated liposomal-delivered doxorubicin, and epirubicin in HL-1 adult cardiomyocytes in culture as well as in the mouse in vivo.

Methods

The cardiomyocytes were incubated with the three anthracyclines (1 µM) to assess reactive oxygen generation, DNA damage and apoptotic cell death. CF-1 mice (10/group) received doxorubicin, epirubicin or non-pegylated liposomal-doxorubicin (10 mg/kg) and cardiac function was monitored by Doppler echocardiography to measure left ventricular ejection fraction (LVEF), heart rate (HR) and cardiac output (CO) both prior to and 10 days after drug treatment.

Results

In HL-1 cells, non-pegylated liposomal-doxorubicin generated significantly less reactive oxygen species (ROS), as well as less DNA damage and apoptosis activation when compared with doxorubicin and epirubicin. Cultured breast tumor cells showed similar sensitivity to the three anthracyclines. In the healthy mouse, non-pegylated liposomal doxorubicin showed a minimal and non-significant decrease in LVEF with no change in HR or CO, compared to doxorubicin and epirubicin.

Conclusion

This study provides evidence for reduced cardiac toxicity of non-pegylated-liposomal doxorubicin characterized by attenuation of ROS generation, DNA damage and apoptosis in comparison to epirubicin and doxorubicin.     

MiR-20a Promotes Cervical Cancer Proliferation and Metastasis In Vitro and In Vivo

MicroRNAs (miRNAs) are small, non-coding RNAs that are critical regulators of various diseases. MicroRNA-20a (miR-20a) has previously significantly altered in a range of cancers. In this study, we detected the relationship between miR-20a and the development of cervical cancer by qRT-PCR, we found that the expression level of miR-20a was significantly higher in cervical cancer patients than in normal controls, the aberrant expression of miR-20a was correlated with lymph node metastasis, histological grade and tumor diameter. Then we successfully established the stable anti-miR-20a cervical cancer cell lines by lentivirus. Inhibited miR-20a prevented tumor progression by modulating cell cycle, apoptosis, and metastasis in vitro and in vivo. TIMP2 and ATG7 were proved to be direct targets of miR-20a, using luciferase assay and western blot. These results indicate that miR-20a suppresses the proliferation, migration and invasion of cervical cancer cell through targeting ATG7 and TIMP2. Our results support the involvement of miR-20a in cervical tumorigenesis, especially lymph node metastasis. We propose that miRNAs might be used as therapeutic agent for cervical cancer.     

Deregulated HOXB7 Expression Predicts Poor Prognosis of Patients with Esophageal Squamous Cell Carcinoma and Regulates Cancer Cell Proliferation In Vitro and In Vivo

Background

We observed abnormal HOXB7 expression in esophageal squamous cell carcinoma (ESCC) previously. This study was to evaluate the prognostic significance of HOXB7 and reveal the potential mechanism.

Methods

Immunohistochemistry was used to confirm the abnormal expression of HOXB7 in ESCC. The prognostic significance of HOXB7 expression was analyzed in two independent cohorts. RNAi was used to establish two stable HOXB7-knockdown cell strains. CCK8 assay, cell growth curve assay, colony formation assay, flow cycle analysis and tumorigenicity assay in nude mice were employed to investigate the effect of HOXB7 on proliferation in vitro and in vivo.

Results

Immunohistochemistry confirmed the abnormal expression of HOXB7 in ESCC compared with paracancerous mucosa (18/23 vs. 9/23, p=0.039). HOXB7 expression was positively correlated with the T stage, lymph node metastasis and TNM stage. The median survival of patients with high HOXB7 expression was significantly shorter than that with low expression (45 months vs. 137 months, p = 0.007 for cohort 1; 19 months vs. 34 months, p = 0.001 for cohort 2). Multivariate survival analysis showed that HOXB7 expression was another independent prognostic factor (HR [95% CI] = 0.573 [0.341–0.963], p = 0.036 for cohort 1; HR [95%CI] = 0.543 [0.350–0.844], p = 0.024 for cohort 2). Experiments in vitro and in vivo showed that after knockdown of HOXB7, the proliferation rate dropped, growth rate descended, colony-formation ability reduced, G1-phase arrest occurred and the tumorigenicity reduced remarkably.

Conclusions

HOXB7 could promote cancer cell proliferation and might be an independent prognostic factor for patients with ESCC.     

AMP-Activated Protein Kinase Suppresses the In Vitro and In Vivo Proliferation of Hepatocellular Carcinoma

AMP-activated protein kinase (AMPK) is a central metabolic sensor and plays an important role in regulating glucose, lipid and cholesterol metabolism. Therefore, AMPK is a key therapeutic target in diabetes. Recent pilot studies have suggested that diabetes drugs may reduce the risk of cancer by affecting the AMPK pathway. However, the association between AMPK and the proliferation of hepatocellular carcinoma (HCC) is unknown. In this study, we investigated the relationship between AMPK activity and the proliferation of HCC in cell lines, nude mice and human clinic samples. We first investigated the relationship between AMPK activity and cell proliferation in two HCC cell lines, PLC/PRF/5 and HepG2, by two AMPK activators, 5-aminoimidazole-4-carboxamide-1-h-D-ribofuranoside (AICAR) and metformain. AICAR and metformin treatment significantly inhibited the proliferation of HCC cells and induced cell cycle arrest at G1-S checkpoint. We then observed that metformin abrogated the growth of HCC xenografts in nude mice. The clinical pathology of AMPK activity in HCC, including cell proliferation, differential grade, tumor size and microvessel density, was studied by using 30 clinical tissue samples. In HCC tissue samples, phosphorylated AMPK was expressed mainly in cytoplasm. AMPK activity decreased significantly in HCC in comparison with paracancerous liver tissues (P<0.05). AMPK activity was negatively correlated with the level of Ki-67 (a marker of cell proliferation), differential degradation and tumor size (P<0.05), but not with microvessel density, hemorrhage or necrosis in HCC. Our findings suggest that AMPK activity inhibits the proliferation of HCC and AMPK might be an effective target for prevention and treatment of HCC.     

Effects of Propyzamide on Tobacco Cell Microtubules In Vivo and In Vitro

Treatment with propyzamide at 2 ? 10^-6 M or at higher concentrationsarrested the cell cycleat metaphase in tobacco BY-2 cells. Metaphasecells having disorganized spindle microtubulesand scatteredchromosomes began to appear within several minutes of the additionof propyzamide. Within 30 min, disrupted spindle microtubulesand dispersed chromosomes were seenin all metaphase cells. Propyzamideat 2 ? 10^-6 M or at higher concentrations also disrupted corticalmicrotubules, but disruption of cortical microtubules requiredmore time than disruption of spindle microtubules. The effectof propyzamide on microtubules was found to be readily reversible.The cells arrested at metaphase by 2 ? 10^-6 M propyzamide resumedmitosis within 2 h from the termination of treatment with propyzamide.Spindle microtubules reappeared within 15 min from the terminationof treatment with propyzamide, and the cortical microtubuleswithin 1 h. Tubulin was isolated from tobacco BY-2 cells bycolumn chromatography on ethyl Nphenylcarbamate-Sepharose 4B.On incubation with EGTA, Mg²⁺ and DMSO, the purified tobaccotubulin polymerized into microtubules. Propyzamide at 1 ? 10^-4M completely inhibitedthe polymerization of tobacco tubulin,but did not inhibit polymerization of bovine braintubulin. Tobaccotubulin was adsorbed onto a column of propyzamide-analogue-linkedSepharose 4B and then purified by chromatography on this column. (Received February 15, 1988; Accepted June 29, 1988)     

Micheliolide Derivative DMAMCL Inhibits Glioma Cell Growth In Vitro and In Vivo

BackgroundThere is no highly effective chemotherapy for malignant gliomas to date. We found that dimethylaminomicheliolide (DMAMCL), a selective inhibitor of acute myeloid leukemia (AML) stem/progenitor cells, inhibited the growth of glioma cells.MethodsThe distribution of DMAMCL in brain was analyzed by an ultraperformance liquid chromatography-mass spectrometry (UPLC-MS/MS) system. The anti-tumor evaluations of DMAMCL in vitro were performed by MTT, FACS and RT-PCR. In vivo, the mixture of C6 cells and matrigel was injected into caudatum, and the anti-tumor activity of DMAMCL was evaluated by tumor growth and rat survival. The toxicity of DMAMCL was evaluated by body weight, daily food intake, hematological or serum biochemical analyses, and histological appearance of tissues.ResultsThe IC₅₀ values of DMAMCL against the C6 and U-87MG cell lines in vitro were 27.18 ± 1.89 μM and 20.58 ± 1.61 μM, respectively. DAMMCL down-regulated the anti-apoptosis gene Bcl-2 and increased apoptosis in C6 and U-87MG cells in a dose-dependent manner. In a C6 rat tumor model, daily administration of DMAMCL for 21 days reduced the burden of C6 tumors by 60% to 88% compared to controls, and more than doubled the mean lifespan of tumor-bearing rats. Distribution analysis showed that the DMAMCL concentration was higher in the brain than in plasma. Evaluations for toxicity revealed that oral administration of DMAMCL at 200 or 300 mg/kg once a day for 21 days did not result in toxicity.ConclusionsThese results suggest that DMAMCL is highly promising for the treatment of glioma.     

Hypoxia Increases the Cyclic AMP Content of the Cat Carotid Body In Vitro

The cyclic AMP content of cat carotid bodies in vitro measured with a radioimmunoassay under control conditions (PO2: 230 torr) was 0.79 +/- 0.10 pmol/carotid body (n = 10). Lowering medium PO2 to 20 torr for 2 min significantly increased cyclic AMP content to 1.13 +/- 0.14 pmol/carotid body (n = 10). This increase was inhibited neither by propranolol (34 microM) nor by propranolol plus haloperidol (27 microM). Inhibition of the cyclic nucleotide phosphodiesterase with 1-methyl-3-isobutylxanthine (0.8 mM) provoked a fast and large increase in cyclic AMP during both control and hypoxic conditions. The cyclic AMP increase induced by hypoxia was still observed when extracellular Ca2+ was absent. Inhibition of the adenylate cyclase by N-(cis-2-phenylcyclopentyl)azacyclotridecan-2-imine hydrochloride (MDL 12330A; 20-1,000 microM) under zero-Ca2+ conditions irreversibly inhibited the cyclic AMP increase produced by hypoxia. Similarly, inhibition of the Ca2(+)-calmodulin complex by trifluoperazine (0.2 mM) or calmidazolium (R 24571; 50-200 microM) prevented the cyclic AMP response. These results suggest that cyclic AMP may be involved in the PO2-sensing mechanism of the carotid body. Hypoxia appears to activate adenylate cyclase directly and independent of any hormone-receptor interactions.     

False In Vitro and In Vivo Elevations of Uric Acid Levels in Mouse Blood

Uric acid (UA) levels in mouse blood have been reported to range widely from 0.1 μM to 760 μM. The aim of this study was to demonstrate false in vitro and in vivo elevations of UA levels in mouse blood. Male ICR mice were anesthetized with pentobarbital (breathing mice) or sacrificed with overdose ether (non-breathing mice). Collected blood was dispensed into MiniCollect® tubes and incubated in vitro for 0 or 30 min at room temperature. After separation of plasma or serum, the levels of UA and hypoxanthine were determined using HPLC. From the non-incubated plasma of breathing mice, the true value of UA level in vivo was 13.5 ± 1.4 μM. However, UA levels in mouse blood increased by a factor of 3.9 following incubation in vitro. This “false in vitro elevation” of UA levels in mouse blood after blood sampling was inhibited by allopurinol, a xanthine oxidase inhibitor. Xanthine oxidase was converted to UA in mouse serum from hypoxanthine which was released from blood cells during incubation. Plasma UA levels from non-breathing mice were 19 times higher than those from breathing mice. This “false in vivo elevation” of UA levels before blood sampling was inhibited by pre-treatment with phentolamine, an α-antagonist. Over-anesthesia with ether might induce α-vasoconstriction and ischemia and thus degrade intracellular ATP to UA. For the accurate measurement of UA levels in mouse blood, the false in vitro and in vivo elevations of UA level must be avoided by immediate separation of plasma after blood sampling from anesthetized breathing mice.     

Arginase Activity is Inhibited by l -NAME,both In Vitro and In Vivo

The present study investigated the ability of the arginine analog L -NAME (N^ω-Nitro- L -arginine methyl ester) to modulate the activity of arginase. L -NAME inhibited the activity of arginase in lysates from rat colon cancer cells and liver. It also inhibited the arginase activity of tumor cells in culture. Furthermore, in vivo treatment of rats with L -NAME inhibited arginase activity in tumor nodules and liver, and the effect persisted after treatment ceased. The effect of L -NAME on arginase requires consideration when it is used in vivo in animal models with the aim of inhibiting endothelial NO-synthase, another enzyme using arginine as substrate.     

Purification and Characterization of the Lectin from Taro (Colocasia esculenta) and Its Effect on Mouse Splenocyte Proliferation In Vitro and In Vivo

Lectins are proteins found in a wide range of organisms, with the ability to bind reversibly to specific carbohydrates. They can display important biological activities, such as the activation of the cell cycle in lymphocytes. Storage proteins with lectin activity have been reported in tuberous plant species, such as Colocasia esculenta, popularly known as taro. A simple strategy based on Cibacron Blue chromatography was used to purify a 12 kDa polypeptide 1.3-fold, with a recovery of 30 %. The purified protein was identified as tarin by mass spectrometry, which indicated that it was present in G1a/G1d isoforms. Tarin exhibited both agglutinating activity against hamster erythrocytes and mitogenic activity in vitro and in vivo toward mouse splenocytes. Optimum cellular proliferation in vitro was achieved by 625 ng of the crude extract or 500 ng of the purified tarin. Total mouse splenocyte proliferation measured after 5 days of intraperitoneal inoculation of purified tarin was increased 3.3-fold in comparison to the control group. Half of the proliferating cells were identified as B lymphocytes by flow cytometry. These results show that this is an efficient and simple strategy to purify tarin and aid in establishing this protein as a new therapeutic drug, able to promote cell proliferation in a murine model.     

Salinomycin Inhibits Proliferation and Induces Apoptosis of Human Hepatocellular Carcinoma Cells In Vitro and In Vivo

The anti-tumor antibiotic salinomycin (Sal) was recently identified as a selective inhibitor of breast cancer stem cells; however, the effect of Sal on hepatocellular carcinoma (HCC) is not clear. This study aimed to determine the anti-tumor efficacy and mechanism of Sal on HCC. HCC cell lines (HepG2, SMMC-7721, and BEL-7402) were treated with Sal. Cell doubling time was determinated by drawing growth curve, cell viability was evaluated using the Cell Counting Kit 8. The fraction of CD133⁺ cell subpopulations was assessed by flow cytometry. We found that Sal inhibits proliferation and decreases PCNA levels as well as the proportion of HCC CD133⁺cell subpopulations in HCC cells. Cell cycle was analyzed using flow cytometry and showed that Sal caused cell cycle arrest of the various HCC cell lines in different phases. Cell apoptosis was evaluated using flow cytometry and Hoechst 33342 staining. Sal induced apoptosis as characterized by an increase in the Bax/Bcl-2 ratio. Several signaling pathways were selected for further mechanistic analyses using real time-PCR and Western blot assays. Compared to control, β-catenin expression is significantly down-regulated upon Sal addition. The Ca²⁺ concentration in HCC cells was examined by flow cytometry and higher Ca²⁺ concentrations were observed in Sal treatment groups. The anti-tumor effect of Sal was further verified in vivo using the hepatoma orthotopic tumor model and the data obtained showed that the size of liver tumors in Sal-treated groups decreased compared to controls. Immunohistochemistry and TUNEL staining also demonstrated that Sal inhibits proliferation and induces apoptosis in vivo. Finally, the role of Sal on in vivo Wnt/β-catenin signaling was evaluated by Western blot and immunohistochemistry. This study demonstrates Sal inhibits proliferation and induces apoptosis of HCC cells in vitro and in vivo and one potential mechanism is inhibition of Wnt/β-catenin signaling via increased intracellular Ca²⁺ levels.