Identification of sphingosine kinase 1 (SphK1) as a primary target of icaritin in hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is a highly aggressive neoplasm. We aim to explore the anti-HCC activity by a natural prenylflavonoid icaritin. Icaritin was cytotoxic and pro-apoptotic when added to established (HepG2, KYN-2 and Huh-7 lines) and primary human HCC cells. At the signaling level, icaritin inhibited sphingosine kinase 1 (SphK1) activity in HCC cells, which led to pro-apoptotic ceramide production and JNK1 activation. SphK1 inhibition or silence (by shRNA/microRNA) mimicked icaritin-mediated cytotoxicity, and almost nullified icaritin's activity in HepG2 cells. Reversely, exogenous over-expression of SphK1 sensitized icaritin-induced HepG2 cell apoptosis. In vivo, oral administration of icaritin dramatically inhibited HepG2 xenograft growth in SCID mice. Further, SphK1 activity in icaritin-treated tumors was largely inhibited. In summary, icaritin exerts potent anti-HCC activity in vitro and in vivo. SphK1 inhibition could be the primary mechanism of its actions in HCC cells.

Aqueous Oldenlandia diffusa extracts inhibits colorectal cancer cells via activating AMP-activated protein kinase signalings.

Here we evaluated the anti-cancer activity of aqueous Oldenlandia diffusa (OD) extracts (ODE) in colorectal cancer (CRC) cells. We showed that ODE exerted potent anti-proliferative, cytotoxic and pro-apoptotic activities against a panel of established CRC lines (HCT-116, DLD-1, HT-29 and Lovo) and primary (patient-derived) human CRC cells. ODE activated AMP-activated protein kinase (AMPK) signaling, which led to subsequent mTORC1 inhibition and Bcl-2/HIF-1α downregulation in CRC cells. In ODE-treated CRC cells, AMPKα1 formed a complex with p53. This might be important for p53 activation and subsequent cancer cell apoptosis. Inhibition of AMPK signaling, though dominant negative (dn) mutation or shRNA/siRNA knockdown of AMPKα1 attenuated ODE-exerted CRC cytotoxicity. In vivo, i.p. administration of ODE inhibited HCT-116 xenograft tumor growth in SCID mice. In addition, AMPK activation, mTORC1 inhibition and p53 activation were observed in ODE-treated HCT-116 xenograft tumors. These results suggest that ODE inhibits CRC cells in vitro and in vivo, possibly via activation of AMPK-dependent signalings.

KU-0060648 inhibits hepatocellular carcinoma cells through DNA-PKcs-dependent and DNA-PKcs-independent mechanisms.

Here we tested anti-tumor activity of KU-0060648 in preclinical hepatocellular carcinoma (HCC) models. Our results demonstrated that KU-0060648 was anti-proliferative and pro-apoptotic in established (HepG2, Huh-7 and KYN-2 lines) and primary human HCC cells, but was non-cytotoxic to non-cancerous HL-7702 hepatocytes. DNA-PKcs (DNA-activated protein kinase catalytic subunit) is an important but not exclusive target of KU-0060648. DNA-PKcs knockdown or dominant negative mutation inhibited HCC cell proliferation. On the other hand, overexpression of wild-type DNA-PKcs enhanced HepG2 cell proliferation. Importantly, KU-0060648 was still cytotoxic to DNA-PKcs-silenced or -mutated HepG2 cells, although its activity in these cells was relatively weak. Further studies showed that KU-0060648 inhibited PI3K-AKT-mTOR activation, independent of DNA-PKcs. Introduction of constitutively-active AKT1 (CA-AKT1) restored AKT-mTOR activation after KU-0060648 treatment in HepG2 cells, and alleviated subsequent cytotoxicity. In vivo, intraperitoneal (i.p.) injection of KU-0060648 significantly inhibited HepG2 xenograft growth in nude mice. AKT-mTOR activation was also inhibited in xenografted tumors. Finally, we showed that DNA-PKcs expression was significantly upregulated in human HCC tissues. Yet miRNA-101, an anti-DNA-PKcs miRNA, was downregulated. Over-expression of miR-101 in HepG2 cells inhibited DNA-PKcs expression and cell proliferation. Together, these results indicate that KU-0060648 inhibits HCC cells through DNA-PKcs-dependent and -independent mechanisms.

C6 ceramide dramatically increases vincristine sensitivity both in vivo and in vitro, involving AMP-activated protein kinase-p53 signaling.

Use of the conventional cancer chemotherapy (i.e. vincristine) is limited in tumor cells exhibiting pre-existing or acquired resistance. Here, we found that C6 ceramide (C6) dramatically sensitized vincristine's activity. In vitro, C6 and vincristine coadministration induced substantial necrosis and apoptosis in multiple human cancer cell lines, which were accompanied by a profound AMP-activated protein kinase (AMPK) activation, subsequent p53 activation, mTORC1 inactivation and Bcl-2/HIF-1α downregulation. Such synergistic effects were attenuated by AMPK inactivation through genetic mutation or short hairpin RNA silencing. Coadministration-activated p53 translocated to mitochondria, and formed a complex with cyclophilin-D, leading to mitochondrial permeability transition pore opening and cell necrosis. Disrupting p53-Cyp-D complexation through pharmacological or genetic means reduced costimulation-induced cytotoxicity. In vivo, a liposomal C6 was synthesized, which dramatically enhanced the antiproliferative activity of vincristine on HCT-116 or A2780 xenografts. Together, C6 sensitizes vincristine-induced anticancer activity in vivo and in vitro, involving activating AMPK-p53 signaling.

Mechanism involved in Danshen-induced fluid secretion in salivary glands.

AIM: Danshen's capability to induce salivary fluid secretion and its mechanisms were studied to determine if it could improve xerostomia. METHODS: Submandibular glands were isolated from male Wistar rats under systemic anesthesia with pentobarbital sodium. The artery was cannulated and vascularly perfused at a constant rate. The excretory duct was also cannulated and the secreted saliva was weighed in a cup on an electronic balance. The weight of the accumulated saliva was measured every 3 s and the salivary flow rate was calculated. In addition, the arterio-venous difference in the partial oxygen pressure was measured as an indicator of oxygen consumption. In order to assess the mechanism involved in Danshen-induced fluid secretion, either ouabain (an inhibitor of Na(+)/K(+) ATPase) or bumetanide (an inhibitor of NKCC1) was additionally applied during the Danshen stimulation. In order to examine the involvement of the main membrane receptors, atropine was added to block the M3 muscarinic receptors, or phentolamine was added to block the α1 adrenergic receptors. In order to examine the requirement for extracellular Ca(2+), Danshen was applied during the perfusion with nominal Ca(2+) free solution. RESULTS: Although Danshen induced salivary fluid secretion, 88.7 ± 12.8 µL/g-min, n = 9, (the highest value around 20 min from start of DS perfusion was significantly high vs 32.5 ± 5.3 µL/g-min by carbamylcholine, P = 0.00093 by t-test) in the submandibular glands, the time course of that secretion differed from that induced by carbamylcholine. There was a latency associated with the fluid secretion induced by Danshen, followed by a gradual increase in the secretion to its highest value, which was in turn followed by a slow decline to a near zero level. The application of either ouabain or bumetanide inhibited the fluid secretion by 85% or 93%, and suppressed the oxygen consumption by 49% or 66%, respectively. These results indicated that Danshen activates Na(+)/K(+) ATPase and NKCC1 to maintain Cl(-) release and K(+) release for fluid secretion. Neither atropine or phentolamine inhibited the fluid secretion induced by Danshen (263% ± 63% vs 309% ± 45%, 227% ± 63% vs 309% ± 45%, P = 0.899, 0.626 > 0.05 respectively, by ANOVA). Accordingly, Danshen does not bind with M3 or α1 receptors. These characteristics suggested that the mechanism involved in DS-induced salivary fluid secretion could be different from that induced by carbamylcholine. Carbamylcholine activates the M3 receptor to release inositol trisphosphate (IP3) and quickly releases Ca(2+) from the calcium stores. The elevation of [Ca(2+)]i induces chloride release and quick osmosis, resulting in an onset of fluid secretion. An increase in [Ca(2+)]i is essential for the activation of the luminal Cl(-) and basolateral K(+) channels. The nominal removal of extracellular Ca(2+) totally abolished the fluid secretion induced by Danshen (1.8 ± 0.8 µL/g-min vs 101.9 ± 17.2 µL/g-min, P = 0.00023 < 0.01, by t-test), suggesting the involvement of Ca(2+) in the activation of these channels. Therefore, IP3-store Ca(2+) release signalling may not be involved in the secretion induced by Danshen, but rather, there may be a distinct signalling process. CONCLUSION: The present findings suggest that Danshen can be used in the treatment of xerostomia, to avoid the systemic side effects associated with muscarinic drugs.

Preventive effect of Actinidia valvata Dunn extract on N-methyl-N'-nitro-N-nitrosoguanidine-induced gastrointestinal cancer in rats.

PURPOSE: This study was conducted to assess the preventive effect of Actinidia valvata Dunn (AVD) extract on an animal model of gastrointestinal carcinogenesis on the basis of changes in tumor incidence, cell proliferation, and apoptosis. MATERIALS AND METHODS: Seventy-five male Wistar rats were divided into five different treatment groups with 15 rats in each group. Group I was given normal feed, whereas Groups II to IV were treated with 10% sodium chloride in the first six weeks and 100 ug/mL of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in drinking water for 24 weeks. Group II was then given normal feed, whereas Group III was given AVD extract (0.24 g/kg/day) for 12 weeks. Group IV was given AVD extract from the first week to the 36th week, whereas Group V was treated with AVD extract alone for 36 weeks. All rats were sacrificed at the end of the 36-week experiment and assessed for the presence of gastrointestinal tumors. The occurrence of cancer was evaluated by histology. Bax, Bcl-2, Caspase-3, and cyclinD1 were determined by immunohistochemical staining and Western blotting. RESULTS: The incidences of gastric cancer were 0% in Group I, 73.3% in Group II, 33.3% in Group III, 26.7% in Group IV, and 0% in Group V. Bcl-2 and cyclinD1 expression was decreased in AVD extract treated groups, whereas Bax and Caspase-3 expression was increased. Comparison with group II revealed significant differences (p<0.01). CONCLUSIONS: AVD extract exhibits an obvious preventive effect on gastrointestinal carcinogenesis induced by MNNG in rats through the regulation of cell proliferation and apoptosis.

Histological changes of gastric mucosa after Helicobacter pylori eradication: a systematic review and meta-analysis.

AIM: To systematically review pathological changes of gastric mucosa in gastric atrophy (GA) and intestinal metaplasia (IM) after Helicobacter pylori (H. pylori) eradication. METHODS: A systematic search was made of PubMed, Web of Science, EMBASE, ClinicalTrials.gov, OVID and the Cochran Library databases for articles published before March 2013 pertaining to H. pylori and gastric premalignant lesions. Relevant outcomes from articles included in the meta-analysis were combined using Review Manager 5.2 software. A Begg's test was applied to test for publication bias using STATA 11 software. χ(2) and I(2) analyses were used to assess heterogeneity. Analysis of data with no heterogeneity (P > 0.1, I (2) < 25%) was carried out with a fixed effects model, otherwise the causes of heterogeneity were first analyzed and then a random effects model was applied. RESULTS: The results of the meta-analysis showed that the pooled weighted mean difference (WMD) with 95%CI was 0.23 (0.18-0.29) between eradication and non-eradication of H. pylori infection in antral IM with a significant overall effect (Z = 8.19; P <0.00001) and no significant heterogeneity (χ(2) = 27.54, I(2) = 16%). The pooled WMD with 95%CI was -0.01 (-0.04-0.02) for IM in the corpus with no overall effect (Z = 0.66) or heterogeneity (χ(2) = 14.87, I(2) =0%) (fixed effects model). In antral GA, the pooled WMD with 95% CI was 0.25 (0.15-0.35) with a significant overall effect (Z = 4.78; P < 0.00001) and significant heterogeneity (χ(2) = 86.12, I(2) = 71%; P < 0.00001). The pooled WMD with 95% CI for GA of the corpus was 0.14 (0.04-0.24) with a significant overall effect (Z = 2.67; P = 0.008) and significant heterogeneity (χ(2) = 44.79, I(2) = 62%; P = 0.0003) (random effects model). CONCLUSION: H. pylori eradication strongly correlates with improvement in IM in the antrum and GA in the corpus and antrum of the stomach.

TLR4 polymorphisms associated with developing gastric pre-cancer lesions in a Chinese Han population.

Helicobacter pylori infection is a risk factor for gastric cancer. In addition, toll-like receptor 4 (TLR4) plays a fundamental role in pathogen recognition and activation of innate immunity. This study investigated the association of TLR4 polymorphisms with a risk of intestinal metaplasia (IM) and intraepithelial neoplasia (IN) in a Chinese Han population. This study analyzed TLR4 gene polymorphisms in 333 patients (IM, 193 cases; IN, 140 cases) and 312 atypia-free controls in a Chinese Han population using a Taqman allelic discrimination assay. The TLR4 single nucleotide polymorphisms +896A/G and +1196C/T were not associated with the risk of IM or IN. However, the single-locus analysis showed that the C allele of TLR4+2856T/C had significantly reduced risk of IM and IN [adjusted odds ratio (OR)=0.42; 95%CI=0.29-0.62 and OR=0.62; 95%CI=0.41-0.93, respectively] compared with the wild-type homozygote (TT). The frequencies of TLR4+2856T/C TC and T carrier were significantly lower in patients with Sydney's slight IM and low grade IN (P<0.01 and P=0.01, respectively), while the TC genotype showed a lower risk of moderate IM compared to healthy controls (P=0.045). In addition, the data revealed that H. pylori infection, heavy alcohol consumption and high salt uptake were associated with a higher susceptibility for developing this neoplasm. TLR4 rs10759932 TC and C carriers were associated with a lower risk in developing precancerous lesions in the stomach in a Chinese Han population.

MicroRNA-451 regulates AMPK/mTORC1 signaling and fascin1 expression in HT-29 colorectal cancer.

The earlier studies have shown that Fascin1 (FSCN1), the actin bundling protein, is over-expressed in colorectal cancers, and is associated with cancer cell progression. Here, we aimed to understand the molecular mechanisms regulating FSCN1 expression by focusing on mammalian target of rapamycin (mTOR) signaling and its regulator microRNA-451. We found that microRNA-451 was over-expressed in multiple colorectal cancer tissues, and its expression was correlated with mTOR complex 1 (mTORC1) activity and FSCN1 expression. In cultured colorectal cancer HT-29 cells, knockdown of FSCN1 by RNAi inhibited cell migration and proliferation. Activation of mTORC1 was required for FSCN1 expression, HT-29 cell migration and proliferation, as RAD001 and rapamycin, two mTORC1 inhibitors, suppressed FSCN1 expression, HT-29 cell migration and proliferation. Meanwhile, forced activation of AMP-activated protein kinase (AMPK), the negative regulator of mTORC1, by its activators or by the genetic mutation, inhibited mTORC1 activation, FSCN1 expression, cell migration and proliferation. In HT-29 cells, we found that over-expression of microRNA-451 inhibited AMPK activation, causing mTORC1 over-activation and FSCN1 up-regulation, cells were with high migration ability and proliferation rate. Significantly, these effects by microRNA-451 were largely inhibited by mTORC1 inhibitors or the AMPK activator AICAR. On the other hand, knockdown of miRNA-451 by the treatment of HT-29 cells with miRNA-451 antagomir inhibited mTORC1 activation and FSCN1 expression. The proliferation and migration of HT-29 cells after miRNA-45 knockdown were also inhibited. Our results suggested that the over-expressed microRNA-451 in colon cancer cells might inhibit AMPK to activate mTORC1, which mediates FSCN1 expression and cancer cell progression.

Activation of AMP-activated protein kinase (AMPK) mediates plumbagin-induced apoptosis and growth inhibition in cultured human colon cancer cells.

Here we report that activation of AMP-activated protein kinase (AMPK) mediates plumbagin-induced apoptosis and growth inhibition in both primary cultured human colon cancer cells and cell lines. Knocking-down of AMPKα by the target shRNA significantly inhibits plumbagin-induced cytotoxicity in cultured colon cancer cells, while forced activation of AMPK by introducing a constitutively active AMPK (CA-AMPK), or by the AMPK activator, inhibits HT-29 colon cancer cell growth. Our Western-blots and immunoprecipitation (IP) results demonstrate that plumbagin induces AMPK/Apoptosis signal regulating kinase 1 (ASK1)/TNF receptor-associated factor 2 (TRAF2) association to activate pro-apoptotic c-Jun N-terminal kinases (JNK)-p53 signal axis. Further, after plumbagin treatment, activated AMPK directly phosphorylates Raptor to inhibit mTOR complex 1 (mTORC1) activation and Bcl-2 expression in colon cancer cells. Finally, we found that exogenously-added short-chain ceramide (C6) enhances plumbagin-induced AMPK activation and facilitates cell apoptosis and growth inhibition. Our results suggest that AMPK might be the key mediator of plumbagin's anti-tumor activity.

CFTR polymorphisms of healthy individuals in two Chinese cities--Changchun and Nanjing.

BACKGROUND AND AIM: Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a chloride channel, cause cystic fibrosis. In order to investigate the polymorphic backgrounds of CFTR genes of healthy populations in different Chinese cities (Changchun and Nanjing), we analyzed 119 blood samples (Changchun 64, Nanjing 55) of randomly selected healthy individuals for poly T, TG-repeats and M470V polymorphisms. We analyzed the differences of CFTR polymorphic distributions between the two Chinese cities from the south and the north. Methods Genomic DNA was extracted from whole blood. DNA fragments of CFTR gene were amplified by polymerase chain reaction (PCR). Poly-T and TG repeats were directly sequenced by auto sequencer (ABI 310). M470V was detected by a HphI restriction enzyme. RESULTS: The T7 allele was the most common haplotype in Changchun (0.938) and Nanjing (0.927) populations. The T5 allele was present in only 7 Changchun and 3 Nanjing subjects. The TG11 and TG12 alleles were dominant haplotypes in Changchun (TG11 0.500, TG12 0.453) and Nanjing (TG11 0.345, TG12 0.609). The frequency of the V470 allele was 0.633 in Changchun, which was higher than that in Nanjing (0.500) (p < 0.05). There were three major haplotypes: T7-TG11-V470, T7-TG12-M470 and T7-TG12-V470. The T7-TG11-V470 was the most common haplotype in Changchun (0.514), while T7-TG12-M470 was the most common haplotype in Nanjing (0.500). CONCLUSION: Though Changchun and Nanjing are in the same country, their polymorphic backgrounds of CFTR gene are very different. Most of the two populations have genotypes that cause lower CFTR function.

Association between polymorphisms of trinucleotide repeat containing 9 gene and breast cancer risk: evidence from 62,005 subjects.

Trinucleotide repeat containing 9 (TNRC9) is a gene located at chromosome 16q12. Although of an uncertain function, it is a newly described risk factor for breast cancer. It contains a putative high-mobility group box motif, suggesting its possible role as transcription factor; it has been implicated in breast cancer metastasis. Published studies on the association between TNRC9 polymorphisms and breast cancer risk remain inconclusive, and a meta-analysis is required to verify the association. This pioneering research performed a meta-analysis of eight studies comprising a total of 25,828 cases and 36,177 controls. Significantly elevated breast cancer risk was associated with TNRC9 rs3803662 polymorphism when all studies were pooled in the meta-analysis (T vs. C allele contrast model: OR 1.18, 95% CI 1.09-1.28; TT vs. CC homozygote codominant model: OR 1.26, 95% CI 1.02-1.55; TT vs. CC+CT recessive model: OR 1.23, 95% CI 1.06-1.42). For TNRC9 rs12443621 polymorphism, no significant association was detected in all genetic models. For TNRC9 rs12443621 polymorphism, meanwhile, no significant association was observed in all comparison models. Conclusively, this meta-analysis suggests that TNRC9 rs3803662 polymorphism was significantly correlated with breast cancer risk and the variant T allele of TNRC9 rs3803662 polymorphism is a low-penetrant risk factor for developing breast cancer. There is no significant association between TNRC9 rs12443621 and rs8051542 polymorphisms and risk of breast cancer in current literature.

Association between mitogen-activated protein kinase kinase kinase 1 rs889312 polymorphism and breast cancer risk: evidence from 59,977 subjects.

Published data on the association between mitogen-activated protein kinase kinase kinase 1 (MAP3K1) gene rs889312 polymorphism and breast cancer risk are inconclusive. To derive a more precise estimation of the relationship, a meta-analysis was performed. Crude ORs with 95% CIs were used to assess the strength of association between them. A total of seven eligible articles including 26,015 cases and 33,962 controls based on the search criteria were involved in this meta-analysis. We observed that the MAP3K1 rs889312 polymorphism was significantly correlated with breast cancer risk from the fixed effects model when all studies were pooled into the meta-analysis (the allele contrast model: OR 1.09, 95% CI 1.07-1.12; the homozygote codominant: OR 1.22, 95% CI 1.15-1.29; the heterozygote codominant: OR 1.07, 95% CI 1.04-1.11; the dominant model: OR 1.10, 95% CI 1.06-1.13; the recessive model: OR 1.18, 95% CI 1.12-1.25). No significant association was found in the BRCA1 mutation carriers in all genetic models. When stratified by BRCA2 mutation carriers status, statistically significantly elevated risk was found in this meta-analysis (C vs. A: OR 1.12, 95% CI 1.01-1.23; CC vs. AA: OR 1.35, 95% CI 1.06-1.71; the recessive model: OR 1.31, 95% CI 1.05-1.65). There was no evidence for significant association between MAP3K1 rs889312 polymorphism and breast cancer risk in BRCA1 and BRCA2 positive cohort for all comparison models. In conclusion, this meta-analysis suggests that the MAP3K1 rs889312 C allele is a low-penetrant risk factor for developing breast cancer, and there is limited evidence to indicate that MAP3K1 rs889312 polymorphism is associated with increased risk of breast cancer in BRCA1 mutation carriers.