Increase In Proportion Of Cells Research Articles

DNA Methylation-Derived Immune Cell Proportions and Cancer Risk in Black Participants.

This study describes associations between immune cell types and cancer risk in a Black population; elevated regulatory T-cell proportions that were associated with increased overall cancer and lung cancer risk, and elevated memory B-cell proportions that were associated with increased prostate and all cancer risk.

Novel sulfonyl-substituted tetrandrine derivatives for colon cancer treatment by inducing mitochondrial apoptosis and inhibiting PI3K/AKT/mTOR pathway

Tetrandrine (TET) possesses multiple pharmacological activities and could suppress tumor proliferation via PI3K pathway inhibition. However, inferior antitumor activity and potential toxicity limit its clinical application. In the present study, a series of 14-sulfonamide and sulfonate TET derivatives were designed, synthesized, and evaluated for biological activities. Through structural-activity relationship studies, compound 3c with α, β-unsaturated carbonyl group exhibited the most potent activity against all tested tumor cell lines (including Hela, HCT116, HepG2, MCF-7, and SHSY5Y), as well as negligible toxicity against normal cell lines LO2 and HEK293. Additionally, compound 3c effectively inhibited HCT116 and CT26 cell proliferation in vitro with increased cell proportion in the G2/M phase, activated the mitochondrial apoptosis pathway, and induced colon cancer cell apoptosis by suppressing the PI3K/AKT/mTOR pathway. The further molecular docking results confirmed that compound 3c is potentially bound to multiple residues in PI3K with a stronger binding affinity than TET. Ultimately, compound 3c dramatically suppressed tumor growth in the CT26 xenograft tumor model, without noticeable visceral toxicity detected in the high-dose group. In summary, compound 3c might present new insights for designing new PI3K inhibitors and be a potential candidate for colon cancer treatment.

Fance deficiency inhibits primordial germ cell proliferation associated with transcription–replication conflicts accumulate and DNA repair defects

Fanconi anemia (FA) gene mutations are critical components in the genetic etiology of premature ovarian insufficiency (POI). Fance−/− mice detected meiotic arrest of primordial germ cells (PGCs) as early as embryonic day (E) 13.5 and exhibited decreased ovarian reserve after birth. However, the mechanism of Fance defect leading to dysgenesis of PGCs is unclear. We aimed to explore the effect of Fance defects on mitotic proliferation of PGCs. Combined with transcriptomic sequencing and validation, we examined the effect of Fance defects on cell cycle, transcription–replication conflicts (TRCs), and multiple DNA repair pathways in PGCs during active DNA replication at E11.5 and E12.5. Results showed Fance defects cause decreased numbers of PGCs during rapid mitosis at E11.5 and E12.5. Mitotic cell cycle progression of Fance−/− PGCs was blocked at E11.5 and E12.5, shown by decreased cell proportions in S and G2 phases and increased cell proportions in M phase. RNA-seq suggested the mechanisms involved in DNA replication and repair. We found Fance−/− PGCs accumulate TRCs during active DNA replication at E11.5 and E12.5. Fance−/− PGCs down-regulate multiple DNA repair pathways at E11.5 and E12.5 including the FA pathway, homologous recombination (HR) pathway, and base excision repair (BER) pathway. In conclusion, Fance defect impaired the mitotic proliferation of PGCs leading to rapidly decreased numbers and abnormal cell cycle distribution. Proliferation inhibition of Fance−/− PGCs was associated with accumulated TRCs and down-regulation of FA, HR, BER pathways. These provided a theoretical basis for identifying the inherited etiology and guiding potential fertility management for POI.

HIV Infection Induces Aberrant Myeloid Lineage Differentiation

AbstractIntroduction: Numerous studies on hematopoietic changes caused by HIV infection have focused on immune cell depletion, while research on other hematopoietic lineages development have been sparse and inconclusive. In this study, we used precise markers to detect changes in the distribution of bone marrow hematopoietic stem/progenitor cells in HIV-infected patients and analyzed the dynamic changes of mature cells of each lineages in 782 patients with disease progression. Furthermore, we sought to explore the potential relationship between abnormal development of these lineage cells and survival of HIV patients. Our study provides evidence for a more comprehensive understanding of the effects of HIV on hematopoiesis. Methods: We included 782 HIV-infected patients who underwent bone marrow aspiration from January 2016 to October 2020 in Zhongnan Hospital, and were followed up until January 2022. Examination including flow cytometry and bone marrow aspiration were performed to detect the development of hematopoietic cells. 509 cases were tested for lymphocyte subsets. According to CD3+CD4+ cell count, 325 patients (63.9%) were in the very low group, 61 patients (12.0%) in the low group, 55 patients (10.8%) in the medium group, and 68 patients (13.4%) in the high group. By January 2022, 84 patients had been lost to follow-up and 425 patients had accurate and complete follow-up data. We analyzed the risk factors for HIV death in 425 patients and established a predictive model. Results: Flow cytometry analysis showed an increased proportion of granulocytemonocyte progenitors (GMP) but a decreased proportion of common lymphoid progenitors (CLP) in HIV patients. In myelogram, the percentage of granulocytes and granulocytes/erythroid cells (G/E) increased with the decrease of CD3+CD4+ count (both P<0.001). In addition to the increase in cell proportion, we also observed that the morphologic abnormalities in granulocyte increased with the progression of the disease, including toxic granulation, cytoplasmic vacuolization, Intracellular and extracellular plasma (P<0.001), Döhle bodies (P=0.033) and increased lobulation in megakaryocyte (P=0.048). Subsequent LASSO binary logistic regression also confirmed that the increased granulocyte ratio was an important factor in patients' disease. Conclusion: HIV infection directly or indirectly induces bias on myeloid lineage differentiation. Increased granulocyte ratio is an important risk factor for disease aggravation and death in HIV patients. Our research provide a new perspective for evaluating the status and prognosis of HIV patients by focusing on the changes of myeloid granulocytes. Disclosures No relevant conflicts of interest to declare.

Exosomes from miR-141-Inhibitor-Modified Bone Marrow Stem Cells Restrain the Proliferation of Prostate Cancer Cells

miR-141 is highly expressed in prostate cancer. miR-141 can down-regulate PTEN to enhance Akt expression, thereby facilitating the malignant features of cells. Bone marrow stem cells (BMSCs) are one type of promising therapeutic carrier for the exosomes. This study assessed the effect of exosomes originating from the miR-141-inhibitor-modified BMSCs on prostate cancer cells. For in vitro experiment, a prostate cancer cell line (DU145 cells) was employed and when they reach a logarithmic growth phase, they were co-cultured with BMSCs and then assigned into miR-141 down group, miR-141 up group, NC group and control group followed by analysis of miR-141 expression by quantitative-PCR, cell proliferation by MTT assay, cell cycle and apoptosis by flow cytometry, and P13K/Akt/mTOR pathway-related proteins expression by western blotting. The exosomes originating from the miR-141-inhibitor-modified BMSCs decreased miR-141 expression, restrained cell proliferation, increased cell proportions in G0/G1 and G2 phases, while decreased cell proportions in S phase. Control group and miR-141 down group exhibited weak expression of PI3K/Akt/mTOR signal proteins, which included p-mTOR, p-P70S6K, p-4E-BP1, p-Akt and p-PTEN. In conclusion, exosomes from the miR-141-inhibitor-modified BMSCs can restrain prostate cancer cell proliferation and increase apoptosis possibly through targeting PTEN to modulate the P13K/Akt/mTOR signal transduction pathway.

Multi-Omics Uncover Neonatal Cecal Cell Development Potentials

Although, the cecum plays vital roles in absorption of water, electrolytes, and other small molecules, and harbors trillions of commensal bacteria to shape large intestine immune functions, it is unknown the cecum development potentials at single cell level during the very crucial neonatal developmental period. Using singe cell RNA-seq and proteomics, we have characterized six major types of cecal cells: undifferentiated cells; immune cells (Ims); cecumocytes (CCs); goblet, Paneth like cells (PLCs), and enteroendocrine cells (EECs) with specific markers. CCs mature with a gradual decrease in proportion of cells; however, Ims develop with a continuing increase in proportion of cells. Meanwhile, goblet and EEC cells reduced in proportion of cells from do to d14 or d21; PLCs increased in proportion of cells from d0 to d7 then decreased at d14 and d21. The cells exhibit specific development and maturation trends controlled by transcriptional factors, ligand-receptor pairs, and other factors. As piglets grow, cecal content and mucosal microbial diversity increases dramatically with population of beneficial microbiota, such as lactobacillus. Moreover, cecal mucosal-associated and cecal content microbiota are positively correlated and both show significant correlation with different types of cecal cells and plasma metabolites. This is the first presentation of neonatal cecal cell development and maturation naturally at single cell level with transcript, protein, microbiota and metabolism perspectives. Furthermore, this study provides an important tool for the determination of novel interventions in cecal drug delivery and metabolism studies.

Dihydroartemisinin induces cell apoptosis through repression of UHRF1 in prostate cancer cells.

Prostate cancer (PCa) seriously jeopardizes men's health worldwide. Dihydroartemisinin, which is an effective antimalarial agent, has shown potential anticancer effects in various human cancer cell lines, including PCa cells. However, the mechanisms underlying the anticancer activity of dihydroartemisinin are not fully understood. Ubiquitin-like with plant homeodomain and ring finger domain 1 (UHRF1) is highly expressed in a variety of tumors and is negatively correlated with the prognosis of various tumors. We reported previously that UHRF1 is downregulated during apoptosis induced by dihydroartemisinin in PC-3 PCa cells. In this study, we transfected PC-3 cells with lentiviruses containing UHRF1 or shRNA-UHRF1. Then, the cells were treated with dihydroartemisinin at different concentrations. Our data showed that overexpression of UHRF1 promoted cell proliferation and migration in PC-3 cells, inhibited cell apoptosis, increased cell proportion in G2 phase, increased DNA methyltransferase 1 and decreased p16INK4A expression at mRNA and protein levels. Downregulation of UHRF1 produces the opposite results. Moreover, the phenomena caused by overexpression of UHRF1 were inhibited after dihydroartemisinin treatment. Compared with control cells, cells overexpressing UHRF1 can resist the proapoptotic and antiproliferative effects of dihydroartemisinin to a certain extent. The effects of UHRF1 knockdown were further aggravated by dihydroartemisinin treatment, but no statistically significant effect was observed with increasing drug concentration. Our results suggested that dihydroartemisinin decreases proliferation and migration but enhances apoptosis of PCa cells, likely by downregulating UHRF1 and upregulating p16INK4A.

Single-cell transcriptome analysis reveals CNS innate immune landscape plasticity in diet-induced obesity and type 2 diabetes

Abstract Midlife obesity and type 2 diabetes are strong risk factors for late-life cognitive impairment and dementia. Previous studies show that obesity is associated with a heightened state of neuroinflammation, indicating that systemic factors profoundly influence brain innate immunity. Here, we reported that the diversity and plasticity of brain myeloid cells are reshaped in middle-aged mice fed with a high-fat diet (HFD). Using single-cell RNA sequencing, we revealed the heterogeneity of brain-resident myeloid cells, including microglia (MG, Tmem119/P2ry12/Hexb) and border-associated macrophages (BAMs, Mrc1/Ms4a7/Pf4). MG from HFD-fed mice exhibited changes of genes involved in oxidative phosphorylation and lipoprotein metabolism, suggesting a modulated cellular metabolism in MG responding to HFD. A subset of MG displayed induced disease-associated MG (DAM) gene signature during HFD feeding. We found that Apoe was predominantly expressed in BAM clusters and up-regulated in HFD-fed MG clusters. Several distinct subsets of BAMs enriched in MHC-II (H2-Eb1, H2-Aa, and Cd74), or leukocyte adhesion and migration (Clec4n, Ccl7, Vcam1) genes, displayed the increased cell proportion in HFD-fed mice. In addition, HFD feeding led to the IL-10 downregulation in MHC-II-expressing BAMs. These data suggested that pro-inflammatory BAMs might incorporate T cell immunity to potentiate cerebrovascular inflammation during chronic HFD consumption. Overall, our results highlighted the heterogeneity and plasticity of the brain myeloid cells in middle-aged obesity. The identified BAM subsets provide insights into crosstalk between CNS innate immune cells and T cells to feature neuroinflammation in obesity-associated cognitive decline.

Cytotoxic and Apoptotic Efficacy of Concanavalins Isolated from Canavalia ensiformis and Canavalia gladiata on Colon Carcinoma Cells

Concanavalin a lectin from seeds of Canavalia spp. is extracted and purified to study antitumor effect in vitro. Canavalia ensiformis and Canavalia gladiata were tested for their cytotoxicity, apoptotic potential and effect on cell cycle phases in HT-29 colon carcinoma cells. It was revealed that both lectins inhibit growth of HT-29 cells in dose dependent manner without affecting normal Human embryonic kidney 293T cells. The half maximal inhibitory concentration values were 14.86 and 10.45 μg/ml for Canavalia ensiformis and Canavalia gladiata respectively. Both lectins induced apoptotic morphology like chromatin condensation, deoxyribonucleic acid fragmentation and apoptotic bodies in HT-29 cells. Moreover, apoptosis was further confirmed in Canavalia ensiformis and Canavalia gladiata by increased proportion of cells in sub-G0/G1 phase. Apoptosis was induced by down regulation via tumor suppressor protein, serine/ threonine kinase and extracellular signal regulated kinase 1/2 pathways as demonstrated by quantitative reverse transcription polymerase chain reaction. The above results indicated that both lectins selectively induce apoptosis in HT-29 cellss.

Knockdown of mediator complex subunit 19 suppresses the proliferation in prostate cancer LNCaP cells

Objective To study the role of mediator complex subunit 19 (Med 19) in prostate cancer by analyzing the effects of lentivirus-mediated suppression of Med 19 expression on LNCaP prostate cancer cells in vitro and explore the mechanism of proliferation. Methods The lentivirus vectors containing small interfering RNA (siRNA) targeting Med 19 gene were constructed and transfected to LNCaP cells. Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) and Western blotting analysis were used to detect the Med 19 expression in the siRNA group (LNCaP-Med 19-siRNA cells)and the control group (LNCaP-Med 19-scRNA cells) at 72 h after the transfection. The influence of Med 19 on the proliferation of prostate cancer cells were assessed using methyl thiazol tetrazolium (MTT), Soft Agar Assay. Cell cycle was analyzed with flow cytometry assay. Results Med 19 relative mRNA level and Med19 protein expressing in the siRNA group were significantly inhibited (t=-69.1413, P<0.01). Compared with the control group, the siRNA group displayed a significant cell proliferation defect by MTT and the number of colonies (8.61±0.51) was significant decreased (t=-11.948, P<0.01; t=-98.775, P<0.01). The siRNA group displayed an increased proportion of cells (67.29±1.70)% in the G0/G1 phase compared with the control group [(56.23±1.46)%, t=-21.906, P<0.01]. Conclusion Knockdown of Med 19 gene increased the number of human prostate cancer LNCaP cells in G0/G1 phase, resulting in significantly inhibited proliferation and growth of prostate cancer. Key words: Mediator complex subunit 19; Prostate carcinoma; Proliferation

CCR5 silencing reduces inflammatory response, inhibits viability, and promotes apoptosis of synovial cells in rat models of rheumatoid arthritis through the MAPK signaling pathway.

Rheumatoid arthritis (RA) is a chronic inflammatory disorder that can, in severe cases, lead to disability. CC chemokine receptor (CCR), an integral membrane protein, has been suggested to play a key role in the RA developmentThis study is to explore the role of CCR5 silencing in inflammatory response, viability, and apoptosis of synovial cells in RA rats by inactivating the mitogen-activated protein kinase (MAPK) signaling pathway. Microarray analysis was conducted to screen out differentially expressed genes from RA-related chips. The rat model was established by injection of siRNA-CCR5 and PD98059 (inhibitor of mitogen-activated protein kinase kinase 1) to evaluate the role of CCR5 silencing in RA, with the involvement of inflammatory response, synovial cell viability, apoptosis, and cycle. CCR5 was predicted to participate in RA by regulating the MARK pathway. In animal experiments, reduction was identified in arthritis index (AI), CCR5 positive expression rate, levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), matrix metalloproteinase (MMP)-1, and MMP-3 in serum of RA rats after CCR5 siRNA and PD98059 injections. RA rats treated with CCR5 siRNA, and PD98059 presented with inhibition in cell viability, promotion of apoptosis, increase in cell proportion in G0/G1 phase, and shortened the S phase. In addition, the treatment of CCR5 siRNA, and PD98059 resulted in downregulated JNK1, ERK1, p38, Cyclin D1, Cyclin E1, Cyclin B1, and Bcl-2 and upregulated Bax and Cas3. These findings reveal that CCR5 silencing suppresses inflammatory response, inhibits viability, and promotes apoptosis of synovial cells in RA rats by inhibiting MAPK pathway. Therefore, CCR5 silencing may provide a novel therapeutic target for RA.

空气细颗粒物PM2.5对HaCaT细胞增殖、细胞周期及凋亡的影响

Objective To evaluate effects of fine particulate matter PM2.5 in ambient air on the proliferation, cell cycle and apoptosis of a human keratinocyte cell line HaCaT. Methods PM2.5 in haze-fog episodes during the heating season was collected in Beijing from 2015 to 2016, and processed into PM2.5 suspensions. HaCaT cells were divided into several groups to be treated with culture medium alone (control group) , PM2.5 suspensions at different concentrations of 100-400 mg/L (experiment groups, 50-800 mg/L for observation of cellular morphology and analysis of cell proliferation) for 24 hours, or cell culture medium without cells or PM2.5 suspensions (blank group) . Cellular morphological changes were observed under an inverted microscope. Cell counting kit-8 (CCK-8) assay was performed to determine cell survival rate, flow cytometry to determine the cell cycle distribution and detect cell apoptosis, and Western blot analysis to determine the protein expression of cyclin A2 and cyclin-dependent kinase1 (CDK1) . Results Along with the increase of PM2.5 concentration, HaCaT cells lost their normal shape gradually, and the number of viable cells gradually decreased. Compared with the control group (100% ± 4.95%) , the 50-mg/L PM2.5 group showed no changes in cell survival rates (P > 0.05) , while the 100-, 200-, 400- and 800-mg/L PM2.5 group showed significantly lower survival rates (91.77% ± 2.04%, 80.01% ± 1.57%, 57.80% ± 1.56%, 21.98% ± 0.86%, respectively, all P < 0.05) . Flow cytometry revealed that the 100-, 200- and 400-mg/L PM2.5 groups showed gradually increased proportion of cells at S phase, but gradually decreased proportion of cells at G2/M phase compared with the control group (all P < 0.05) . As Western blot analysis showed, the protein expression of cyclin A2 and CDK1 significantly decreased in the 100-, 200- and 400-mg/L PM2.5 groups compared with the control group, which was lowest in the 200-mg/L PM2.5 group (all P < 0.05) . In addition, the 100-, 200- and 400-mg/L PM2.5 groups showed significantly higher total apoptosis rates (9.98% ± 0.21%, 12.56% ± 0.74%, 16.74% ± 1.48%, respectively) compared with the control group (6.24% ± 0.17%, all P < 0.05) . Conclusion PM2.5 can inhibit cell proliferation and promote apoptosis of HaCaT cells, likely by downregulating the expression of cyclin A2 and CDK1 and arresting HaCaT cells at S phase. Key words: Cell proliferation; Apoptosis; Particulate matter; Air; HaCaT cells; PM2.5

BCL2L10 inhibits growth and metastasis of hepatocellular carcinoma both in vitro and in vivo.

BCL2L10 is an apoptosis-related member of the BCL-2 protein family. The role of BCL2L10 in the pathogenesis of hepatocellular carcinoma (HCC) is poorly understood. This study was aimed to investigate the function and underlying mechanisms of BCL2L10 in HCC. BCL2L10 expression in human HCC and corresponding adjacent normal tissues was investigated by quantitative real-time PCR and Western blot. The biological functions of BCL2L10 in HCC cell lines were determined by cell viability, colony formation, cell apoptosis, cell cycle, and cell metastasis assays, and in vivo by tumorigenicity and lung metastasis assays in nude mice. Human cancer pathway PCR array was employed to explore the genes regulated by BCL2L10 in HCC. BCL2L10 was down-regulated in human HCC tissues compared to their adjacent non-tumor tissues. Ectopic expression of BCL2L10 in HepG2 and Huh7 cells suppressed cell growth as evidenced by cell viability and colony formation assay, and induced cell apoptosis. HCC cells transfected with BCL2L10 revealed an increased cell proportion arrested at G2/M phase, concomitant with a reduction in the cell proportion in S-phase as compared with control cells. Additional, BCL2L10 repressed cell migration and angiogenesis. Over-expression of BCL2L10 also restrained the tumorigenecity and lung metastasis capacity in nude mice. The activation of JAK-STAT3 signaling was suppressed by BCL2L10 in HCC. BCL2L10 was down-regulated in human HCC tissues compared to adjacent normal tissues. BCL2L10 suppressed HCC progression through inhibiting cell growth and metastasis. Thus, BCL2L10 functions as a tumor-suppressor in HCC. © 2016 Wiley Periodicals, Inc.

Chalcone derivatives cause accumulation of colon cancer cells in the G2/M phase and induce apoptosis

AimsChalcones, naturally occurring open-chain polyphenols abundant in plants, have demonstrated antiproliferative activity in several cancer cell lines. In the present study, the potential anticancer activity of two synthetic analogues named Ch1 and Ch2 in colon cancer cell line was investigated. Main methodsAntiproliferative activities of both synthetic analogues were assessed by Growth Inhibition Assay (MTT) and xCELLigence cell analysis. Apoptosis was assessed by annexin V/PI staining (early stage) or by DNA fragmentation (final stage). To study the cell death mechanism induced by tested substances, we assessed a series of assays including measurements of the caspase 3 activity, membrane mitochondrial potential (MMP) changes, reactive oxygen species (ROS) production by flow cytometry and expression of important apoptosis-related genes by realtime PCR. Key findingsWe found concentration and time-dependent cytotoxicity, inhibition of proliferation of Caco-2 cells after Ch1 and Ch2 treatment in parallel with G2/M phase cell cycle arrest and increased cell proportion in subG0/G1 population with annexin V positivity. We demonstrated that both Ch1 and Ch2 induced caspase-dependent cell death associated with increased ROS production, suppressed Bcl-2 and Bcl-xL and enhanced Bax expression. Treatment of Ch1 also suppressed α-, α1- and β5-tubulins, on the other hand Ch2 only suppressed α-tubulin expression. SignificancePresented chalcones induce apoptosis by intrinsic pathways, and therefore may be an interesting strategy for cancer therapy.

Cytotoxicity and DNA damage in mouse macrophages exposed to silica nanoparticles.

Silica (SiO2) nanoparticles are being progressively applied in various applications, including cosmetics, food technology, and medical diagnostics. Although crystalline SiO2 is a known carcinogen, the carcinogenicity of SiO2 nanoparticles remains unclear. Here, we assessed the cytotoxic effects and DNA injury induced by exposure to various dosages of SiO2 nanoparticles at 0-2400 mg/mL (0-3200 mg/mL microscale SiO2 as positive control) for 24 h using RAW264.7 cells, followed by methyl tetrazolium (MTT) assay. Cells were also treated by 31.25, 125, and 500 mg/mL SiO2 nanoparticles (500 mg/mL microscale SiO2 as positive control) for 24 h and examined by single cell gel electrophoresis assay (SCEG) and flow cytometry. Outstanding dose-related decline in cell viability was observed with enhancing dosages of SiO2 nanoparticles by MTT assay. The inhibitory concentration 50% of SiO2 nanoparticles and microscale SiO2 was 16690 and 5080 mg/mL, respectively. The comet rate (comet%), length of tail, the percentage in DNA tail (TDNA%) and olive tail moment (OTM) induced by SiO2 nanoparticles were significantly increased in comparison with control and microscale SiO2 at 500 mg/mL. 500 mg/mL SiO2 nanoparticles and microscale SiO2 caused a significant increase in apoptosis rate, decreased proliferation index and increased cell proportions in G0/G1 phases by contrast to the negative control (P < 0.05). This indicates that SiO2 nanoparticles are more cytotoxic than microscale SiO2 particles; they induce DNA injury, increase apoptosis, and decrease the proliferation index in RAW264.7 cells. DNA injury and apoptosis may be involved in reducing cell proliferation.

Specific cell’s nuclei DNA content and cellular heterogeneity of colon adenocarcinoma

Definition of properties and fine pathogenesis mechanisms is a direct condition for improving the effectiveness of the fight against cancer, including colon cancer. Aim. In order to assess the cellular composition heterogeneity of colon adenocarcinoma (AC) on the specific DNA content in the cells’ nuclei 141 biopsies were studied using histological, histochemical, morphological, densitometric and mathematical methods. Methods and results. It was established that in AC with grade increase (G) there is an increase in the relative proportion of cells with nuclei which are most appropriate for functional activity and vice versa. Conclusion. This suggests that AC cellular heterogeneity is not linear, and the function approximates to the parabolic one.

TR-07 * LB100, A NOVEL PROTEIN PHOSPHATASE 2A INHIBITOR, ENHANCES CISPLATIN AND RADIATION MEDIATED CYTOTOXICITY AND OVERCOME CISPLATIN RESISTANCE IN-VITRO

INTRODUCTION: Medulloblastoma is the most common malignant brain tumors. Current therapeutic strategies combining surgery, chemotherapy and radiation offer high survival rate but confer significant treatment-related comorbidity. LB100, a novel small molecule Protein Phosphatase 2A (PP2A) inhibitor, has demonstrated efficacy in a number of non-intracranial tumors. The objective of this study is to assess LB100 effectiveness for treatment of medulloblastoma. METHODS: Experiments were performed using DAOY and D341-MED cell lines: XTT assay was performed to assess for cell viability, immunohistochemistry was performed to assess for DNA double-strand breaks and mitotic catastrophe using gamma H2X and alpha tubulin staining respectively. Flow cytometry was performed for cell cycle analysis. A cisplatin - resistant D341 cell line was developed by constant exposure to escalating concentration of cisplatin (up to 4 uM) resulting in a stably resistant cell line with 8-fold increases in IC50. RESULTS: LB100 enhances cisplatin-mediated cytotoxicity. XTT assay showed the IC50 of cisplatin decreased from ∼7uM to < 2uM and from ∼3uM to <0.5 uM with 5uM LB100 in DAOY and D341 respectively after 48 hours of drug exposure. In DAOY, LB100 enhances radiation induced DNA double-strand breaks with ∼5 fold increase in gamma H2X foci and ∼4 fold increase in proportion of cells undergoing mitotic catastrophe on immunohistochemistry with 5uM LB100. In D341, cell cycle analysis demonstrates ∼ 2 fold increase in percentage of cells in G2/M with 10uM LB100 consistent with increased mitotic catastrophe. There is no difference in sensitivity to increasing concentration of LB100 between wide-type D341 and cisplatin resistant D341 on cell viability suggesting that LB100 has equal potency against wild type and cisplatin resistant cell line. CONCLUSIONS: LB100 treatment is an effective radiation and chemotherapy sensitizing agent in-vitro. Further in-vivo study will provide the preclinical data needed to consider LB100 as an adjuvant agent for treatment of medulloblastoma.

Biological activities of fusarochromanone: a potent anti-cancer agent.

BackgroundFusarochromanone (FC101) is a small molecule fungal metabolite with a host of interesting biological functions, including very potent anti-angiogenic and direct anti-cancer activity.ResultsHerein, we report that FC101 exhibits very potent in-vitro growth inhibitory effects (IC50 ranging from 10nM-2.5 μM) against HaCat (pre-malignant skin), P9-WT (malignant skin), MCF-7 (low malignant breast), MDA-231 (malignant breast), SV-HUC (premalignant bladder), UM-UC14 (malignant bladder), and PC3 (malignant prostate) in a time-course and dose-dependent manner, with the UM-UC14 cells being the most sensitive. FC101 induces apoptosis and an increase in proportion of cells in the sub-G1 phase in both HaCat and P9-WT cell lines as evidenced by cell cycle profile analysis. In a mouse xenograft SCC tumor model, FC101 was well tolerated, non-toxic, and achieved a 30% reduction in tumor size at a dose of 8 mg/kg/day. FC101 is also a potent anti-angiogenenic agent. At nanomolar doses, FC101 inhibits the vascular endothelial growth factor-A (VEGF-A)-mediated proliferation of endothelial cells.ConclusionsOur data presented here indicates that FC101 is an excellent lead candidate for a small molecule anti-cancer agent that simultaneously affects angiogenesis signaling, cancer signal transduction, and apoptosis. Further understanding of the underlying FC101’s molecular mechanism may lead to the design of novel targeted and selective therapeutics, both of which are pursued targets in cancer drug discovery.

Role of Med19 in the proliferation and tumorigenicity of human bladder cancer cells

Objective To study the role of Med19 in bladder cancer by analyzing the effects of lentivirus-mediated suppression of Med19 expression on T24 bladder cancer cells in vitro.Methods The lentivirus vectors containing a small hairpin RNA (shRNA) to target Med19 were constructed.After T24 bladder cancer cells were infected,real-time PCR and Western-blotting were used to study the Med19 expressions in the CON group (non-infected cells),the NC group (Lv-NC-infected cells) and the KD group (Lv-shMed19-infected cells).The influence of Med19 on the proliferation of bladder cancer cells were assessed using MTT,BrdU,colony formation assay and tumorigenicity experiment in mice.Cell cycle was analyzed with flow cytometry assay.Results Med19 relative mRNA level (0.35 ± 0.03) and Med19 protein expressing in the KD group were significantly inhibited (P ＜ 0.05).The KD group displayed an increased proportion of cells (77.50 ± 0.29)％ in the G0/G1 phase compared with the CON group (69.81 ± 0.81)％and NC group (67.53 ± 0.67) ％ (P ＜ 0.05).Compared with the CON group and the NC group,the KD group displayed a significant cell proliferation defect by MTT and BrdU assay and the number of colonies (91.33 ± 6.11) was significant decreased (P ＜ 0.05).On the day 24,the tumor volume (596.64 ± 485.36) mm3 and weight (0.57 ± 0.44) g of the KD group mice were decreased after inoculation into nude mice (P ＜ 0.05).Specific lentivirus-mediated knockdown of Med19 significantly impacted the cell cycle and proliferation of bladder cancer cells.Infected T24 cells nearly lost their tumorigenicity when being inoculated into nude mice.Conclusion Our results provide new evidence of an important role for Med19 in the development of bladder cancer,suggesting that lentiviruses delivering shRNA against Med19 may be a promising tool for bladder cancer therapy. Key words: Med19; Bladder cancer; RNAi; Proliferation

Abstract 201: DD3/PCA3 non-coding RNA regulates prostate cancer cell survival and modulates AR signaling

Abstract The prostate cancer antigen 3 (DD3/PCA3) is a non-coding RNA (ncRNA) specifically expressed in prostate tissues and overexpressed in prostate cancer (PCa) tumors. Although widely applied as a diagnostic marker for PCa, to date nothing has described about its role in PCa biology. We used herein small interfering RNA (siRNA) in order to knockdown DD3 mRNA message as an approach to elucidate DD3 functional roles in PCa cells. LNCaP cell line was been used herein as an in vitro model for DD3 functional assays. siRNA sequences were specifically designed for DD3 exon 4 mRNA sequences (siDD3), as well an scrambled siRNA (siScr), as negative control. LNCaP cells were transiently transfected with siDD3 or siScr and DD3 expression was analysed by real time PCR (qRT-PCR) using DD3 specific oligonucleotides. LNCaP cells transfected with siDD3 demonstrated a marked decrease in cell proliferation and viability, as compared to siScr transfected cells. Further, LNCaP cells in which DD3 was knocked-down presented a significant increase in proportion of cells in SubG0/G1 phase of cell cycle and presenting pyknotic nuclei, indicative of cells undergoing apoptosis. In order to investigate the putative mechanisms underlying the decrease of LNCaP cell survival as a result of DD3 knockdown, we then evaluated the involvement of DD3 on androgen receptor (AR) pro-survival signaling. DD3 expression was significantly uregulated as a result of LNCaP treatment with dihydrotestosterone (DHT), the active androgen metabolite. This effect was reverted by the addition of the AR antagonist, flutamide. Consistent to an AR activation by DHT treatment, LNCaP cells presented a significant upregulation of AR target genes. Notably, siDD3/LNCaP transfected cells significantly inhibited the expression of tested AR responsive genes. Besides, DD3 knockdown was able to counteract DHT stimulatory effects over AR target gene expression. Despite negatively modulating the transcription of AR target genes, DD3 knockdown did not alter Akt and ERK phosphorylation, suggesting that DD3 is mainly controlling the expression of signaling pathways downstream to AR activation. In summary, our findings indicate that DD3 is a ncRNA whose expression is AR regulated and is involved on the control of PCa cell survival and proliferation, in part by modulating the AR signaling pathway and its target genes. These [[Unsupported Character - &amp;#64257;]]ndings correspond to the first description of DD3 roles on PCa cells and could provide new insights into understanding prostate carcinogenesis, besides opening new prospects to use DD3 not only as a biomarker for PCa, but also as an specific target for therapeutic approaches aiming to inhibit PCa growth by negatively modulating AR pro-survival signal and their target genes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 201. doi:1538-7445.AM2012-201