Aberrant peribiliary gland niche exacerbates fibrosis in primary sclerosing cholangitis and a potential therapeutic strategy.

Primary sclerosing cholangitis (PSC) is a rare but progressive and fatal autoimmune disease without clear pathogenesis and effective therapies. Peribiliary macrophage recruitment and peribiliary gland (PBG) proliferation and expansion have been associated with various cholangiopathies. This study aimed to evaluate the involvement of the PBG niche and macrophages in PSC progression, potential treatment strategies, and the underlying mechanism in acute and chronic experimental PSC. First, the upregulation of chemokines and fibrosis in PSC patients was confirmed via RNA-seq analysis. In vivo data illustrated that inflammation and fibrosis are the main characteristics, and recession of these can effectively interfere with PSC. Histopathological staining and RT-PCR revealed that more significant ductular reaction (DR) and PBG proliferation in the chronic PSC model, in which fibrosis mainly accumulated in the peribiliary area. In vitro, a transwell migration experiment showed that MCP-1 secreted by cholangiocytes in PBG niche, which recruited monocyte-derived macrophages (MoMFs) to the peribiliary area and promoted inflammation and fibrosis. Then, the luciferase assay and EMSA showed that POU6F1 could activate MCP-1 transcription. Furthermore, 18ß-Glycyrrhetinic acid (GA) reduced macrophages and fibrosis accumulated in the peribiliary, space and reduced PBG proliferation to benefit acute and chronic PSC models. Collectively, our results indicated that POU6F1 transcriptionally activates MCP-1, promoting the recruitment and infiltration of MoMFs and fibrosis into the PBG niche in PSC mouse models, and GA effectively suppressed the above phenotypes. These findings provide potential targets and a theoretical basis for the clinical treatment of PSC.

Aristolochic acids exposure was not the main cause of liver tumorigenesis in adulthood.

Aristolochic acids (AAs) have long been considered as a potent carcinogen due to its nephrotoxicity. Aristolochic acid I (AAI) reacts with DNA to form covalent aristolactam (AL)-DNA adducts, leading to subsequent A to T transversion mutation, commonly referred as AA mutational signature. Previous research inferred that AAs were widely implicated in liver cancer throughout Asia. In this study, we explored whether AAs exposure was the main cause of liver cancer in the context of HBV infection in mainland China. Totally 1256 liver cancer samples were randomly retrieved from 3 medical centers and a refined bioanalytical method was used to detect AAI-DNA adducts. 5.10% of these samples could be identified as AAI positive exposure. Whole genome sequencing suggested 8.41% of 107 liver cancer patients exhibited the dominant AA mutational signature, indicating a relatively low overall AAI exposure rate. In animal models, long-term administration of AAI barely increased liver tumorigenesis in adult mice, opposite from its tumor-inducing role when subjected to infant mice. Furthermore, AAI induced dose-dependent accumulation of AA-DNA adduct in target organs in adult mice, with the most detected in kidney instead of liver. Taken together, our data indicate that AA exposure was not the major threat of liver cancer in adulthood.

Transcytosis mechanisms of cell-penetrating peptides: Cation-independent CC12 and cationic penetratin.

Cell-penetrating peptides (CPPs) can aid in intracellular and in vivo drug delivery. However, the mechanisms of CPP-mediated penetration remain unclear, limiting the development and further application of CPPs. Flow cytometry and laser confocal fluorescence microscopy were performed to detect the effects of different endocytosis inhibitors on the internalization of CC12 and penetratin in ARPE-19 cells. The co-localization of CPPs with the lysosome and macropinosome was detected via an endocytosis tracing experiment. The flow cytometry results showed that chlorpromazine, wortmannin, cytochalasin D, and the ATP inhibitor oligomycin had dose-dependent endocytosis-inhibitory effects on CC12. The laser confocal fluorescence results showed that oligomycin had the most significant inhibitory effect on CC12 uptake; CC12 was co-located with the lysosome, but not with the macropinosome. For penetratin, cytochalasin D and oligomycin had obvious inhibitory effects. The laser confocal fluorescence results indicated that oligomycin had the most significant inhibitory effect on penetratin uptake; the co-localization of penetratin with the lysosome was higher than that with the macropinosome. Cation-independent CC12 and cationic penetratin may be internalized into cells primarily through caveolae and clathrin-mediated endocytosis, and they are typically dependent on ATP. The transport of penetratin could be partly achieved through the direct transmembrane pathway, as the positive charge of penetratin interacts with the negative charge of the cell membrane, and partly through the endocytic pathway.

Kupffer cells play a crucial role in monocrotaline-induced liver injury by producing TNF-α.

Monocrotaline (MCT), an unsaturated pyrrolizidine alkaloid (PA) in plants, is mainly toxic to the lung and liver of mammals. As a commonly used tool for liver injury model, the mechanism of MCT hepatoxicity has still not been fully clarified. Kupffer cells (KCs) are the liver-resident macrophages and have various responses to different toxicants and liver damage. However, the role of KCs in MCT-induced liver injury remains controversial. In current work, we investigated the effects of KCs on MCT-induced liver injury, especially on MCT-induced hepatocyte death. KCs were depleted in Balb/c mice by liposome-entrapped clodronate (Lip/Clo) (0.2 mL/mouse, i.p.) or GdCl3 (0.7 mg/kg, i.p.) before MCT administration (300 mg/kg, i.p.), we found that the Lip/Clo group showed higher efficiency in KCs depletion and stronger hepatoprotective effects against MCT. We also found TNF-α was remarkably decreased after KCs depletion, the experiment of administering anti-TNF-α antibody (20 µg/mouse, i.p.) to MCT-treated animals generated the similar results. To further elaborate the function of KCs, we compared the ALT levels released from co-culturing murine hepatic parenchymal cells (HPCs) and RAW264.7 cells with that from HPCs alone. After the treatment of MCT, the released ALT levels in co-culture system were shown to be dependent on the number of RAW264.7 cells, while the anti-TNF-α antibody could suppress it. Finally, we discovered RIPK3/MLKL pathway might be activated by TNF-α released from KCs, and subsequently induced hepatocyte necrosis. Noteworthy, the known mechanisms including ER stress and NF-κB pathways were also found to be involved in the activation of KCs. In conclusion, our study reveals a further mechanism to MCT-induced hepatoxicity mediated directly by KCs via producing TNF-α.

Profiling pharmacokinetics of double-negative T cells and cytokines via a single intravenous administration in NSG mice.

This study was intended to delineate the profile of double-negative T cells (DNTs) in NOD.Cg-Prkdcscid Il2rgtm1wj /SzJ mice and cytokines released from DNTs in vivo and in vitro. Total 4 × 107 cells of RC1012 injection per mice were intravenously infused. IFN-γ, TNF-α, IL-1ß, IL-2, IL-4, IL-6, IL-10 were measured in vivo and in vitro. A quantitative polymerase chain reaction (PCR) was employed to determine the gene copies of Notch2-NLA per DNT cell from collected organs. Cytokines were significantly increased in vitro (4 h) and in vivo (3 h). DNT cells were distributed into the lung, liver, heart, and kidney earlier, and redistributed to lymphocyte homing spleen and bone marrow, which seemed to frame a two-compartment pharmacokinetics (PK) model but more data are needed to confirm this, and the clearance of DNT cells fell into first-order kinetics.

ZEB1 Mediates Bone Marrow Mesenchymal Stem Cell Osteogenic Differentiation Partly via Wnt/ß-Catenin Signaling.

Zinc finger E-box-binding homebox 1 (ZEB1) is a zinc-finger transcription factor best known for its role in promoting the epithelial-mesenchymal transition, which is also related to osteogenesis. Here, ZEB1 was investigated for its role in the commitment of bone marrow mesenchymal stem cells (BMSCs) to osteoblasts. In vitro, ZEB1 expression decreased following osteogenic differentiation. Furthermore, silencing of ZEB1 in BMSCs promoted osteogenic activity and mineralization. The increase in osteogenic differentiation induced by si-ZEB1 could be partly rescued by the inhibition of Wnt/ß-catenin (si-ß-catenin). In vivo, knockdown of ZEB1 in BMSCs inhibited the rapid bone loss of ovariectomized (OVX) mice. ZEB1 expression has also been negatively associated with bone mass and bone formation in postmenopausal women. In conclusion, ZEB1 is an essential transcription factor in BMSC differentiation and may serve as a potential anabolic strategy for treating and preventing postmenopausal osteoporosis (PMOP).

Aristolochic acid I promoted clonal expansion but did not induce hepatocellular carcinoma in adult rats.

Aristolochic acid I (AAI) is a well-known nephrotoxic carcinogen, which is currently reported to be also associated with hepatocellular carcinoma (HCC). Whether AAI is a direct hepatocarcinogen remains controversial. In this study we investigated the association between AAI exposure and HCC in adult rats using a sensitive rat liver bioassay with several cofactors. Formation of glutathione S-transferase placental form-positive (GST-P+) foci was used as the marker for preneoplastic lesions/clonal expansion. We first conducted a medium-term (8 weeks) study to investigate whether AAI had any tumor-initiating or -promoting activity. Then a long-term (52 weeks) study was conducted to determine whether AAI can directly induce HCC. We showed that oral administration of single dose of AAI (20, 50, or 100 mg/kg) in combination with partial hepatectomy (PH) to stimulate liver proliferation did not induce typical GST-P+ foci in liver. In the 8-week study, only high dose of AAI (10 mg · kg-1 · d-1, 5 days a week for 6 weeks) in combination with PH significantly increased the number and area of GST-P+ foci initiated by diethylnitrosamine (DEN) in liver. Similarly, only high dose of AAI (10 mg· kg-1· d-1, 5 days a week for 52 weeks) in combination with PH significantly increased the number and area of hepatic GST-P+ foci in the 52-week study. No any nodules or HCC were observed in liver of any AAI-treated groups. In contrast, long-term administration of AAI (0.1, 1, 10 mg· kg-1· d-1) time- and dose-dependently caused death due to the occurrence of cancers in the forestomach, intestine, and/or kidney. Besides, AAI-DNA adducts accumulated in the forestomach, kidney, and liver in a time- and dose-dependent manner. Taken together, AAI promotes clonal expansion only in the high-dose group but did not induce any nodules or HCC in liver of adult rats till their deaths caused by cancers developed in the forestomach, intestine, and/or kidney. Findings from our animal studies will pave the way for further large-scale epidemiological investigation of the associations between AA and HCC.

MiR-629-5p promotes the invasion of lung adenocarcinoma via increasing both tumor cell invasion and endothelial cell permeability.

Tumor invasion underlies further metastasis, the leading cause for cancer-related deaths. Deregulation of microRNAs has been identified associated with the malignant behavior of various cancers, including lung adenocarcinoma (LUAD), the major subtype of lung cancer. Here, we showed the significantly positive correlation between miR-629-5p level and tumor invasion in LUAD specimens (n = 49). In a human LUAD metastasis mouse model, H1650 cells (high level of miR-629-5p) were more aggressive than A549 cells (low level of miR-629-5p) in vivo, including higher incidence of vascular invasion and pulmonary colonization. Ectopic expression of miR-629-5p in A549 cells also increased their invasive capability. Then we identified that miR-629-5p promotes LUAD invasion in a mode of dual regulation via tumor cells invasion and endothelial cells permeability, respectively. In tumor cells, miR-629-5p enhanced motility and invasiveness of tumor cells by directly targeting PPWD1 (a cyclophilin), which clinically related to tumor invasion in LUAD specimens. Restoring PPWD1 protein significantly attenuated the invasion-promoting effects of miR-629-5p. Besides, exosomal-miR-629-5p secreted from tumor cells could be transferred to endothelial cells and increased endothelial monolayers permeability by suppressing CELSR1 (a nonclassic-type cadherin), which had a low level in the endothelial cells of invasive LUAD specimens. Activating the expression of CELSR1 in endothelial cells markedly blocked the effect of miR-629-5p. Our study suggests the dual roles of miR-629-5p in tumor cells and endothelial cells for LUAD invasion, implying a therapeutic option to targeting miR-629-5p using the "one stone, two birds" strategy in LUAD.

(5R)-5-hydroxytriptolide ameliorates liver lipid accumulation by suppressing lipid synthesis and promoting lipid oxidation in mice.

AIMS: (5R)-5-hydroxytriptolide (LLDT-8) is a triptolide analog with excellent capability against cancers, cerebral ischemic injury and rheumatoid arthritis. Here, we discovered its hepatoprotective effects in a mouse model of non-alcoholic fatty liver disease (NAFLD) by ameliorating liver lipid accumulation. MAIN METHODS: Male C57BL/6J mice were fed with a high-fat/high-fructose (HFHFr) diet for 29 weeks to induce the pathological phenomena of NAFLD. Then the mice were treated with LLDT-8 (0.5mg/kg and 1mg/kg) or Vehicle for 8 weeks. Finally, the serum biochemical indexes, liver histological features, fatty acids (FAs) profile and related gene expression in liver were detected to investigate the effect of LLDT-8 on lipid accumulation and its possible mechanism. KEY FINDINGS: LLDT-8 treatment significantly inhibited hepatic injury featured by the decrease of serum alanine aminotransferase (ALT) and aspartate transaminase (AST), the lessening of hepatic ballooning and macrovesicular steatosis. Moreover, LLDT-8 could downregulate the expression of stearoyl-CoA desaturase 1 (SCD1), which further led to the lower ratios of C16:1/C16:0 and C18:1/C18:0 and thus inhibited lipid synthesis. LLDT-8 treatment also could upregulate liver peroxisome proliferator-activated receptor α (PPARα), carnitine palmitoyltransferase 1a (Cpt1a), peroxisomal acyl-CoA oxidase 1 (Acox1), long-chain acyl-CoA dehydrogenase (Acadl) and medium-chain acyl-CoA dehydrogenase (Acadm) expression levels involved in fatty acids oxidation (FAO) and markedly promoted lipolysis. SIGNIFICANCE: Our results provide a novel application of LLDT-8 in improving NAFLD.

MiR-1205 functions as a tumor suppressor by disconnecting the synergy between KRAS and MDM4/E2F1 in non-small cell lung cancer.

Activated KRAS is frequently observed and paralleled by inactivating of tumor suppressors in lung cancer, while the mechanisms remained elusive. Here, our study revealed a microRNA was involved in KRAS overexpression, activation of KRAS signaling and its synergy with inactivating of tumor suppressor genes. miR-1205 was selected by its sequence-dependent inhibition on KRAS and negative clinical correlation with KRAS. A549 and H460 cells carrying mutant KRAS, were sensitive to the growth inhibition and G1/S arrest induced by miR-1205. Target analysis revealed that miR-1205 could simultaneously downregulate the expression levels of MDM4 and E2F1, which were downstream of KRAS and synergistic with KRAS. Silencing of MDM4 or E2F1 inhibited cellular proliferation. MiR-1205 decreased the protein levels of MDM4 and E2F1 via directly binding to the coding sequence of E2F1 and 3'UTR of MDM4. Meanwhile, blocking RAS-MAPK signaling using KRAS siRNA or ERK1/2 inhibitor exerted similar inhibitory effects on MDM4 and E2F1. Forced expression of KRAS partially restored the inhibition of miR-1205 on MDM4 and E2F1. Overexpression of KRAS, MDM4 or E2F1 could partially rescued the growth inhibition of miR-1205 in vitro. More importantly, miR-1205 strongly inhibited the tumor growth of A549 xenografts in nude mice and decreased the protein levels of KRAS, MDM4 and E2F1 in tumor tissues. Together, our study firstly confirmed a potential synergy between KRAS and MDM4/E2F1 which are p53/RB inactivators in non-small cell lung cancer, and identified miR-1205 as a potent destructor of this synergy, making miR-1205 function as a tumor suppressor in vitro and in vivo.

The dual-inhibitory effect of miR-338-5p on the multidrug resistance and cell growth of hepatocellular carcinoma.

Chemotherapeutic treatments against hepatocellular carcinoma (HCC) are necessary for both inoperable patients to improve prospects for survival and surgery patients to improve the outcome after surgical resection. However, multidrug resistance (MDR) is a major obstacle to obtaining desirable results. Currently, increasing the chemotherapy sensitivity of tumor cells or discovering novel tumor inhibitors is an effective therapeutic strategy to solve this issue. In the present study, we uncovered the dual-inhibitory effect of miR-338-5p: on the one hand, it could downregulate ABCB1 expression and sensitize HCC cells to doxorubicin and vinblastine by directly targeting the 3'-untranslated region (3'-UTR) of ABCB1, while, on the other hand, it could suppress the proliferation of HCC cells by directly targeting the 3'-UTR of EGFR and reducing EGFR expression. Since EGFR regulates ABCB1 levels, the indirect action of miR-338-5p in ABCB1 modulation was revealed, in which miR-338-5p inhibits ABCB1 expression by targeting the EGFR/ERK1/2 signaling pathway. These data indicate that the miR-338-5p/EGFR/ABCB1 regulatory loop plays a critical role in HCC, and a negative correlation between miR-338-5p and EGFR or ABCB1 was also detected in HCC clinical samples. In conclusion, these findings reveal a critical role for miR-338-5p in the regulation of MDR and proliferation of HCC, suggesting the potential therapeutic implications of miR-338-5p in HCC treatment.

The miR-491-3p/Sp3/ABCB1 axis attenuates multidrug resistance of hepatocellular carcinoma.

As one of main obstacles in the treatment and prognosis of hepatocellular carcinoma (HCC), multidrug resistance (MDR) is usually associated with the overexpression of the drug efflux pump P-glycoprotein (P-gp/ABCB1) which is responsible for reducing the intracellular concentration of chemotherapeutic agents. In current work, we discovered the novel role of miR-491-3p in ABCB1-mediated multidrug resistance in HCC and revealed the underlying mechanism in which miR-491-3p downregulated the expression of ABCB1 and its transcription factor Sp3 by directly targeting their 3'-UTR. Moreover, overexpressing ABCB1 or Sp3 reversed the sensitivity to chemotherapeutics in Hep3B cells induced by miR-491-3p, confirming miR-491-3p/Sp3/ABCB1 regulatory loop plays an important role in enhancing the drugs sensitivity of HCC. Meanwhile, the discovery of that the expression level of miR-491-3p was inversely correlated with that of ABCB1 and Sp3 in HCC cell lines and clinical samples pointed out the possibility of miR-491-3p in clinical use. In summary, our results reveal a pivotal role of miR-491-3p in the regulation of MDR in HCC, and suggest the potential application of miR-491-3p as a therapeutic strategy for modulating MDR in cancer cells.

MiR-1254 suppresses HO-1 expression through seed region-dependent silencing and non-seed interaction with TFAP2A transcript to attenuate NSCLC growth.

MicroRNAs (miRNAs) are a class of small non-coding RNAs, which direct post-transcriptional gene silencing (PTGS) and function in a vast range of biological events including cancer development. Most miRNAs pair to the target sites through seed region near the 5' end, leading to mRNA cleavage and/or translation repression. Here, we demonstrated a miRNA-induced dual regulation of heme oxygenase-1 (HO-1) via seed region and non-seed region, consequently inhibited tumor growth of NSCLC. We identified miR-1254 as a negative regulator inhibiting HO-1 translation by directly targeting HO-1 3'UTR via its seed region, and suppressing HO-1 transcription via non-seed region-dependent inhibition of transcriptional factor AP-2 alpha (TFAP2A), a transcriptional activator of HO-1. MiR-1254 induced cell apoptosis and cell cycle arrest in human non-small cell lung carcinoma (NSCLC) cells by inhibiting the expression of HO-1, consequently suppressed NSCLC cell growth. Consistently with the in vitro studies, mouse xenograft studies validated that miR-1254 suppressed NSCLC tumor growth in vivo. Moreover, we found that HO-1 expression was inversely correlated with miR-1254 level in human NSCLC tumor samples and cell lines. Overall, these findings identify the dual inhibition of HO-1 by miR-1254 as a novel functional mechanism of miRNA, which results in a more effective inhibition of oncogenic mRNA, and leads to a tumor suppressive effect.

Fbxw7 haploinsufficiency loses its protection against DNA damage and accelerates MNU-induced gastric carcinogenesis.

Fbxw7, a subunit of the SCF E3 ubiquitin ligase, recognizes oncoprotein substrates and leads to their proteasomal degradation. Fbxw7 acts as a tumor suppressor via inducing apoptosis and growth arrest in various kinds of tumors. To clarify the initiating role in gastric carcinogenesis as well as the histologic characterization of tumor in Fbxw7 allele haploinsufficient mice, we generated Fbxw7 heterozygous knockout mice (Fbxw7+/-) and treated them with chemical carcinogen N-methyl-N-nitrosourea (MNU) at 5-6 weeks of age. We also treated mouse embryo fibroblasts (MEFs) from Fbxw7+/- and Fbxw7+/+ mice with MNU and examined cell DNA damage via comet assay. The protein expression of Fbxw7 and its substrate c-Myc from mouse tumors, as well as human tumors sampled from six patients, were detected by Western blot. As results, the tumor incidence was obviously higher in Fbxw7+/- mice (13/20) than in Fbxw7+/+ mice (6/20) after 35-week observation. Intestinal metaplasia (P = 0.013) and dysplasia (P = 0.036) were more severe in Fbxw7+/- mice than in Fbxw7+/+ mice. The repair potential of DNA damage was suppressed in MEFs from Fbxw7+/- mice after MNU exposure. Increased c-Myc expression was accompanied by decreased Fbxw7 protein expression in tumor tissues from mouse and patients. In conclusion, Fbxw7 haploinsufficiency increased the risk of gastric carcinogenesis induced by MNU, which is associated with the accumulation of DNA damage as well as c-Myc oncoprotein.

1,2:5,6-dianhydrogalactitol inhibits human glioma cell growth in vivo and in vitro by arresting the cell cycle at G2/M phase.

1,2:5,6-dianhydrogalactitol (DAG) is a hexitol epoxide with marked antitumor activity against multiple types of cancer cells, but the molecular mechanisms by which DAG functions as an antitumor agent is largely unknown. In this study, we investigated the inhibitory effects of DAG on human glioma cell growth in vitro and in vivo and uncovered the underlying molecular mechanisms. Treatment with DAG (120 µmol/L) dose-dependently inhibited the proliferation and colony formation in human glioma cell lines LN229, U251, and U87MG in vitro. DAG (1, 2, 5 µmol/L) induced cell cycle arrest at G2/M phase in the 3 glioma cell lines in a dose-dependent manner. The signaling pathways involved in DAG-caused cell cycle arrest was further analyzed in LN229 cells, which revealed that DAG dose-dependently activated two parallel signaling cascades, ie, the p53-p21 cascade and the CDC25C-CDK1 cascade. DAG also significantly enhanced the radiosensitivity of LN229 cells as shown in the clonogenic assay. In nude mice bearing subcutaneously xenografted LN229 glioma, administration of DAG (5 mg/kg, iv, twice per week for 6 weeks) effectively suppressed the growth of xenografted tumors: the relative tumor growth rate (T/C) was reduced to 22.38%, and the tumor growth inhibitory rate (TGI) was 83.58% (P<0.01). In addition, DAG administration significantly activated the CDC25C-CDK1 cascade in the xenografted tumors. In conclusion, DAG inhibited human glioma cell growth in vitro and in vivo by inducing cell cycle arrest at G2/M phase. Two parallel cascades are activated and involved in the cell cycle arrest.

MicroRNA-1304 suppresses human non-small cell lung cancer cell growth in vitro by targeting heme oxygenase-1.

Previous studies have shown that microRNA-1304 (miR-1304) is dysregulated in certain types of cancers, including non-small cell lung cancer (NSCLC), and might be involved in tumor survival and/or growth. In this study we investigated the direct target of miR-1304 and its function in NSCLC in vitro. Human lung adenocarcinoma cell lines (A549 and NCI-H1975) were studied. The cell proliferation and survival were investigated via cell counting, MTT and colony-formation assays. Cell apoptosis and cell cycle were examined using annexin V-PE/7-AAD and PI staining assays, respectively. The dual-luciferase reporter assay was used to verify post-transcriptional regulation of heme oxygenase-1 (HO-1) by miR-1304. CRISPR/Cas9 was used to deplete endogenous miR-1304. Overexpression of MiR-1304 significantly decreased the number and viability of NSCLC cells and colony formation, and induced cell apoptosis and G0/G1 phase cell cycle arrest. HO-1 was demonstrated to be a direct target of miR-1304 in NSCLC cells. Restoration of HO-1 expression by hemin (20 µmol/L) abolished the inhibition of miR-1304 on cell growth and rescued miR-1304-induced apoptosis in A549 cells. Suppression of endogenous miR-1304 with anti-1304 significantly increased HO-1 expression and promoted cell growth and survival in A549 cells. In 17 human NSCLC tissue samples, miR-1304 expression was significantly decreased, while HO-1 expression was significantly increased as compared to normal lung tissues. MicroRNA-1304 is a tumor suppressor and HO-1 is its direct target in NSCLC. The results suggest the potential for miR-1304 as a therapeutic target for NSCLC.

miR-145 sensitizes breast cancer to doxorubicin by targeting multidrug resistance-associated protein-1.

Multidrug resistance-associated protein 1 (MRP1) is an important efflux transporter and overexpression of MRP1 usually leads to chemoresistance in breast cancer. Here, we found MRP1 overexpressed in human breast cancer tissues and breast cancer cell lines (compared with normal breast tissues and cell line, respectively). And MRP1 level increased in doxorubicin resistant MCF-7 cells compared with parental MCF-7 cells. Increasing evidences suggest microRNAs (miRNAs) influence chemotherapy response. We found miR-145 level decreased in human breast cancer tissues, breast cancer cell lines and doxorubicin resistant MCF-7 cells, and inversely correlated with MRP1 expression level. In the process of constructing MCF-7 doxorubicin resistant cell line, escalating doxorubicin markedly decreased miR-145 level, following by increased MRP1 level. Further study showed, miR-145 suppressed MRP1 expression by directly targeting MRP1 3'-untranslated regions. Overexpression of miR-145 sensitized breast cancer cells to doxorubicin in vitro and enhanced to doxorubicin chemotherapy in vivo through inducing intracellular doxorubicin accumulation via inhibiting MRP1. Taken together, our study revealed miR-145 sensitizes breast cancer to doxorubicin by targeting MRP1 and indicated the potential application in developing MRP1 inhibitor.

Dephosphorylation of Tak1 at Ser412 greatly contributes to the spermatocyte-specific testis toxicity induced by (5R)-5-hydroxytriptolide in C57BL/6 mice.

(5R)-5-Hydroxytriptolide (LLDT-8), a novel triptolide derivative, will proceed to phase II clinical trials for the treatment of rheumatoid arthritis and cancer. However, the selection of disease and patients is largely limited by the testis toxicity, yet toxicity mechanisms are still poorly understood. In this study, LLDT-8 dose and time-dependently decreased the testes weight, germinal cell layers and induced abnormal spermatid development. Analysis of the germ cell-specific marker showed that spermatocytes were more sensitive to LLDT-8, which was confirmed by the in vitro sensitivity assay with spermatocyte-like GC-2spd and sertoli-like TM4 cells. In GC-2spd, LLDT-8 induced G1/S arrest and apoptosis. MAPK activity screening identified that TGF-ß activated kinase 1 (Tak1) is critical in LLDT-8 induced apoptosis. LLDT-8 reduced the Tak1 protein and dephosphorylated Tak1 at Ser412 in GC-2spd and the testes, but not in TM4. RNAi mediated depletion or pharmacologic inhibition of Tak1 induced apoptosis in GC-2spd. Meanwhile, activating Tak1 rescued up to 50% of the GC-2spd cells from the apoptosis induced by LLDT-8. Altogether, our study firstly revealed the important role of Tak1 in the survival of spermatocytes, and dephosphorylation of Tak1 at Ser412 may contribute to the spermatocyte-specific testis toxicity induced by LLDT-8.

Baicalin Protects Mice from Aristolochic Acid I-Induced Kidney Injury by Induction of CYP1A through the Aromatic Hydrocarbon Receptor.

Exposure to aristolochic acid I (AAI) can lead to aristolochic acid nephropathy (AAN), Balkan endemic nephropathy (BEN) and urothelial cancer. The induction of hepatic CYP1A, especially CYP1A2, was considered to detoxify AAI so as to reduce its nephrotoxicity. We previously found that baicalin had the strong ability to induce CYP1A2 expression; therefore in this study, we examined the effects of baicalin on AAI toxicity, metabolism and disposition, as well as investigated the underlying mechanisms. Our toxicological studies showed that baicalin reduced the levels of blood urea nitrogen (BUN) and creatinine (CRE) in AAI-treated mice and attenuated renal injury induced by AAI. Pharmacokinetic analysis demonstrated that baicalin markedly decreased AUC of AAI in plasma and the content of AAI in liver and kidney. CYP1A induction assays showed that baicalin exposure significantly increased the hepatic expression of CYP1A1/2, which was completely abolished by inhibitors of the Aromatic hydrocarbon receptor (AhR), 3',4'-dimethoxyflavone and resveratrol, in vitro and in vivo, respectively. Moreover, the luciferase assays revealed that baicalin significantly increased the luciferase activity of the reporter gene incorporated with the Xenobiotic response elements recognized by AhR. In summary, baicalin significantly reduced the disposition of AAI and ameliorated AAI-induced kidney toxicity through AhR-dependent CYP1A1/2 induction in the liver.

Absence of mature microRNAs inactivates the response of gene expression to carcinogenesis induced by N-ethyl-N-nitrosourea in mouse liver.

This study aims to evaluate the role of microRNAs (miRNAs) in chemical tumorigenesis by evaluating genomic gene expression in miRNA knockout mice. Previous studies showed that mice without mature miRNAs due to hepatocyte-specific Dicer1 knockout (KO) had a much higher liver tumor incidence than wild-type mice. In this study, Dicer1 KO or the wild-type mice were treated intraperitoneally with genotoxic carcinogen N-ethyl-N-nitrosourea (ENU) at a single dose (150 mg kg(-1) that resulted in liver tumorigenesis) or the vehicle at 3 weeks of age. The animals were killed 2 weeks after treatment and the liver samples were collected for the gene expression study. Principal components analysis and hierarchical cluster analysis showed that gene expression was globally altered by the Dicer1 KO and ENU exposure. There were 5621, 3286 and 2565 differentially expressed genes for Dicer1 disruption, ENU treatment in wild-type mice and ENU treatment in Dicer1 KO mice, respectively. Functional analysis of the differentially expressed genes suggests that the Dicer1 KO mouse liver lost their capability to suppress the carcinogenesis induced by ENU exposure in genomic level. In addition, the miRNA-mediated BRCA1 and P53 signaling pathways were identified as the main pathways responsible for the tumorigenesis. We conclude that the mouse livers in the absence of mature miRNAs could not appropriately respond to carcinogenic insults from ENU treatment, indicating that miRNAs play a critical role in chemical carcinogenesis.