Selective inhibition of HDAC6 promotes bladder cancer radiosensitization and mitigates the radiation-induced CXCL1 signalling.

BACKGROUND: Although trimodality therapy resecting tumours followed by chemoradiotherapy is emerged for muscle-invasive bladder cancer (MIBC), chemotherapy produces toxicities. Histone deacetylase inhibitors have been identified as an effective strategy to enhance cancer radiotherapy (RT). METHODS: We examined the role of HDAC6 and specific inhibition of HDAC6 on BC radiosensitivity by performing transcriptomic analysis and mechanism study. RESULTS: HDAC6 knockdown or HDAC6 inhibitor (HDAC6i) tubacin exerted a radiosensitizing effect, including decreased clonogenic survival, increased H3K9ac and α-tubulin acetylation, and accumulated γH2AX, which are similar to the effect of panobinostat, a pan-HDACi, on irradiated BC cells. Transcriptomics of shHDAC6-transduced T24 under irradiation showed that shHDAC6 counteracted RT-induced mRNA expression of CXCL1, SERPINE1, SDC1 and SDC2, which are linked to cell migration, angiogenesis and metastasis. Moreover, tubacin significantly suppressed RT-induced CXCL1 and radiation-enhanced invasion/migration, whereas panobinostat elevated RT-induced CXCL1 expression and invasion/migration abilities. This phenotype was significantly abrogated by anti-CXCL1 antibody, indicating the key regulator of CXCL1 contributing to BC malignancy. Immunohistochemical evaluation of tumours from urothelial carcinoma patients supported the correlation between high CXCL1 expression and reduced survival. CONCLUSION: Unlike pan-HDACi, the selective HDAC6i can enhance BC radiosensitization and effectively inhibit RT-induced oncogenic CXCL1-Snail-signalling, thus further advancing its therapeutic potential with RT.

Effects of group density, hunting, and temperature on the singing patterns of eastern hoolock gibbons (Hoolock leuconedys) in Gaoligongshan, Southwest China.

Many non-human primates produce species-specific loud calls to communicate within and between groups over long distances. Understanding these calling patterns can provide insights into how individuals modify their behavior in response to environmental variables as well as help to design efficient bioacoustic survey techniques. Eastern hoolock gibbons in Gaoligongshan inhabit the coldest habitat of all gibbon populations, but both conservation and research efforts on this population have been minimal. We studied singing patterns of two habituated and two unhabituated groups at two sites in Gaoligongshan between July 2010 and June 2015. We systematically collected data of their calls, and its relationship to temperature, group density, and hunting pressure over at least 1 year for each group. Our goal was to elucidate how these factors affect singing patterns of eastern hoolock gibbons. We found that adult pairs coordinated their singing to produce duet bouts that lasted for an average of 25.5 min. The singing rate (number of bouts/number on monitoring days*100%: 7.5-31.4%) was notably lower than other gibbon populations, presumably due to low group density (about 0.5 groups/km(2) ) and prevalence of hunting at the study site. Cold temperature also affected gibbons' singing behavior. Our study groups called, on average, 2.5 hr after sunrise, probably foraging first in the early morning after long nights in this cold habitat delayed singing. Furthermore, mean temperatures in the morning (8:00-12:00 am) were higher on singing days than on non-singing days, and one group called less frequently when monthly mean temperature was below 10°C. Our findings indicate that both hunting pressure from humans and low temperatures suppress calling behavior in hoolock gibbons. Such information is critical in evaluating the use of duetting as a monitoring technique for this endangered gibbon species. Am. J. Primatol. 78:861-871, 2016. © 2016 Wiley Periodicals, Inc.

Observations on the expression of human papillomavirus major capsid protein in HeLa cells.

BACKGROUND: The goal of this study was to identify the nature of the inclusion bodies that have been found in HeLa cells (cervical cancer immortal cell line) by electron microscope and to determine whether the major capsid protein (L1) of human papillomavirus (HPV) can be expressed in HPV-positive uterine cervix cancer cells. METHODS: HPV L1 protein expression in HeLa cells was detected with anti-HPV L1 multivalent mice monoclonal antibody and rabbit polyclonal anti-HPV L1 antibody by ELISA, light microscope immunohistochemistry, electron microscope immunocytochemistry and Western blotting assays. Reverse transcriptional PCR (RT-PCR) was performed to detect the transcription of L1 mRNA in HeLa cells. The immortalized human keratinocyte HeCat was used as the negative control. RESULTS: HPV L1 proteins reacted positively in the lysate of HeLa cells by ELISA assays. HRP labeled light microscope immunohistochemistry assay showed that there was a strong HPV L1 positive reaction in HeLa cells. Under the electron microscope, irregular shaped inclusion bodies, assembled by many small and uniform granules, had been observed in the cytoplasm of some HeLa cells. These granules could be labeled by the colloidal gold carried by HPV L1 antibody. The Western blotting assay showed that there was a L1 reaction strap at 80-85 kDa in the HeLa cell lysates, hence demonstrating the existence of HPV18 L1 in HeLa cells. RT-PCR assay showed that the L1 mRNA was transcribed in HeLa cells. CONCLUSIONS: The inclusion bodies found in the cytoplasm of HeLa cells are composed of HPV18 L1 protein. Since HeLa cell line is a type of cervical cancer cells, this implies that HeLa cells have the ability to express HPV L1 proteins.

DNA methylome and transcriptome landscapes of cancer-associated fibroblasts reveal a smoking-associated malignancy index.

Unlike the better-studied aberrant epigenome in the tumor, the clinicopathologic impact of DNA methylation in the tumor microenvironment (TME), especially the contribution from cancer-associated fibroblasts (CAFs), remains elusive. CAFs exhibit profound patient-to-patient tumorigenic heterogeneity. We asked whether such heterogeneity may be exploited to quantify the level of TME malignancy. We developed a robust and efficient methylome/transcriptome co-analytical system for CAFs and paired normal fibroblasts (NFs) from non-small-cell lung cancer patients. We found 14,781 CpG sites of CAF/NF differential methylation, of which 3,707 sites showed higher methylation changes in ever-smokers than in nonsmokers. Concomitant CAF/NF differential gene expression analysis pointed to a subset of 54 smoking-associated CpG sites with strong methylation-regulated gene expression. A methylation index that summarizes the ß values of these CpGs was built for NF/CAF discrimination (MIND) with high sensitivity and specificity. The potential of MIND in detecting premalignancy across individual patients was shown. MIND succeeded in predicting tumor recurrence in multiple lung cancer cohorts without reliance on patient survival data, suggesting that the malignancy level of TME may be effectively graded by this index. Precision TME grading may provide additional pathological information to guide cancer prognosis and open up more options in personalized medicine.

Pharmacophore-based virtual screening for the identification of the novel Src inhibitor SJG-136 against lung cancer cell growth and motility.

Aberrant elevated Src activity is related to lung cancer growth and metastasis. Therefore, the development of potent small molecule inhibitors to target Src kinase is a potential therapeutic strategy for lung cancer. This study aimed to develop a computational model for the in silico screening of Src inhibitors and then assess the suppressive effect of candidate compounds on cellular functions. A 3D-quantitative structure-activity relationship (QSAR) pharmacophore model consisting of two hydrogen bond acceptors and two hydrophobic regions was constructed by using 28 structurally diverse compounds with IC50 values spanning four orders of magnitude. A National Cancer Institute (NCI) compound dataset was employed for virtual screening by applying the pharmacophore model and molecular docking. Candidate compounds were chosen from the top 20% of scored hits. Among these compounds, the suppressive effects of 30 compounds available in the NCI on Src phosphorylation were validated by using an enzyme-linked immunosorbent assay. Among these compounds, SJG-136, a pyrrolobenzodiazepine dimer, showed a significant inhibitory effect against Src activity in a dose-dependent manner. Further investigations showed that SJG-136 can inhibit lung cancer cell proliferation, clonogenicity, invasion and migration in vitro and tumour growth in vivo. Furthermore, SJG-136 also had an inhibitory effect on Src-related signaling pathways, including the FAK, paxillin, p130Cas, PI3K, AKT, and MEK pathways. In conclusion, we have established a pharmacophore-based virtual screening approach to identify novel Src inhibitors that can inhibit lung cancer cell growth and motility through suppressing Src-related pathways. These findings may contribute to the development of targeted drugs for lung cancer treatment, such as lead compounds.

Large optical Stark shifts in single quantum dots coupled to core-shell GaAs/AlGaAs nanowires.

Nanowire quantum dots (NW-QDs) can be used for future compact and efficient optoelectronic devices. Many efforts have been made to control the QD states by inserting the QDs in doped structures and applying an electric field in a nanowire system. In this paper, we use down-conversion and up-conversion photoluminescence excitations to explore the optical and electronic properties of single quantum dots in GaAs/AlGaAs core-shell nanowires. We investigate a large optical Stark shift in this system as a new method to tune the QD states. When the tunable laser lies within the spectral bandwidth of ZB/WZ GaAs (780 nm-860 nm), we observe an extremely large optical Stark shift of 1.3 nm (0.5 nm) with increasing excitation power at a resonant wavelength of 800 nm (840 nm) in GaAs states. The ability to in situ control the energy states of self-catalyzed NW-QDs should open a new way for quantum light sources and nonlinear optics in a nanowire system.

[Effects of serum derived from rats undergone auricular acupuncture intervention on expression of TNF-alpha mRNA, cell adhesion factor-1 and vascular intercellular adhesion molecule-1 proteins of incubated cerebral microvascular endotheliocytes with diabetic injury].

OBJECTIVE: To observe the protective effect of serum derived from rats undergone auricular electroacupuncture (EA) stimulation on the incubated cerebral microvascular endotheliocytes with diabetic injury so as to investigate the underlying mechanism of cholinergic anti-inflammatory action. METHODS: SD rats were randomized into normal group (n = 10), diabetic model group (n = 6), auricular EA group (n = 8), vagotomy + EA group (n = 7, received ipsilateral vagotomy before auricular EA stimulation), atropine + EA group (n = 8), hexamethonium + EA group (n = 7) and alpha-bungarotoxin + EA group (n = 7). Diabetic mellitus model was established by feeding the rats with high sugar, high fat forage and intraperitoneal injection of 1% streptozotocin injection (STZ, 35 mg/kg). EA was applied to ipsilateral "Yi-Dan"-point and "Er-Shenmen" for 30 min, once daily for 10 days. Atropine (0.1 mg/kg, an anticholinergic drug), hexamethonium (10 mg/kg, an antagonist of the nicotinic acetylcholine receptors located in sympathetic and parasympathetic ganglia) and alpha-bungarotoxin (1.0 microg/kg, a type of alpha-neurotoxin that is known to bind irreversibly and competitively to the nicotinic acetylcholine receptors) were given to the rats by tail venous injection, respectively, before ipsilateral auricular EA intervention, once daily for 10 days. Blood samples from rats of each group were then collected. Normally cultured rat brain microvascular endotheliocytes were randomly divided into the same 7 groups. The diabetic-like damage model of cerebral microvascular endotheliocytes was established in the 6 groups except the normal group by adding the fluid containing glucose (20 mmol/L), insulin (100 mU/L) and oxidized low density lipoprotein (200 mg/L) to the culture medium. After 48 hours' incubation, the conditional culture solutions were collected for filtration and degerming. Morphological changes and cellular ultra-microstructure were examined using light microscope and transmission electron microscope, respectively. Tumor necrosis factor-alpha (TNF-alpha) mRNA expression of the cultured microvascular endotheliocytes was assayed using RT-PCR, and the soluble cell adhesion factor-1 (sICAM-1) and soluble vascular intercellular adhesion molecule-1 (sVCAM-1) concentrations in 1 mL culture fluid were measured using ELISA. RESULTS: Compared to the normal control group, the cultured cerebral microvascular endotheliocytes in the model group displayed a cluster-like or floating state, enlargement of the space, and increase of refractivity under light microscope, and showed swelling of the mitochondria with broken cristae and expansion of the space and even with membrane fusion or disappearance under electron microscope. This situation was relatively lighter in both auricular EA and atropine groups, and severe in the vagotomy, hexamethonium and alpha-bungarotoxin groups. TNF-alpha mRNA expression and sICAM-1 and sVCAM-1 concentrations were significantly higher in the model group than in the normal group, but significantly lower in both auricular EA group and atropine group than in the model group (P < 0.01, P < 0.05). No remarkable diffe-rences were found among the model, vagotomy, hexamethonium and alpha-bungarotoxin groups in the levels of TNF-alpha mRNA expression and sICAM-1 and sVCAM-1 concentrations (P > 0 05). CONCLUSION: Auricular EA intervention rat serum can lighten diabetic cellular injury, suppress TNFalpha mRNA expression and reduce ICAM-1 and sVCAM-1 concentrations of rat cerebral microvascular endotheliocytes, which is closely associated with the intact vagus nerve and normal nicotinic acetylcholine receptors in rats.

Chemosystematics of tea trees based on tea leaf polyphenols as phenetic markers.

This study examined the polyphenols of tea leaves as chemotaxonomic markers to investigate the phenetic relationship between 89 wild (the small-leaved C.sinensis var. sinensis and large-leaved C. sinensis var. assamica), hybrid, and cultivated tea trees from China and Japan. (-)-Epigallocatechin 3-O-gallate, EGCG (1); (-)-epigallocatechin, EGC (2); (-)-epicatechin 3-O-gallate, ECG (3); (-)-epicatechin, EC (4); (+)-catechin, CA (5); strictinin, STR (6); and gallic acid, GA (7) were used as polyphenolic markers. Of the 13 polyphenol patterns observed, Principal Component Analysis (PCA) indicated that the structure-types of the flavonoid B-rings, such as the pyrogallol-(EGCG (1) and EGC (2)) and catechol-(ECG (3) and EC (4)) types, greatly influenced the classification. Ward's minimum-variance cluster analysis was used to produce a dendrogram that consisted of three sub-clusters. One sub-cluster (A) was composed of old tea trees 'Gushu' cha (C. sinensis var. assamica) and cv 'Taidi' cha, suggesting that relatively primitive tea trees contain greater amounts of compounds 3 and 4 and lower amounts of compounds 1 and 2. The other two sub-clusters B and C, made up of Chinese hybrids (sub-cluster B) and Japanese and Taiwanese tea trees (sub-cluster C), had lower contents of 3 and 4 than sub-cluster A. Therefore, PCA and cluster analysis indicated that the greater the amounts of 1 and 2 (and the lower of 3 and 4), the more recent the origin of the tea line. Based on morphological characteristics, geographical information, and the historical information on tea trees, these results show good agreement with the current theory of tea tree origins, and this suggests that the Xishuangbanna district and Puer City are among the original sites of the tea tree species.