Feril and co-workers examined the correlation between ultrasound parameters and induction of apoptosis . The authors reported that applying the low-intensity ultrasound with intensity as low as 0.3 W/cm2 at a frequency of 1 MHz provided the highest apoptotic rate for U937 IGF 1 LR3 supplier (leukemia cell lines). No traces of free radical were detected at this intensity. It might indicate that the observed bio-effects (cell lysis and apoptosis) were result of sono-mechanical trauma rather than sono-chemical reactions. The role of mechanical stress in triggering apoptosis was investigated by Mayr and co-workers . The data of the study demonstrated that the biomechanical stress induced the elevation of p53 level and launching caspase cascade (through Bax activation) resulted in the cell death. P53 plays a crucial role in tumor suppression and protection from mutations  and . The degree of p53 expression can affect the induction of apoptosis. Abdollahi and colleagues reported about inhibition of proliferation and induction the apoptosis in Tk6 lymphoblasts . It was found out that ultrasound-elicited apoptosis occurred more frequently in p53+ lymphoblasts than in p53 negative counterpart. The up-regulation of p53 in Tk6 cells was detected in post-sonication period. These effects were observed in the range of applied sound energy between <0.1 and >10 MPa. In another study 24 kHz ultrasound at pressures of 0.36 and 0.54 MPa was exploited to identify apoptotic processes in cells (DU 145 prostate cancer line) in post-sonication time . The authors used the method of flow cytometry with fluorescein isothiocyante (FITC)-labeled Annexin V, Propidium Iodide and calcein as indicators of apoptosis, viability and intracellular uptake, respectively. The acquired data demonstrated that the augmentation of the acoustic pressure from 0.36 up to 0.54 MPa resulted in significant increase of apoptotic cell population. The Fluo-4 AM (Ca2+ specific indicator) has been used to examine the correlation between high level of intracellular Ca2+ and ultrasound-induced apoptosis. It was found out that annexin V-positive (apoptotic) cells had a high level of intracellular Ca2+, which supports the hypothesis that ultrasound-elicited apoptosis is associated with an increase of Ca2+.
ACTB (¦Â-actin)TTGCCGACAGGATGCAGAAGCCGATCCACACGGAGTACT Full-size table Table optionsView in workspaceDownload as CSV 2.10. Quantitative real-time RT-PCR (qRT-PCR) qRT-PCR was performed by using ABI Prism 7900HT (Applied Biosystems, Grand Island, NY) with 2¡Á Quantifast SYBR Green PCR Master mix (Qiagen) with 1 mM primers. Specific primer sets for HIF1¦Á, HIF2¦Á and ACBT (¦Â-actin, as internal control) were listed in Table 1. Each target gene was analyzed in three independent qRT-PCR assays. Thermal cycling was started with D-Biotin initial activation step for 5 min at 95 ¡ãC, followed by 40 cycles of 95 ¡ãC for 10 s and 60 ¡ãC for 30 s. Relative expression of the target gene was analyzed by the ¦¤¦¤Ct method (the reference gene: ¦Â-actin). Results were normalized relative to the non-treatment control. 2.11. Statistical analysis Statistical significance was determined by one-way analysis of variance followed by Fisher¡¯s probable least-squares difference analysis as a Post Hoc test for data from phagokinetic track assay, and by two-sided Student¡¯s t-test for data from invasion assays. In all statistical comparisons, P < 0.01 was used to indicate statistically significant difference.
Liver cancer stem HOBt (LCSCs) can drive and maintain hepatocellular carcinoma (HCC) growth, metastasis, and recurrence. Therefore, they are potentially responsible for the poor prognosis of HCC. Oxygen and nutrient deficiencies are common characteristics of the tumor microenvironment. However, how LCSCs adapt to oxygen- and nutrient-deprived conditions is unclear. Here, we used immunofluorescent staining and flow cytometry analysis to show that CD133+ cells were significantly enriched after hypoxia and nutrient starvation (H/S) in the human HCC cell line Huh7. Sorted CD133+ cells showed higher survival, less apoptosis, and possess higher clonogenic ability under H/S compared to the CD133? population. Under H/S, electron microscopy revealed more advanced autophagic vesicles in CD133+ cells. Additionally, CD133+ cells had higher autophagy levels as measured by both RT-qPCR and Western blotting. CD133+ cells had more accumulated GFP-LC3 puncta, which can be detected by fluorescence microscopy. The autophagic inhibitor chloroquine (CQ) significantly increased apoptosis and decreased the clonogenic capacity of CD133+ cells under H/S. Pre-culturing in H/S enhanced the sphere-forming capacity of CD133+ cells. However, CQ significantly impaired this process. Therefore, autophagy is essential for LCSCs maintenance. CD133+ cells were also found to have a higher tumor-forming ability in vivo, which could be inhibited by CQ administration. Collectively, our results indicate that the involvement of autophagy in maintenance of CD133+ LCSCs under the oxygen- and nutrient-deprived conditions that are typical of the tumor microenvironment in HCC. Therefore, autophagy inhibitors may make LCSCs more sensitive to the tumor microenvironment and be useful in improving anti-cancer treatments.
Although it HOBt has been demonstrated that miR-519c , miR-520h  and miR-328  could target the 3¡äUTR of BCRP and negatively regulate BCRP expression, these reports all lacked the supporting evidence of in vivo research results. It is necessary to carry out in vivo experiments to verify in vitro results. In the present study, following Jin Hou¡¯s methods , when the average tumor size reached approximately 125 mm3, we intratumorally injected 1 nmol of miR-487a agmir at multiple sites in the xenograft mice and/or injected 1.5 mg/kg MX by tail vein twice a week for a total of 5 injections. We found that injecting miR-487a agmir increased miR-487a expression in xenograft tumors, suggesting multiple sites of injection could maintain high expression of miR-487a in vivo (Fig. 6A). Additionally, the results revealed that ectopic miR-487a expression decreased the expression of BCRP in xenograft tumors (Fig. 6B¨CD) and enhanced the antitumor activity of MX (Fig. 7A¨CC). This in vivo study powerfully supports our hypothesis that miR-487a is an important target in regulating BCRP expression and drug sensitivity in breast cancer and also offers a practical way to study miRNA transfection in vivo.
2.4. X-ray and micro-computed tomography (¦Ì-CT) analysis Tibias removed from mice were scanned with X-ray (MX-20, Faxitron X-ray, WI, USA) and a high HOBt microtomographic system, ¦Ì-CT 40 (Scanco Medical, Switzerland). The tibia specimens were measured at room temperature and placed inside the X-ray chamber of the X-ray machine. The voltage and exposure time of the X-ray were 32 kV and 10 s, respectively. Then the samples were exposed to the ¦Ì-CT. Each three-dimensional image data were consisted of approximately 500 micro-CT slide image (8 ¦Ìm/slide) starting from the growth plate of tibial interface and moving down the tibia. The bone density was expressed as percentage of BV/TV, which was generated and compared with each groups using the formula: (Bone volume/Tissue volume) ¡Á 100% . 2.5. Histology Lungs and livers were fixed in 10% buffered formalin for 7 days at room temperature. As for the tibia, after fixed in 10% buffered formalin, the tibia was decalcified in decalcification buffer (14% EDTA w/v in distilled water, pH 6.8¨C7.2) for 21 days. Then samples were paraffin embedded, sectioned longitudinally at 5 ¦Ìm, and stained with H&E. Stained sections were examined and photographed using an Olympus IX71 microscope (Japan) and were analyzed using SPOT advanced (version 3.5.6) software. Tumor burden, defined as the tumor area, was calculated from the section of the lung or liver and expressed as an average tumor area per group in absolute units (mm2).
Given that Nm23H1 inhibited RasGV-induced tumorigenesis in nude mice assays, our proposed mechanism by which RGS19 suppresses Ras signaling through Nm23H1/KSR  appears to hold true in NIH3T3 cells. Nevertheless, it remains possible that other pathways may also contribute toward Ras suppression. In particular, constitutive activation of c-Jun N-terminal kinase (JNK) has been implicated in Ras-induced neoplastic transformation and this activity can be suppressed by the tumor suppressor protein, Ras association domain-containing protein 1A . Elevated expression of RGS19 in HEK293 HOBt has been shown to impair the responsiveness of JNK and p38 mitogen-activated protein kinase (MAPK) as well as their upstream GTPases Rac1 and Cdc42 . Interestingly, Rac1 activation by its guanine nucleotide exchange factor Tiam1 is negatively regulated by Nm23H1 . Hence it is conceivable that by up-regulating Nm23, RGS19 can attenuate the activity of Rac1/JNK and contribute towards the suppression of RasGV-induced tumorigenesis. Other precedents for Ras inactivation have also been demonstrated for a variety of signaling components including RIG1 (retinoid-inducible gene 1; ), RalA GTPase , and PC4 (a positive co-activator of AP-2; ). It is not known if RGS19 can affect the expression or function of these proteins, but Nm23H1 apparently has widespread inhibitory actions on small GTPases ranging from Ras  to Rac1 , Cdc42 , and Rad , and thus it would not be surprising if RalA can be negatively regulated by Nm23H1.
The development of genetically engineered mouse models with pancreatic cancer  and , specifically the progress in PanIN and PDAC mouse models ,  and , has significantly contributed to our understanding of the HOBt of pancreatic neoplasia  and . Previous studies have isolated cell lines from the pancreas of genetically engineered mutant mice with PanIN and PDAC  and . In this study, mouse pancreatic neoplasia tissues and PanIN cell line isolated from the mutant mouse were employed to study the acoelomates biological features and molecular genetic alterations of PanIN. 2. Materials and methods 2.1. Patient and tissue samples PDAC and peritumoral normal tissue samples were obtained from 86 patients at Ruijin Hospital in Shanghai. None of the patients had previously received radiotherapy or chemotherapy. After surgery, each tissue sample was fixed in formalin and embedded in paraffin. Histological diagnoses were performed by two independent senior pathologists in the Department of Pathology at Ruijin Hospital.
Fig. 4. Small-molecule Hsp70 inhibitor strongly induced apoptosis and inhibited cell growth of MM cell lines. (a) EC50 curves of HOBt panel of MM cell lines treated with Ver-155008 for 24 h as determined by WST-1 assay. (b) Dose- and time-dependent response of four human MM cell lines treated with Ver-155008. (c) Annexin V/PI staining assay for cell survival following 24-h treatment of four MM cell lines using Ver-155008. (a¨Cc) Graphs represent mean values taken from triplicate determinations, error bars represents standard deviation. (d) Western blot analysis on KMS11 and RPMI8226 cells treated with 15 Ver-155008 for 24 h. (e) Dose dependent response of primary MM and normal peripheral blood mononuclear cells treated with Ver-155008 for 24 h as determined by WST-1 assay. Graph represents mean values taken from all cell samples with duplicate readings. Error bars represents standard deviation.Figure optionsView in workspaceDownload full-size imageDownload high-quality image (216 K)Download as PowerPoint slide
It is conceivable that physiological differences between young and old patients, first, DAPT structure levels may be responsible for the different roles of the two isoforms in tumor growth regulation. Since only ER¦Â1 can bind estrogens, it is possible that under physiological conditions of high estrogen levels in young patients, the decreased portion of this isoform may contribute to tumor growth. In contrast, in older patients with established low estrogen levels, the ER¦Â2 isoform (which does not bind estrogens) may be the major repressor of ER activity. Thus, the observed effects of ER¦Â1 and ER¦Â2 (although measured as mRNA) on tumor growth in young and elder age may be connected with hormonal status. While it was previously shown that both, ER¦Â1 and ER¦Â2 inhibit or at least attenuate the activity of ER¦Á on ERE driven promoters , ,  and , the mechanism by which these two isoforms affect ER¦Á is different  and . Omoto and coworkers showed that ER¦Â1 inhibits ER¦Á activity mainly by competing for binding sites by formation of ER¦Â1:ER¦Â1 homo- or ER¦Á:ER¦Â1 hetero-dimers in stably transfected MCF7 cells . Similarly, ER¦Â2 repression of ER¦Á activity was reported to occur via formation of inefficient ER¦Á:ER¦Â2 heterodimers  and  and by decreasing available ER¦Á protein through activation of its proteasome degradation . We also found an inverse correlation of ER¦Â2 mRNA with ER¦Á protein levels determined by IHC in the analyzed tumors (data not shown), which is in line with the latter mechanism.
3.4. WNT activation induces expression of EMT activators Increased invasive potential of cancer HOSu sequence in solid tumors has been associated with EMT of the migrating tumor cell population  and . To determine if induction of EMT in GBM-derived cell cultures may explain their increased invasion following WNT pathway activation, we analyzed mRNA expression levels of EMT-related transcription factors ZEB1, Twist, Snail and Slug. Indeed, increased levels of ZEB1 and Twist (both 2¨C3.5¡Á) could be detected. KK showed the strongest induction of Snail (3.7¡Á). These alterations could be proved also on protein level as shown in Fig. 4A. Another important feature of EMT is an increased ratio of N-Cadherin to E-Cadherin. The expression of E-Cadherin could not be observed in any of the GBM cultures. Nevertheless N-Cadherin transcript levels were clearly enhanced in cells with activated WNT/¦Â signaling (up to 3¡Á for NCH421k and over 2¡Á for U87, Fig. 4A). Fig. 4. Activation of WNT/¦Â-catenin signaling promotes the expression of EMT activators and CD133. S33Y transfected cell cultures show induction of epithelial-to-mesenchymal (EMT) activators ZEB1, Twist, Snail as well as N-Cadherin as showed at mRNA and protein levels (A), whereas the inhibition of intrinsic WNT/¦Â-catenin signaling led to their decreased transcription and translation (B). Also a putative brain tumor stem cell marker CD133 appeared to up- and down regulated in response to WNT/¦Â-catenin pathway modulation with the highest transcript and protein level following WNT activation (C,D) (A,B Student t-Test, p-value ? 0.05).Figure optionsView in workspaceDownload full-size imageDownload high-quality image (505 K)Download as PowerPoint slide