Search

Extra copies of centrosomes are frequently observed in cancer cells. To survive and proliferate, cancer cells have developed strategies to cluster extra-centrosomes to form bipolar mitotic spindles. The aim of this study was to investigate whether centrosome clustering (CC) inhibition (CCi) would preferentially radiosensitize non–small cell lung cancer (NSCLC). Griseofulvin (GF; FDA-approved treatment) inhibits CC, and combined with radiation treatment (RT), resulted in a significant increase in the number of NSCLC cells with multipolar spindles, and decreased cell viability and colony formation ability in vitro. In vivo, GF treatment was well tolerated by mice, and the combined therapy of GF and radiation treatment resulted in a significant tumor growth delay. Both GF and radiation treatment also induced the generation of micronuclei (MN) in vitro and in vivo and activated cyclic GMP-AMP synthase (cGAS) in NSCLC cells. A significant increase in downstream cGAS-STING pathway activation was seen after combination treatment in A549 radioresistant cells that was dependent on cGAS. In conclusion, GF increased radiation treatment efficacy in lung cancer preclinical models in vitro and in vivo. This effect may be associated with the generation of MN and the activation of cGAS. These data suggest that the combination therapy of CCi, radiation treatment, and immunotherapy could be a promising strategy to treat NSCLC.

Figure S1 shows basal centrosome amplification in cell lines. Figure S2 shows the effect of GF on cell viability. Figure S3 shows HSET expression in cell lines. Figure S4 shows the effect of GF on clonogenic potential after RT. Figure S5 shows the induction of micronuclei after GF and/or RT. Figure S6 shows the expression of IFN-β after GF and/or RT.

Extra copies of centrosomes are frequently observed in cancer cells. To survive and proliferate, cancer cells have developed strategies to cluster extra-centrosomes to form bipolar mitotic spindles. The aim of this study was to investigate whether centrosome clustering (CC) inhibition (CCi) would preferentially radiosensitize non–small cell lung cancer (NSCLC). Griseofulvin (GF; FDA-approved treatment) inhibits CC, and combined with radiation treatment (RT), resulted in a significant increase in the number of NSCLC cells with multipolar spindles, and decreased cell viability and colony formation ability in vitro. In vivo, GF treatment was well tolerated by mice, and the combined therapy of GF and radiation treatment resulted in a significant tumor growth delay. Both GF and radiation treatment also induced the generation of micronuclei (MN) in vitro and in vivo and activated cyclic GMP-AMP synthase (cGAS) in NSCLC cells. A significant increase in downstream cGAS-STING pathway activation was seen after combination treatment in A549 radioresistant cells that was dependent on cGAS. In conclusion, GF increased radiation treatment efficacy in lung cancer preclinical models in vitro and in vivo. This effect may be associated with the generation of MN and the activation of cGAS. These data suggest that the combination therapy of CCi, radiation treatment, and immunotherapy could be a promising strategy to treat NSCLC.

Supplementary Materials & Methods

Background and aims Peroxisome proliferator-activated receptor-gamma (PPARγ) coactivator-1α (PGC1α) is a key regulator of mitochondrial biogenesis and respiration. PGC1α is involved in the carcinogenesis, progression, and metabolic state of cancer. However, its role in the progression of hepatocellular carcinoma (HCC) remains unclear. Approach and results In this study, we observed that PGC1α was down-regulated in human HCC. A clinical study showed that low levels of PGC1α expression were correlated with poor survival, vascular invasion, and larger tumor size. PGC1α inhibited the migration and invasion of HCC cells with both in vitro experiments and in vivo mouse models. Mechanistically, PGC1α suppressed the Warburg effect through down-regulation of pyruvate dehydrogenase kinase isozyme 1 (PDK1) mediated by the WNT/β-catenin pathway, and inhibition of the WNT/β-catenin pathway was induced by activation of PPARγ. Conclusions Low levels of PGC1α expression indicate a poor prognosis for HCC patients. PGC1α suppresses HCC metastasis by inhibiting aerobic glycolysis through regulating the WNT/β-catenin/PDK1 axis, which depends on PPARγ. PGC1α is a potential factor for predicting prognosis and a therapeutic target for HCC patients.

Lung cancer is the most common cancer and the leading cause of cancer deaths worldwide. In addition to coding genes, the contribution of long noncoding RNA (lncRNA) to non-small cell lung cancer (NSCLC) remains unclear. Here, we explored lncRNA expression profiles by Affymetrix Gene Chip Human Transcriptome Array 2.0 in 37 paired samples of tumorous NSCLC tissues and adjacent nontumorous tissues. We showed that LHFPL3-AS2 is a novel lncRNA, significantly decreased in NSCLC tissues. LHFPL3-AS2 was further validated in an additional 93 paired samples of NSCLC. Low levels of LHFPL3-AS2 expression were highly correlated with poor overall survival, TNM stage, and metastasis of NSCLC patients. Enhanced expression of LHFPL3-AS2 inhibited NSCLC invasion and metastasis in vitro and in vivo. Moreover, downregulation of LHFPL3-AS2 reduced its specific interaction with SFPQ, resulting in more SFPQ binding to the promoter of TXNIP and causing the transcriptional repression of TXNIP, thus finally promoting the migration and invasion of NSCLC cells. Furthermore, LHFPL3-AS2 was shown to be regulated by EGR1 under hypoxia.

Long noncoding RNAs (lncRNAs) are implicated as novel drivers in hepatocellular carcinoma (HCC), but the underlying mechanisms of this relationship with hepatocarcinogenesis are unknown. We report a novel, liver-specific lncRNA LINC01093 that shows significant downregulation in HCC tissues. LINC01093 expression is inversely correlated with cancer embolus and HCC TNM stage and as a prognostic predictor for HCC patients. LINC01093 overexpression significantly suppresses HCC cell proliferation and metastasis in vitro and in vivo. Conversely, its knockdown promotes HCC progression. Mechanistic analyses indicate that LINC01093 directly binds insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1), interfering with interaction between IGF2BP1 and glioma-associated oncogene homolog 1 (GLI1) mRNA. The result is degradation of GLI1 mRNA, further affecting expression of GLI1 downstream molecules involved in HCC progression. The liver-enriched lncRNA LINC01093 is a promising prognostic indicator for HCC patients, and the newly identified LINC01093-IGF2BP1-GLI1 axis shows potential for therapeutic targets in HCC.

Metastasis is the main reason for high recurrence and poor survival of hepatocellular carcinoma (HCC). The molecular mechanism underlying HCC metastasis remains unclear. In this study, we found that argininosuccinate synthase 1 (ASS1) expression was significantly decreased and down-regulation of ASS1 was closely correlated with poor prognosis in HCC patients. DNA methylation led to the down-regulation of ASS1 in HCC. Stable silencing of ASS1 promoted migration and invasion of HCC cells, whereas overexpression of ASS1-inhibited metastasis of HCC cells in vivo and in vitro. We also revealed that ASS1-knockdown increased the phosphorylation level of S727STAT3, which contributed to HCC metastasis by up-regulation of inhibitor of differentiation 1 (ID1). These findings indicate that ASS1 inhibits HCC metastasis and may serve as a target for HCC diagnosis and treatment.

Emerging evidence has indicated that deregulation of long non-coding RNAs (lncRNAs) can contribute to the progression and metastasis of human cancer, including hepatocellular carcinoma (HCC). However, the roles of most lncRNAs in HCC remain largely unknown. Here we found a long noncoding RNA termed SchLAH (seven chromosome locus associated with HCC; also called BC035072) was generally downregulated in HCC. Low expression of SchLAH was significantly correlated with shorter overall survival of HCC patients. In vitro and in vivo assays indicated that overexpression of SchLAH inhibited the migration and lung metastasis of HCC cells. Knockdown of SchLAH by siRNA pool promoted the migration of HCC cells. RNA pull-down and RNA immunoprecipitation assays demonstrated SchLAH physically interacted with fused in sarcoma (FUS). PCR array analysis showed that RhoA and Rac1 were the downstream effector molecules of SchLAH during HCC metastasis. Knockdown of FUS rescued the mRNA levels of RhoA and Rac1 that were repressed by SchLAH. These results suggest that SchLAH may suppress the metastasis of HCC cells by interacting with FUS, which indicates potential of SchLAH for the prognosis and treatment of HCC.

Alterations in cellular metabolism are one of the characteristics in cancer. They are not only the result of tumor progression but also the cause of cancer initiation. Pyruvate dehydrogenase kinase 4 (PDK4) is a key metabolic enzyme, which regulates cell metabolism by inhibiting pyruvate dehydrogenase (PDH). However, the function and regulating mechanism of PDK4 in HCC remain unclear. Here, we found that the expression of PDK4 was significantly decreased in HCC tissues, and its downregulation could predict poor prognosis of HCC patients. Silencing PDK4 significantly facilitated proliferation and migration of HCC cells. Knockdown of PDK4 didn't influence the oxidative phosphorylation and glycolysis capacity of HCC cells in vitro. However, knockdown of PDK4 increased expression of key lipogenic enzymes, fatty acid synthase (FASN) and stearoyl-CoA desaturase (SCD), which finally induced lipogenesis. These data suggest that PDK4 inhibits proliferation and migration of HCC cells probably via suppressing lipogenesis.

Resveratrol, a natural polyphenolic compound found in foods and beverages, has attracted increasing attention in recent years because of its potent chemopreventive and anti-tumor effects. In this study, the effects of resveratrol on the expression of P-glycoprotein/multi-drug resistance protein 1 (P-gp/MDR1), and the underlying molecular mechanisms, were investigated in oxaliplatin (L-OHP)-resistant colorectal cancer cells (HCT116/L-OHP). Resveratrol downregulated MDR1 protein and mRNA expression levels and reduced MDR1 promoter activity. It also enhanced the intracellular accumulation of rhodamine 123, suggesting that resveratrol can reverse multi-drug resistance by downregulating MDR1 expression and reducing drug efflux. Resveratrol treatment also reduced nuclear factor-κB (NF-κB) activity, reduced phosphorylation levels of IκBα, and reduced nuclear translocation of the NF-κB subunit p65. Moreover, downregulation of MDR1 expression and promoter activity was mediated by resveratrol-induced AMP-activated protein kinase (AMPK) phosphorylation. The inhibitory effects of resveratrol on MDR1 expression and cAMP-responsive element-binding protein (CREB) phosphorylation were reversed by AMPKα siRNA transfection. We found that the transcriptional activity of cAMP-responsive element (CRE) was inhibited by resveratrol. These results demonstrated that the inhibitory effects of resveratrol on MDR1 expression in HCT116/L-OHP cells were closely associated with the inhibition of NF-κB signaling and CREB activation in an AMPK-dependent manner.

In lung cancer and other malignancies, the so-called "liquid biopsy" is quickly moving into clinical practice. Its full potential has not yet been fully identified, but the "liquid biopsy" is no longer a promise but has become a reality that allows for better treatment selection and monitoring of lung cancer. This emerging field has significant potential to make up for the limitations of the traditional tissue-derived biomaterials. Exosomes are spherical nano-sized vesicles with a diameter of 40-100 nm and a density of 1.13-1.19 g/ml. In both physiological and pathological conditions, exosomes can be released by different cell types, including immune cells, stem cells and tumor cells. These small molecules may serve as promising biomarkers in lung cancer "liquid biopsy" as they can be easily obtained from most body fluids. In addition, the lipid bilayer of exosomes allows for stable cargoes which are relatively hard to degrade. Furthermore, the composition of exosomes reflects that of their parental cells, suggesting that exosomes are potential surrogates of the original cells and, therefore, are useful for understanding cell biology. Previous studies have demonstrated that exosomes play important roles in cell-to-cell communication. Moreover, tumor-derived exosomes are evolved in tumor-specific biological process, including tumor proliferation and progression. Recently, a growing number of studies has focused on exosomal cargo and their use in lung cancer genesis and progression. In addition, their utility as lung cancer diagnostic, prognostic and predictive biomarkers have also been studied. The current review primarily summaries lung cancer-related exosomal biomarkers that have recently been identified and discusses their potential in clinical practice.

Cyclosporin A (CsA) is a widely used immunosuppressive agent that also causes hypertension. However, the molecular basis for CsA-induced hypertension remains elusive. In this study, we established an in vitro model for CsA-induced pathological changes in vasculature. Rat mesenteric arteries were incubated with CsA for 24 hr in an organ culture system. Both vasocontraction and vasorelaxation in mesenteric artery were studied using myograph technology, and mRNA expressions of the contractile receptors were determined by real-time PCR. The results showed that CsA increased the mRNA expression and contractile function of several G-protein-coupled receptors (GPCRs), such as endothelin receptor type B (ETB ), 5-hydroxytryptamine type 1B (5-HT1B ) and 1D (5-HT1D ), in vascular smooth muscle cells. In addition, both nitric oxide (NO)-mediated vasorelaxation and endothelium-independent relaxation were impaired by CsA. In summary, this study showed the enhanced GPCRs-mediated contraction and reduced vasorelaxation in mesenteric artery after organ culture with CsA, which mimicked the CsA-induced pathological changes in the vascular system in vivo. Furthermore, this organ culture system would be an ideal in vitro tool to study the molecular mechanisms of CsA-induced hypertension.

Myasthenia gravis (MG) are T-cell dependent antibody-mediated autoimmune disorders, microRNAs are important regulators of human autoimmune disease pathogenesis. Here, we investigated the miRNAs expression profiles in MG for the first time and found that miR-320a was significantly downregulated in MG patients compared to normal healthy people. Meanwhile, pro-inflammatory cytokins in MG patients were overexpressed. Furthermore, we identified MAPK1 as a direct target of miR-320a. Downregulation of miR-320a induced the overexpression of pro-inflammatory cytokins through promoting COX-2 expression. This process was modulated by ERK/ NF-κB pathways. Taken together, our findings suggested that miR-320a could play a role in modulation of inflammatory cytokins production.

Myasthenia gravis (MG) is a T cell-dependent and B cell-mediated autoimmune disease of neuromuscular junctions and cytokines may play a crucial role in the pathogenesis and perpetuation of MG. MicroRNAs (miRNAs) have been implicated as fine-tuning regulators controlling diverse biological processes at the level of posttranscriptional repression. Dysregulation of miRNAs has been described in various disease states. In this study, miRNA microarrays identified let-7 family to be decreased in peripheral blood mononuclear cells (PBMCs) from MG patients compared to the healthy controls. We next demonstrated the differential expression of let-7 family in larger samples by quantitative real-time PCR. Using a combination of bioinformatics and molecular approaches, we confirmed IL-10 as a target for let-7c. IL-10 expression also showed a negative correlation with let-7c expression in PBMCs from MG patients. Further experiments revealed that induced levels of IL-10 were inversely related to let-7c levels. We also showed that let-7c could regulate IL-10 expression in Jurkat cells. In summary, our results suggest that abnormal expression/regulation of microRNAs may contribute to or be indicative of the initiation and progression of MG.

Renal pathological changes in cirrhotic rat have not been extensively reported. The aim of this study was to investigate whether Xiayuxue decoction (XYXD) could attenuate renal injury induced by bile duct ligation (BDL), with special focus on the mechanisms promoting renal macrophage apoptosis. The rats were treated with BDL for 5 weeks and administered 0.36 g/kg XYXD intragastrically from day 1 of initiating BDL. Renal tissue was monitored by hematoxylin-eosin and Sirius red staining. Macrophage infiltration and proinflammatory cytokines such as tumor necrosis factor and chemokine ligand 2 were detected by quantitative polymerase chain reaction. Macrophage apoptosis was detected by double immunofluorescence staining. Blood urea nitrogen, creatinine, and glomerulus diameter increased significantly after a 5-week BDL treatment in XYXD (BDL-XYXD) rats. CD68 and pro-inflammatory cytokine mRNA increased in the kidneys of control (BDL-water) rats. Fluorescence microscopy analysis showed that XYXD promoted apoptosis in renal CD68+ macrophages. Collogen1 (Col 1), pro-fibrogenic cytokines, and α-smooth muscle actin in kidneys of BDL-water rats increased significantly compared to the sham group. XYXD inhibited Col 1 and pro-fibrotic factors in BDL-XYXD rats. Our results demonstrated that XYXD significantly reduced renal injury by, at least in part, promoting macrophage apoptosis in rats with damaged renal histopathology due to BDL-induced cirrhosis.

Background/Aims: Circulating long non coding RNAs (lncRNAs) have emerged recently as major players in tumor biology and may be used for cancer diagnosis, prognosis, and as potential therapeutic targets. We explored circulating lncRNA as a predictor for the tumorigenesis of non-small-cell lung cancer (NSCLC). Methods: In this study, we applied a lncRNA microarray to screen for a potential biomarker for NSCLC, utilizing RT-PCR (ABI 7900HT). A multi-stage validation and risk score formula detection analysis was used. Results: We discovered that three lncRNAs (RP11-397D12.4, AC007403.1, and ERICH1-AS1) were up regulated in NSCLC, compared with cancer-free controls, with the merged area under the curve in the training and validation sets of 0.986 and 0.861. Furthermore, the positive predictive value and negative predictive value of the three merged factors were 0.72 and 0.87. We confirmed stable detection of the three lncRNAs by three cycles of freezing and thawing. Conclusions: RP11-397D12.4, AC007403.1, and ERICH1-AS1 may be potential biomarkers for predicting the tumorigenesis of NSCLC in the future.

Background Hepatic stellate cell (HSC) activation is activated mainly by endotoxin and transforming growth factor (TGF-β1) in chronic liver injury, consequently, can be important therapeutic targets. Xia-yu-xue decoction (XYXD), a classical recipe used in China to treat liver fibrosis, and has been revealed to inhibit hepatic fibrosis in animal models, the mechanism of action of XYXD remains elusive. In the present study, we evaluated whether XYXD reduced endotoxin and pro-fibrogenic pathways induced by lipopolysaccharide (LPS) and TGF-β1 in HSCs. Methods The in vivo effect of XYXD on fibrosis progression was assessed in mice model induced by carbon tetrachloride (CCl4), The in vitro effect of XYXD on mice GFP-Col-HSC cells was evaluated using LPS and TGF-β1 stimulation. Results XYXD treatment reduced CCl4-induced liver fibrosis and decreased hepatic hydroxyproline (Hyp) content, the mRNA levels of smooth muscle actin (α-SMA) and Col 1(α1) in fibrotic liver. XYXD suppressed nuclear factor-κB (NF-κB) activation induced by LPS and TGF-β1 assessed by using NF-κB-luciferase reporter. The expression of NF-κB target genes, chemokine (C-C motif) ligand 2 (CCL2) and chemokine (C-X-C motif) ligand 2 (CXCL2) induced by LPS was suppressed after XYXD treatment. The expression of TGF-β1 targets genes, Col1(α1) and tissue inhibitor of metalloproteinases (TIMP1) induced by TGF-β1 was inhibit after XYXD treatment. Conclusion XYXD treatment attenuates liver fibrosis by inhibiting HSC activation via inhibition of NF-κB and TGF-β1 signaling pathway, thereby blocking the synthesis of Col1 (α1) and TIMP-1. These findings from present study suggest that XYXD may be a therapeutic decoction for liver fibrosis in which NF-κB and TGF-β1 are thought to take part.

Hypertension is one of the most frequent complications of solid organ transplantation, and cyclosporin A (CsA) plays a predominant role in the pathophysiology of post-transplant hypertension. However, the exact molecular mechanisms of CsA-induced hypertension remain obscure. We previously showed that CsA increased the mRNA expression and contractile function of endothelin B (ETB) receptor in vascular smooth muscle cells. The present study was designed to investigate the underlying mechanisms of CsA-induced upregulation of ETB receptor in vasculature. Rat mesenteric arteries were incubated with CsA in an organ culture system, and results showed that CsA enhanced ETB receptor mRNA in the time- and dose-dependent manner, and increased protein expression levels of ETB receptor after treatment with CsA 10(-5)M for 6h. Furthermore, CsA induced phosphorylation of extracellular regulated protein kinases 1 and 2 (ERK1/2), p38, and translocation of nuclear factor-kappaB (NF-κB) p65 in vasculature. Blocking ERK1/2, p38, or NF-κB activation with their specific inhibitors markedly attenuated CsA-induced upregulation of ETB receptor mRNA expression and protein levels, and ETB receptor-mediated contraction. In summary, this study showed that mitogen-activating protein kinases (ERK1/2 and p38) and the downstream transcriptional factor NF-κB pathways were involved in CsA-induced upregulation of ETB receptor in arterial smooth muscle cells.

Previous studies have reported that 8-c [6-(2-(2-(dimethylamino)ethylamino)ethylamino)-2-octyl-1H-benzo[de]isoquinoline-1,3(2H)-dione], a novel amonafide analogue, was generated as a new anticancer candidate. However, little is known about its activity in chemoresistant cells. In this study, the antitumor effects of 8-c on the multi-drug-resistant human colorectal carcinoma cancer cell lines HCT-116/L-OHP and HCT-8/VCR have been investigated for the first time. 8-c showed similar concentration-dependent inhibitory activities against multi-drug-resistant cells and corresponding parental cell lines by the MTT assay after 48 h of treatment. 8-c treatment resulted in the induction of apoptosis, as evidenced by fluorescent staining analysis, comet assay data, and the increase in the number of apoptotic cells as detected by flow cytometry. Western blot, qPCR, and siRNA techniques were used to elucidate the molecular mechanism. Our study suggested that the apoptotic effect of 8-c can be attributed to the upregulation of p53, caspase-3, and cleaved poly(ADP-ribose) polymerase (PARP) and the downregulation of Bcl-2. Furthermore, ERCC1 is essential for nucleotide excision repair. ERCC1 expression was correlated with sensitivity to chemotherapy in various colon cancer cell lines. It is intriguing that decreases in ERCC1 protein and mRNA levels were also observed in the HCT-116/L-OHP and HCT-8/VCR cells after exposure to 8-c. Further transient transfection of multi-drug-resistant cells with ERCC1 siRNA enhanced 8-c-induced cytotoxicity. In contrast, epidermal growth factor-induced increase in ERCC1 protein levels was shown to rescue cell viability upon 8-c treatment. These findings suggest that 8-c has a strong potential to be developed as a new antitumor agent for the treatment of multi-drug-resistant colon cancer cells, and is worthy of further studies.

Photodynamic therapy (PDT) is a regulatory-approved modality for treating a variety of malignant tumors. It induces tumor tissue damage via photosensitizer-mediated oxidative cytotoxicity. The heat shock protein 70 (HSP70-1) is a stress protein encoded by the HSPA1A gene and is significantly induced by oxidative stress associated with PDT. The aim of this study was to identify the functional region of the HSPA1A promoter that responds to PDT-induced oxidative stress and uses the stress responsiveness of HSPA1A expression to establish a rapid and cost-effective photocytotoxic assessment bioassay to evaluate the photodynamic potential of photosensitizers. By constructing luciferase vectors with a variety of hspa1a promoter fractions and examining their relative luciferase activity, we demonstrated that the DNA sequence from −218 to +87 of the HSPA1A gene could be used as a functional promoter to detect the PDT-induced oxidative stress. The maximal relative luciferase activity level of HSPA1A (HSP70-1) induced by hypericin-PDT was nearly nine times that of the control. Our results suggest that the novel reporter gene assay using a functional region of the HSP70A1A promoter has significant advantages for the detection of photoactivity in terms of both speed and sensitivity, when compared with a cell viability test based on ATP quantification and ROS levels. Furthermore, phthalocyanine zinc and methylene blue both induced significantly elevated levels of relative luciferase activity in a dose-dependent manner.