Expression Of Proliferation Marker Ki-67 Research Articles

Loss of miR-199b Impairs Active Cycling of Hematopoietic Stem Cells during Steady-State Hematopoiesis

Several recent studies have showed that dysregulation of microRNA (miRNA) expression in hematopoietic stem cells (HSC) can affect self-renewal of HSCs, and indicated a role for miRNAs in development of acute myeloid leukemia (AML). We and others have reported a significant down-regulation of miR-199b in AML patients. Recently we found that miR-199b is enriched in long-term hematopoietic stem cells (LT-HSC), suggesting that miR199-b may regulate HSCs function. Therefore, to understand the physiologic role of miR-199 in hematopoiesis during homeostasis, we evaluated various hematopoietic stem and progenitor cells (HSPC) populations in mice harboring genetic deletion of miR199b-5p using CRISPR/Cas method. We found that ablation of miR199b resulted in markedly increased frequencies of primitive HSC and MPPs, and analyses of distribution pattern in myeloid progenitor populations showed reduced numbers of common myeloid progenitors (CMPs) biased toward granulocyte-monocyte (GMPs) linage with no changes in megakaryocytic-erythroid progenitors (MEPs). The elevated numbers of HSC and MPPs may indicate that increased proportion of HSC population is actively cycling, thus we analyzed LSK populations for expression of proliferation marker Ki67 along with DNA staining. We found that miR-199b deletion reduces proportion of primitive HSC and MPPs in cell-cycle, which may affect HSC cell self-renewal. Futher cell-cycle analyses revealed that miR-199b null HSCs leave G0 faster to accumulate in G1, but rather do not progress into mitosis, which was recovered upon 5-fluorouracil-induced cytokine burst. These results indicate that loss of miR-199b increases cell cycle duration. To verify that the absence of miR-199b influences proliferation of HSCs we pulsed miR-199b KO and WT mice with BrdU for 16 hours. We found the difference in the cell cycle distribution between HSCs and progenitors, namely reduction of BrdU-positive HSC and MPPs and progression of GMP compartment. These results show that miR-199b deletion decreases HSC active cell cycle by prolonging cell cycle transition during steady-state hematopoiesis and promotes proliferation of myeloid cells. Because quiescent cells only become susceptible to 5-FU during hematopoietic stress, driving them into cycle, we injected 5-FU into miR-199 KO and WT mice once per week until hematopoietic failure occurred. We found that miR199-b KO mice died soon after two subsequent injections, most likely due to the faster HSC exhaust as compared to WT mice. These results show that loss of miR199b produces HSC with reduced quiescence and prolonged cell cycle, however upon stress these cells progress into cell cycle, making them more susceptible for 5-FU treatment. These results demonstrate that miR-199b intrinsically regulates active cycling of HSCs. CFU-S assays showed that miR-199b KO donors showed decreased colonies in spleen, suggesting that miR-199b deletion affects short-term repopulation. In long-term repopulation assay, we observed a significant reduction of HSCs compartment, but elevated numbers of MPPs in host mice transplanted with BM from miR-199 KO mice. This data indicates that loss of miR-199b causes defects in HSC self-renewal and alters HSCs reconstitution potential. To identify potential miR-199b targets in HSCs under steady-state hematopoiesis, we performed a gene profiling in SLAM-HSCs. mRNA levels of several putative miR-199b targets were markedly elevated in miR-199b KO HSCs. These genes are known to be involved in cell adhesion, cell cycle, transcription regulation and chromatin remodeling including Klf12, Tox3 and Cdk18. Our findings reveal a novel functional role for miR-199b in governing HSC maintenance. DisclosuresNo relevant conflicts of interest to declare.

Role of PI3K-Akt and MAPK Signaling in Uranyl Nitrate-Induced Nephrotoxicity.

Uranium is a heavy metal of considerable environmental and occupational concern. It is well-known that the kidney is the major target organ of uranium exposure. Elucidating the mechanistic basis of uranium interactions is essential for monitoring the health risk. In the present study, we investigated the cellular mechanisms involved in uranyl nitrate-induced nephrotoxicity. Male Swiss albino mice were administrated with a single intraperitoneal dose of 2 and 4mg/kg of uranyl nitrate at different time points 1, 3, 5, 7, 14, and 28days. Uranyl nitrate intoxication-induced apoptosis in the kidney tissue was observed by TUNEL assay. To assess the proliferation, immunohistochemistry was performed using Ki67 proliferative marker followed by western blotting to confirm the involvement of key signaling molecules. The number of TUNEL positive nuclei peaked at third day after uranyl nitrate insult. The increased expression of proliferation marker Ki67 suggests the enhanced DNA repair process prominently at seventh day. Uranyl nitrate administration also resulted in activation of extracellular signal-regulated kinases (ERK), Akt, and c-Jun N-terminal kinases (JNK) expression. All these changes were found to be time-dependent. The result of the current study suggests that uranyl nitrate induces acute renal injury by activation of apoptosis through JNK pathway, while the early activation of signaling molecules Akt and ERK promotes the tubular cell proliferation and cell survival.

Evaluation of diagnostic utility of imprint cytology in paediatric renal tumours with special references to Ki 67 proliferative marker.

BACKGROUND:Pediatric renal neoplasms comprise about 7%–8% of all neoplasms in children. Wilms tumour (WT) is the most common among pediatric renal tumours.AIMS AND OBJECTIVES:The study was undertaken to study the epidemiological occurrence of pediatric renal tumours in a tertiary care hospital and to ascertain the validity and reliability of touch smear imprint cytology in intraoperative diagnosis of renal tumours and correlate with subsequent histopathological diagnosis and to assess the expression of proliferation marker Ki-67 in different components and stages of WT.MATERIALS AND METHODS:It was a single-institution-based prospective and observational study, conducted for 2 years (from October 2013 to September 2015) in the department of pathology at our hospital. A total of fifty cases were enrolled in this study, all were below 15 years of age.RESULTS:Imprint cytology showed sensitivity, specificity, and diagnostic accuracy of 83%, 98%, and 95.74%, respectively, in diagnosing benign and malignant renal tumours. There was statistically significant correlation of imprint cytology with confirmatory histopathological examination of excision specimen (P < 0.001). Immunohistochemical analysis of Ki-67 was done in all WT cases. Epithelial component had higher proliferative index than blastemal component with P = 0.0082, which was highly statistically significant.CONCLUSION:Imprint cytology is found to be a less expensive, simple, and rapid method, which can be used as an adjunct to histopathology. Correlation between proliferation index as measured with Ki-67 antibody and tumour stage was found. Ki-67 is thus a relevant marker for assessing the proliferative activity.

Red (635 nm), Near-Infrared (808 nm) and Violet-Blue (405 nm) Photobiomodulation Potentiality on Human Osteoblasts and Mesenchymal Stromal Cells: A Morphological and Molecular In Vitro Study.

Photobiomodulation (PBM) has been used for bone regenerative purposes in different fields of medicine and dentistry, but contradictory results demand a skeptical look for its potential benefits. This in vitro study compared PBM potentiality by red (635 ± 5 nm) or near-infrared (NIR, 808 ± 10 nm) diode lasers and violet-blue (405 ± 5 nm) light-emitting diode operating in a continuous wave with a 0.4 J/cm2 energy density, on human osteoblast and mesenchymal stromal cell (hMSC) viability, proliferation, adhesion and osteogenic differentiation. PBM treatments did not alter viability (PI/Syto16 and MTS assays). Confocal immunofluorescence and RT-PCR analyses indicated that red PBM (i) on both cell types increased vinculin-rich clusters, osteogenic markers expression (Runx-2, alkaline phosphatase, osteopontin) and mineralized bone-like nodule structure deposition and (ii) on hMSCs induced stress fiber formation and upregulated the expression of proliferation marker Ki67. Interestingly, osteoblast responses to red light were mediated by Akt signaling activation, which seems to positively modulate reactive oxygen species levels. Violet-blue light-irradiated cells behaved essentially as untreated ones and NIR irradiated ones displayed modifications of cytoskeleton assembly, Runx-2 expression and mineralization pattern. Although within the limitations of an in vitro experimentation, this study may suggest PBM with 635 nm laser as potential effective option for promoting/improving bone regeneration.

RANKL/RANK Pathway and Its Inhibitor RANK-Fc in Uterine Leiomyoma Growth.

Uterine leiomyomas are the most common type of gynecologic tumor in women. To determine the role of the cytokine receptor activator of nuclear factor κ-Β ligand (RANKL); its receptor, receptor activator of nuclear factor κ-Β (RANK); and the RANKL/RANK pathway inhibitor RANK-Fc in leiomyoma growth. Messenger RNA (mRNA) or protein levels of RANKL, RANK, and proliferation markers cyclin D1 and Ki67 were assessed in various leiomyoma tissues and cell populations. Human xenograft experiments were performed to determine the effects of RANK-Fc on leiomyoma growth in vivo. Research laboratory. Twenty-four regularly cycling premenopausal women (age 28 to 49 years) who were not receiving hormone therapy. None. Tumor growth in a murine xenograft model following targeting of the RANKL/RANK pathway with RANK-Fc. RANKL mRNA levels in leiomyoma were significantly higher than those in myometrial tissues. The highest RANK levels were found in the leiomyoma stem cell population, which is deficient in progesterone receptor (PR). Conversely, the highest RANKL levels were found in the PR-rich leiomyoma intermediate cell (LIC) population. R5020, a PR agonist, specifically increased RANKL expression in LICs. RANK-Fc blocked RANKL-induced expression of the proliferative gene cyclin D1. Treatment with RANK-Fc also significantly decreased tumor growth in vivo and diminished the expression of proliferation marker Ki67 in tumors (P < 0.01; n = 4). Treatment with the RANKL/RANK pathway inhibitor RANK-Fc significantly decreased human leiomyoma cell proliferation and tumor growth. This suggests that the RANKL/RANK pathway could serve as a potential target for the prevention and treatment of uterine leiomyoma.

Self-protection of type III fibrocytes against severe 3-nitropropionic-acid-induced cochlear damage in mice.

After intense sound exposure, the lack of obvious degeneration in type III fibrocytes suggests that they might protect themselves against acoustic trauma. However, it is unknown whether and how type III fibrocytes play this role in other cochlear damage models. In this study, we investigated the self-protection of type III fibrocytes against severe cochlear energy failure induced by local administration of 3-nitropropionic acid to the inner ear. We detected that the type III fibrocytes did not degenerate significantly after 500 mM 3-nitropropionic acid application, and showed increased expression of proliferation marker Ki67. Moreover, low immunoreactivity for inducible nitric oxide synthase and cleaved caspase-3 was observed in type III fibrocytes 2 days after damage. These results indicate that after severe cochlear energy failure type III fibrocytes possess obvious proliferation activity, as well as strong antioxidant and antiapoptotic capacity, which can protect them from degeneration.

Inhibition of mircoRNA-34a Enhances Survival of Human Bone Marrow Mesenchymal Stromal/Stem Cells Under Oxidative Stress.

BackgroundMesenchymal stromal/stem cells (MSCs) are broadly used for many diseases, but the efficacy of MSC engraftment is very low due to low viability and high cell death rate under a stressful microenvironment. The present study aimed to investigate whether microRNA-34a (miR-34a), which is a downstream target of P53, is involved in H2O2-induced MSC cell death.Material/MethodHuman bone marrow MSCs (hMSCs) were purchased from Lonza and were cultured as previously described. hMSCs were transfected with miR-34a inhibitor and exposed to H2O2. Cell proliferation assay was used to assess the survival rate of hMSCs. Real-time PCR and Western blot analysis were used to examine proliferation and survival ability of hMSCs.ResultsH2O2 exposure significantly increased miR-34a expression in human bone marrow MSCs. H2O2 challenge induced massive MSC cell death along with reduction of expression of proliferation marker Ki67 and survival-related genes Bcl-2 and Survivin. Transfection of miR-34a inhibitor anti-34a led to a significant protective effect and rescued MSC cell death triggered by H2O2 exposure by 50%. Moreover, anti-34a dramatically increased Bcl-2 and Ki67 mRNA expression levels by over 10-fold compared to the mock control group under H2O2 exposure. The protein levels of Bcl-2 and Survivin were also rescued by anti-34a treatment by 50%.ConclusionsOur results suggest that miR-34a plays a key role in oxidative stress-induced MSC cell death, and targeting miR-34a might be a promising strategy to enhance the survival rate of engrafted stem cells, which may improve therapeutic outcome.

Effects of siRNA Silencing of TUG1 and LCAL6 Long Non-coding RNAs on Patient-derived Xenograft of Non-small Cell Lung Cancer.

The aim of the present study was to establish a patient-derived xenograft (PDX) mouse model of non-small cell lung cancer (NSCLC) and investigate the anti-tumor efficacy of silencing of TUG1 and LCAL6 long non-coding RNA in the PDX model. PDXs were established by subcutaneously implanting NSCLC surgical tumor fragments into immunodeficient mice. PDX characterization was performed by histopathological, immunohistochemical and real-time polymerase chain reaction (RT-PCR) analyses for NSCLC subtype-specific markers and expression of LCAL6 and TUG1. Anti-tumor efficacy of siRNA silencing of TUG1 and LCAL6 was also investigated in the PDX model. The effect of TUG1 and LCAL6 silencing on protein expression of proliferation marker Ki67 and HOX-gene family HOXB7 in the tumors was assessed by immunohistochemical staining and Western blotting. Establishment of NSCLC PDX models resulted in 9 of 26 cases (34.6%). Lung squamous cell carcinomas (SCC) had a higher engraftment rate (58.3%) than lung adenocarcinomas (ADC) (18.2%) (p<0.05). Comparative analysis indicated these established PDX models of NSCLC closely resembled the original tumors with regard to NSCLC subtype-specific markers TTF-1, napsin A, p63 and expression of LCAL6 and TUG1. The tumor volume and weight were significantly reduced in the TUG1-silenced group as compared to the control group (p<0.05). However, no significant tumor growth inhibition was found in the LCAL6-silenced group (p>0.05). Expression of both TUG1and LCAL6 was reduced by siRNA treatment. Expression of Ki67 and HOXB7 was significantly suppressed in both the TUG1- and LCAL6-silenced groups compared to the control group (p<0.01). The TUG1-silenced group showed more reduced Ki67 expression than the LCAL6-silenced group (p<0.05). PDX NSCLC models were established with a high degree of similarity with the original tumor with regard to histological, immunohistochemical features and RNA expression of TUG1 and LCAL6. Silencing of TUG1 inhibited both tumor growth and expression of the proliferation marker Ki67 and HOX-gene family HOXB7 in the PDX model of NSCLC.

Abstract 2040: Superior therapeutic efficacy of nanoparticle albumin-bound paclitaxel over cremophor-bound paclitaxel in experimental esophageal adenocarcinoma

Abstract Introduction: Esophageal adenocarcinoma (EAC) has become the dominant type of esophageal cancer in United States. EAC is the fastest growing cancer in the western world and the overall 5 year survival rate of EAC is below 20 percent. Most patients with EAC present with locally advanced or widespread metastatic disease, where current treatment is largely ineffective. Prognosis for EAC patients remains poor even with combination therapies due to high resistance to chemotherapy. Therefore, new therapeutic approaches are urgently needed. Paclitaxel (PTX) has been used in combination with carboplatin (CP) as a standard combination therapy for advanced EAC. PTX required emulsification with solvents to allow intravenous administration which has resulted in hypersensitivity reactions and potentially dramatic side effects in patients. Nanoparticle albumin-bound (nab) PTX is an albumin-stabilized, cremophor-free and water soluble nanoparticle formulation of PTX. Nab-PTX is a novel microtubule-inhibitory cytotoxic agent and the potential role of nab-PTX has not been tested yet in experimental EAC. Methods: We explored the antiproliferative and antitumor efficacy with survival advantage following CP, PTX and nab-PTX as monotherapy and in combinations in in-vitro, and in murine subcutaneous xenograft and peritoneal metastatic survival models of human EAC. Results: Nab-PTX inhibited in-vitro cell proliferation with significantly lower IC50 (0.25 µM in OE19 and 49 nM in OE33) than that of PTX (0.74 µM in OE19 and 98 nM in OE33) and CP (5.21 µM in OE19 and 1.05 µM in OE33) in OE19 and OE33 EAC cell lines. Nab-PTX treatment resulted in significantly higher antitumour efficacy and survival benefit compared with PTX or CP treatment. After two-week nab-PTX, PTX, CP, nab-PTX+CP or PTX+CP treatments, the average in-vivo local tumor growth inhibition rate was 73, 60, 35, 81 and 68 percent respectively (p=0.025). Nab-PTX treatment increased expression of the mitotic-spindle associated phospho-stathmin, decreased expression of proliferative marker Ki-67 and enhanced apoptosis as confirmed by increased expression of cleaved-PARP and cleaved caspase-3. There was an increase in median animal survival after nab-PTX treatment (65 days) compared to controls (46 days, p=0.0023), PTX (57 days, p=0.0034) or to CP therapy (53 days, p=0.0034). Conclusion: In conclusion, the present study demonstrates that nab-PTX had stronger antiproliferative and antitumor activity in experimental EAC than the current standard chemotherapeutic agents. This strong antitumor activity supports the rationale for clinical evaluation of nab-PTX as promising microtubule-inhibitory agent in EAC. Citation Format: Md Sazzad Hassan, Niranjan Awasthi, Roderich E. Schwarz, Margaret A. Schwarz, Urs von Holzen. Superior therapeutic efficacy of nanoparticle albumin-bound paclitaxel over cremophor-bound paclitaxel in experimental esophageal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2040. doi:10.1158/1538-7445.AM2017-2040

Vascular-Associated Muc4/Vwf Co-Localization in Human Conjunctival Malignant Melanoma Specimens—Tumor Metastasis by Migration?

ABSTRACTPurpose: To investigate whether vascular differentiation marker von Willebrand factor (vWf) and proliferation marker KI67 expression correlate with MUC4 localization around stromal tumor vascularization in human conjunctival malignant melanoma (CMM).Materials and methods: For the purposes of this study, we analyzed samples from human CMMs (n = 4), conjunctival compound nevi (n = 7), and samples from healthy conjunctiva (n = 7) for MUC1, 4, and 16 by immunohistochemistry. To test CMM vessel association of MUC4, we investigated the co-localization of MUC4 with vWf or KI67 in human CMM specimens (n = 10) by immunohistochemistry. Also, we investigated the MUC4 localization around vessels of healthy conjunctiva (n = 10).Results: The immunohistochemical analysis demonstrated membrane-associated mucin expression in epithelia of CMM, nevi and healthy conjunctiva, whereas only MUC4 was localized perivascular in CMM tissue in this preliminary analysis. Co-staining analysis with vWf and KI67 demonstrated MUC4 localization around stromal vessels in human CMM specimens. In contrast, no MUC4 localization has been seen around healthy conjunctiva stroma vessels.Conclusions: MUC4 was detected around vWf/KI67-positive CMM stromal vascular tissue, but not around healthy conjunctival stroma vessels. Therefore, we assume that MUC4 might play a role in tumor cell migration toward vessels inducing metastasis.

The inhibitory effect of MEG3/miR-214/AIFM2 axis on the growth of T-cell lymphoblastic lymphoma

T-cell lymphoblastic lymphoma (T-LBL) is an aggressive malignancy with poor prognosis and high recurrence rate. Long non-coding RNA (lncRNA)-MEG3 is an important tumor suppressor in various cancers. The present study investigated the potential role of maternally expressed gene 3 (MEG3) in the progression of T-LBL. Suppressed expression of MEG3 was detected in T-LBL tissues compared with adjacent histologically normal tissues. Down-regulated level of MEG3 was also found in three T-LBL cell lines (CCRF-CEM, Jurkat and SUP-T1) compared with human T-cell line H9. The proliferation of T-LBL cells was inhibited and cell apoptosis rate was largely promoted when MEG3 was upregulated by a lentiviral vector. Further research revealed that microRNA (miRNA)-214 is a direct target of MEG3. The expression of miR-214 was increased in T-LBL tissues and cell lines compared with control groups. Besides, decreased level of miR-214 was elevated adding miR-214 mimic in SUP-T1 cells transfected with LncRNA-MEG3. Similarly, upregulated level of miR-214 was downregulated adding miR-214 inhibitor in SUP-T1 cells transfected with MEG3 siRNA. Luciferase activity assay further confirmed the targeting relationship between MEG3 and miR-214. Moreover, AIFM2 protein was predicted as a target of miR-214. The expression of AIFM2 was increased by MEG3 and was downregulated by miR-214 mimic. miRNA-214 reversed the effect of MEG3 on inhibiting cell proliferation and inducing cell apoptosis and cell cycle arrest in SUP-T1 cells. Moreover, relative expression of AIFM2 had a positive correlation with the expression of MEG3 and was negatively affected by miR-214. In vivo, MEG3 effectively suppressed tumor growth and the expression of proliferation markers Ki-67 and proliferating cell nuclear antigen (PCNA). Taken together, our research revealed that MEG3 worked as an anti-oncogene in T-LBL, and the MEG3-miR-214-AIFM2 pathway regulated the growth of T-LBL, providing potential prognosis markers as well as new potential targets for T-LBL treatment.

Synergistic effects of coralyne and paclitaxel on cell migration and proliferation of breast cancer cells lines.

Breast cancer is one of the most frequently diagnosed cancer in woman. Triple-negative breast cancer (TNBC) is most aggressive form of breast cancer. There is a growing interest in the use of natural products in combinational chemotherapy to improve the effectiveness in combating proliferation of cancer cells. Here, we hypothesized that coralyne in combination with paclitaxel may exhibit synergistic effect on inhibition of proliferation, migration and induction of apoptosis in MCF-7 and MDA-MB-231 breast cancer cell lines. MTT and BrdU incorporation assays were performed to study the effect of drugs alone and in combination on cell cytotoxicity and proliferation of the breast cancer cell lines, respectively. Adhesion and wound healing assays were performed to study the cell and extracellular matrix interactions. In addition, expression of proliferation marker ki-67 and apoptotic markers Bax and Bcl-2 was determined to study the effect of coralyne in combination with paclitaxel by reverse transcriptase PCR and confirmed by Western blot. The results indicated the synergism between coralyne and paclitaxel on proliferation and migration of breast cancer cell lines. This study also showed that combinational drug treatment decreased the expression of ki-67 and there was an increase in pro apoptotic factor Bax with decreased in expression of anti-apoptotic factor Bcl-2 in breast cancer cell lines with negligible effect on normal breast cell line. Overall, our data described the promising therapeutic potential of coralyne in combination with paclitaxel in treating breast cancer at lower effective dose.

Increased Levels of STAT1 Protein in Blood CD4 T Cells from Systemic Lupus Erythematosus Patients Are Associated with Perturbed Homeostasis of Activated CD45RA-FOXP3hi Regulatory Subset and Follow-Up Disease Severity.

In murine systemic lupus erythematosus (SLE), aberrant regulation of interferon (IFN)-alpha-STAT1 signaling and perturbed homeostasis of CD4+FOXP3+ regulatory T cells (Tregs) were described. In the present study, STAT1 signaling and circulating Treg subsets were assessed by flow cytometry in 39 SLE patients and their potential association with disease course was examined during long-term follow-up. Levels of STAT1 protein as measured by median fluorescence intensity (MFI) were significantly increased in SLE CD4 T cells when compared with rheumatoid arthritis patients and healthy controls and were positively correlated with disease activity. The highest STAT1 MFI was found in CD45RA-FOXP3hi-activated Treg (aTreg) subset, which demonstrated the highest STAT1 phosphorylation responses among SLE CD4 T cells and significant decrease in proliferation marker Ki-67 expression after IFN-alpha stimulation. Percentage of Ki-67+ aTregs was significantly decreased in SLE patients and was negatively correlated with CD4 T cell STAT1 MFI. A subgroup of SLE patients characterized by lower aTreg counts experienced more severe relapsing disease course during 1,000 days of follow-up. Mean CD4 T cell STAT1 MFI in follow-up samples from SLE patients was negatively correlated with mean of follow-up aTreg counts. Our findings indicate that augmented STAT1 signaling may be involved in perturbed aTreg homeostasis, which could represent a possible marker of SLE disease severity.

Abstract P3-04-03: S100β as a predictive biomarker and monitoring tool in endocrine resistant breast cancer

Abstract In estrogen receptor positive breast cancer, endocrine therapy is the standard line of treatment and even though it results in reduced recurrence and mortality, a significant number of patients will eventually relapse. Early detection of metastatic disease would significantly enhance management of endocrine resistant breast cancer. Here we investigate the potential of the calcium-binding protein S100β as a predictive biomarker and monitoring tool in endocrine treated patients. Furthermore, the efficacy of S100β inhibition as therapy in patients that fail first line endocrine therapy was examined. Primary tumor tissue expression of S100β protein was assessed in a retrospective cohort of endocrine treated breast cancer patients. Expression of S100β indicated a significant reduction in time to disease recurrence (n=509, Wilcoxon p&amp;lt;0.0001, hazard ratio 2.43, 95% C.I. is 1.607 to 3.69, p&amp;lt;0.0001, Cox proportional hazard model). S100β protein is also detectable in serum of breast cancer patients and elevated levels of serum S100β prior to removal of primary tumor is associated with poor disease free survival in endocrine treated patients (n=190, Wilcoxon p=0.0367, hazard ratio 2.68, 95% C.I. is 1.12 to 6.41, p=0.026, Cox proportional hazard model). Serum levels of S100β are significantly reduced after primary tumor resection (n=19, p=0.0003). In serial samples taken during the treatment period, elevated levels of S100β significantly associated with disease progression and with the emergence of metastatic disease (p=0.0031). In an in-vivo model of endocrine resistant breast cancer, raised levels of S100β marked the emergence of disease progression. The oncogene steroid receptor co-activator 1 (SRC1) and its interaction with homeobox protein (HOXC11) regulates S100β production in a src-kinase dependent manner. Here, src-kinase inhibition reduced tumor burden with a concomitant reduction in serum S100β. We also observed a marked reduction in expression of proliferative marker Ki67 and S100β protein following the treatment of endocrine resistant patient tumor explants with src-kinase inhibitor. Associations between elevated levels of serum S100β and subsequent disease progression in endocrine treated patients, suggests S100β as a monitoring tool for early detection of disease progression. Additionally high level of S100β can be used as a potential companion diagnostic tool for stratifying patients on endocrine therapy suitable for treatment with small molecule src-kinase inhibitor. Citation Format: Charmsaz S, Hughes É, Byrne C, Bane F, Tibbitts P, McIlroy M, Hill AD, Young LS. S100β as a predictive biomarker and monitoring tool in endocrine resistant breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-04-03.

Overexpression of A disintegrin and metalloprotease 10 promotes tumor proliferation, migration and poor prognosis in hypopharyngeal squamous cell carcinoma.

The aim of this study was to determine the effect of A disintegrin and metalloprotease 10 (ADAM10) protein expression on the progression, migration and prognosis of hypopharyngeal squamous cell carcinoma (HSCC). Immunohistochemistry and western blot analysis were performed to detect ADAM10 expression in human HSCC specimens. Cell Counting Kit-8 (CCK-8) assay, flow cytometry analysis and wound-healing assay were employed to investigate the effects of ADAM10 knockdown (ADAM10-RNAi) on major oncogenic properties of FaDu cells. We detected that ADAM10 was overexpressed in HSCC specimens and its expression level was associated with differentiation (p<0.001), tumor size (p=0.019), lymph node metastasis (p=0.001), clinical stage (p<0.001), proliferation marker Ki-67 expression (P=0.001) and overall survival (p<0.046). ADAM10-RNAi in FaDu cells resulted in the inhibition of proliferation and the decrease in migration. Moreover, mechanistic experiments revealed that ADAM10-RNAi resulted in an increase in E-cadherin and a decrease in N-cadherin and vimentin expression. Our study implies that high expression of ADAM10 promotes the proliferation and migration of HSCC. These findings may help to provide a method for treatment of HSCC.

Profile of mRNA Expression of Ki-67 in Breast Cancer Patients Pre- and Post- Chemotherapy

Background: Proliferation is a distinct hallmarks of cancer. Ki-67 designated as a marker of proliferation in solid tumors. The proliferative activity of tumor demonstrated by expression of Ki-67 in breast cancer has been associated with a poor prognosis. Changes in the relative proportions of Ki-67 can be observed during chemotherapy and may correlated with clinical response in breast cancer. Purpose: Evaluate changes in mRNA expression of proliferation marker Ki-67 in breast cancer patients pre- and post-chemotherapy in relation with clinical response to chemotherapy. Method: This is a longitudinal study, 30 subjects breast cancer tissue samples pre- and post-chemotherapy using cyclophosphamide, adriamycin, 5FU regiment. We using qRT-PCR techniques to detect mRNA expression of Ki-67. Chemotherapy response is calculated using RECIST criteria. Results: Mean value of Ki-67 mRNA expression on breast cancer patients pre-chemotheraphy was 11.837±0.360. Mean value of Ki-67 mRNA expression on breast cancer patients post-chemotheraphy was 11.241±1.971. There was no significant correlation between expression of Ki-67 mRNA prechemotherapy with clinical response to chemotherapy, p = 0.862 (<i>p</i> ≥0.05). There is a positive correlation between velocity of Ki-67 mRNA expression with clinical response with value of r = 0.378, this correlation was significant with <i>p</i> = 0.020 (<i>p</i><0.05). Conclusion: Chemotherapy cause decrease in mRNA expression of Ki-67. There is insignificant correlation between expression of mRNA Ki-67 baseline with chemotherapy response. Velocity of Ki-67 mRNA expression has significant correlation with clinical response to chemotherapy.

Electroacupuncture improves neurovascular unit reconstruction by promoting collateral circulation and angiogenesis.

Acupuncture at Shuigou (GV26) shows good clinical efficacy for treating stroke, but its mechanism remains poorly understood. In this study, a cerebral infarction model of ischemia/reperfusion injury received electroacupuncture at GV26 (15 Hz and 1 mA, continuous wave [biphasic pulses], for 5 minutes). Electroacupuncture effectively promoted regional cerebral blood flow on the infarct and non-infarct sides, increased infarct lesions, lectin, and number of blood vessels, upregulated von Willebrand factor and cell proliferation marker Ki67 expression, and diminished neurological severity score. These findings confirm that electroacupuncture at GV26 promotes establishment of collateral circulation and angiogenesis, and improves neurological function.

Abstract 93: The Role of the Stretch-responsive Genes Mlf1 and Rnd1 for Cardiac Physiology and Pathophysiology

Cardiac remodeling is a typical change in function and morphology of the heart in response to pathological biomechanical stress. Despite substantial research in this area several underlying mechanisms remain unclear. We thus devised a screening experiment of dynamic stretch of neonatal cardiomyocytes (NRVCM) which revealed several genes which have not yet been implicated in cardiac remodeling before. In further analyses we focused on Mlf1 and Rnd1 , both showing significant expression in heart as well as in skeletal and liver. Conformational qRT-PCR revealed upregulation of Rnd1 mRNA (5.94x) after 24 h of stretch. Mlf1 mRNA was significantly downregulated (qRT-PCR 0.34x). Stimulation with phenylephrine (PE) induced Rnd1 mRNA (3.98x), while Mlf1 mRNA levels remained unchanged. Furthermore, in line with the in vitro results we found upregulation of Rnd1 (2.1x, p&lt;0.001) and downregulation of the cardiac Mlf1 expression (var. 1 0.45x, var. 2 0.36x, both p&lt;0.001) in mice after transverse aortic constriction. Adenoviral overexpression of Mlf1 in NRVCM induced the fetal gene program (e.g., nppa , nppb ). However, a significant decrease in cell size was found. We noticed the consistent findings after stretch or PE-stimulation. Rnd1 overexpression also led to induction of nppa , nppb and Rcan1.4, but caused an increase in cell size. To analyze whether Mlf1 and Rnd1 also influenced cell proliferation, we analyzed Mlf1/Rnd1-infected cells for the expression of proliferation markers Ki67 and phosphohistone H3 (PHH3). Mlf1 overexpression led to an increase in Ki67+ cells of about 5.92 fold, and in PHH3+ cells of 2.48 fold (p&lt;0.001), Rnd1 overexpression showed an increase in Ki67+ cells of about 6.9 fold and in PHH3+ cells of 5,25 fold (p&lt;0.001). Taken together, we could identify two stretch-sensitive genes which influence the process of cardiac hypertrophy and cell proliferation. Current work focuses on a knockdown of both genes in vivo and on the identification of potential protein-protein interactions to complete the characterization of Mlf1 and Rnd1.

Response of Primary Human Bone Marrow Mesenchymal Stromal Cells and Dermal Keratinocytes to Thermal Printer Materials In Vitro.

Advancement in thermal three-dimensional printing techniques has greatly increased the possible applications of various materials in medical applications and tissue engineering. Yet, potential toxic effects on primary human cells have been rarely investigated. Therefore, we compared four materials commonly used in thermal printing for bioengineering, namely thermally printed acrylonitrile butadiene styrene, MED610, polycarbonate, and polylactic acid, and investigated their effects on primary human adult skin epidermal keratinocytes and bone marrow mesenchymal stromal cells (BM-MSCs) in vitro. We investigated indirect effects on both cell types caused by potential liberation of soluble substances from the materials, and also analyzed BM-MSCs in direct contact with the materials. We found that even in culture without direct contact with the materials, the culture with MED610 (and to a lesser extent acrylonitrile butadiene styrene) significantly affected keratinocytes, reducing cell numbers and proliferation marker Ki67 expression, and increasing glucose consumption, lactate secretion, and expression of differentiation-associated genes. BM-MSCs had decreased metabolic activity, and exhibited increased cell death in direct culture on the materials. MED610 and acrylonitrile butadiene styrene induced the strongest expression of genes associated to differentiation and estrogen receptor activation. In conclusion, we found strong cell-type-specific effects of the materials, suggesting that materials for applications in regenerative medicine should be carefully selected not only based on their mechanical properties but also based on their cell-type-specific biological effects.Electronic supplementary materialThe online version of this article (doi:10.1007/s40846-016-0118-z) contains supplementary material, which is available to authorized users.

Clinical-morphological features of lobular, nodular and diffuse forms of adenomyosis

The purpose of the study was to investigate clinical, morphological and immunohistochemical features and pathogenetic interrelationship between focal, nodular and diffuse forms of adenomyosis. There was investigated surgical samples of removed uterines and their fragments; patients were divided into 3 groups, the 1st group was consisted of 16 cases with focal adenomyosis, the 2nd - 24 females with nodular form of adenomyosis, the 3rd - 21 female with diffuse form of adenomyosis. All patients were underwent general clinical examination, instrumental and laboratory observation with the aim of the verification of the diagnosis. There were executed macro- and microscopic immunohistochemical investigations of fragments of the tissue from the zone of endometrioid heterotopias, surrounding myometrium and eutopic endometrium. There were used antibodies: Ki-67, PTEN, ER, PR (“DAKO”), p16 (“Roshe”). There was established the significant increase in the expression of proliferation marker Ki-67, in parallel with a decrease in the expression level of PTEN, ER, PR from the focal to the nodular and diffuse forms of adenonomyosis. Thus it seems to be possible to consider focal, nodular and diffuse forms of adenomyosis as forms-phases of adenomyosis, that according both to histological characteristics of adenomyosis and immunohistochemical markers offocal and nodular forms may antedate the diffuse form of adenomyosis, that is also confirmed by the detection of a combination of all of these forms. Diffuse form of adenomyosis can be assumed to have the highest potential for the growth and dissemination of the pathological process in the uterine.