Effects of Pyrrole-Imidazole Polyamides Targeting Human TGF-?1 on the Malignant Phenotypes of Liver Cancer Cells.
ABSTRACT: Synthetic pyrrole-imidazole (PI) polyamides bind to the minor groove of double-helical DNA with high affinity and specificity, and inhibit the transcription of corresponding genes. In liver cancer, transforming growth factor (TGF)-? expression is correlated with tumor grade, and high-grade liver cancer tissues express epithelial-mesenchymal transition markers. TGF-?1 was reported to be involved in cancer development by transforming precancer cells to cancer stem cells (CSCs). This study aimed to evaluate the effects of TGF-?1-targeting PI polyamide on the growth of liver cancer cells and CSCs and their TGF-?1 expression. We analyzed TGF-?1 expression level after the administration of GB1101, a PI polyamide that targets human TGF-?1 promoter, and examined its effects on cell proliferation, invasiveness, and TGF-?1 mRNA expression level. GB1101 treatment dose-dependently decreased TGF-?1 mRNA levels in HepG2 and HLF cells, and inhibited HepG2 colony formation associated with downregulation of TGF-?1 mRNA. Although GB1101 did not substantially inhibit the proliferation of HepG2 cells compared to untreated control cells, GB1101 significantly suppressed the invasion of HLF cells, which displayed high expression of CD44, a marker for CSCs. Furthermore, GB1101 significantly inhibited HLF cell sphere formation by inhibiting TGF-?1 expression, in addition to suppressing the proliferation of HLE and HLF cells. Taken together, GB1101 reduced TGF-?1 expression in liver cancer cells and suppressed cell invasion; therefore, GB1101 is a novel candidate drug for the treatment of liver cancer.
Project description:Cancer stem cells (CSCs) niche in the tumor microenvironment is responsible for cancer recurrence and therapy failure. To better understand its molecular and biological involvement in hepatocellular carcinoma (HCC) progression, one can design more effective therapies and tailored then to individual patients. While sorafenib is currently the only approved drug for first-line treatment of advanced stage HCC, its role in modulating the CSC niche is estimated to be small. By contrast, transforming growth factor (TGF)-? pathway seems to influence the CSC and thus may impact hallmarks of HCC, such as liver fibrosis, cirrhosis, and tumor progression. Therefore, blocking this pathway may offer an appealing and druggable target. In our study, we have used galunisertib (LY2157299), a selective ATP-mimetic inhibitor of TGF-? receptor I (TGF?I/ALK5) activation, currently under clinical investigation in HCC patients. Because the drug resistance is mainly mediated by CSCs, we tested the effects of galunisertib on stemness phenotype in HCC cells to determine whether TGF-? signaling modulates CSC niche and drug resistance. Galunisertib modulated the expression of stemness-related genes only in the invasive (HLE and HLF) HCC cells inducing a decreased expression of CD44 and THY1. Furthermore, galunisertib also reduced the stemness-related functions of invasive HCC cells decreasing the formation of colonies, liver spheroids and invasive growth ability. Interestingly, CD44 loss of function mimicked the galunisertib effects on HCC stemness-related functions. Galunisertib treatment also reduced the expression of stemness-related genes in ex vivo human HCC specimens. Our observations are the first evidence that galunisertib effectiveness overcomes stemness-derived aggressiveness via decreased expression CD44 and THY1.
Project description:Hepatocellular carcinoma (HCC) is a major public health problem due to increased incidence, late diagnosis and limited treatment options. TGF-? is known to provide cytostatic signals during early stages of liver damage and regeneration, but exerts tumor promoting effects in onset and progression of liver cancer. To understand the mechanistic background of such a switch, we systematically correlated loss of cytostatic TGF-? effects with strength and dynamics of its downstream signaling in 10 HCC cell lines. We demonstrate that TGF-? inhibits proliferation and induces apoptosis in cell lines with low endogenous levels of TGF-? and Smad7 and strong transcriptional Smad3 activity (PLC/PRF/5, HepG2, Hep3B, HuH7), previously characterized to express early TGF-? signatures correlated with better outcome in HCC patients. TGF-? dependent cytostasis is blunted in another group of cell lines (HLE, HLF, FLC-4) expressing high amounts of TGF-? and Smad7 and showing significantly reduced Smad3 signaling. Of those, HLE and HLF exhibit late TGF-? signatures, which is associated with bad prognosis in HCC patients. RNAi with Smad3 blunted cytostatic effects in PLC/PRF/5, Hep3B and HuH7. HCC-M and HCC-T represent a third group of cell lines lacking cytostatic TGF-? signaling despite strong and prolonged Smad3 phosphorylation and low Smad7 and TGF-? expression. Inhibitory linker phosphorylation, as in HCC-T, may disrupt C-terminally phosphorylated Smad3 function. In summary, we assort 10 HCC cell lines in at least two clusters with respect to TGF-? sensitivity. Cell lines responsive to the TGF-? cytostatic program, which recapitulate early stage of liver carcinogenesis exhibit transcriptional Smad3 activity. Those with disturbed TGF-?/Smad3 signaling are insensitive to TGF-? dependent cytostasis and might represent late stage of the disease. Regulation of this switch remains complex and cell line specific. These features may be relevant to discriminate stage dependent TGF-? functions for the design of efficient TGF-? directed therapy in liver cancer.
Project description:Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related mortality worldwide. Current standard practices for treatment of HCC are less than satisfactory because of cancer stem cells (CSCs)-mediated post-surgical recurrence. For this reason, targeting the CSCs or the cancer cells with CSCs-like properties has become a new approach for the treatment of HCC. GLA exhibits anti-tumor effects in that it attenuates the proliferation, migration, invasion, and angiogenesis of human cancer cells. However, the functions of GLA in the regulation of CSCs-like properties in HCC cells, and the molecular mechanisms underlying in remain obscure. Here we found that GLA attenuated the CSCs-like properties by the microRNA-148a (miR-148a)-mediated inhibition of transforming growth factor beta (TGF-?)/SMAD2 signal pathway in HCC cell lines (HepG2, Huh-7, and MHCC97H). Indeed, GLA inhibited the activations/expressions of both TGF?-induced and the endogenous SMAD2. Further, GLA improved the expression of miR-148a in a dose/time-dependent manner. MiR-148a, which targeted the SMAD2-3'UTR, decreased the expression and function of SMAD2. Knockdown of miR-148a abolished the GLA-induced inhibition of TGF-?/SMAD2 signal pathway and the CSCs-like properties in HCC cells. Our study found a novel mechanism that GLA inhibits the CSCs-like properties of HCC cells by miR-148a-mediated inhibition of TGF-?/SMAD2 signal pathway, which may help to identify potential targets for the therapies of HCC.
Project description:Liver fibrosis is a major endpoint of patients with chronic liver diseases. The molecular mechanisms behind liver fibrosis remain largely unknown. Many studies have indicated the role of microRNA (miRNA) in hepatic tumorigenesis. But the role of miRNA in liver fibrosis is little known. Activated hepatic stellate cells (HSCs) can secret extracellular matrix proteins (ECM) and are the major contributors to liver fibrosis/cirrhosis. Here, a microarray assay of quiescent and transforming growth factor ?1 (TGF-?1) activated HSCs indicated that miR-98 might play a crucial role in liver fibrosis. We found that miR-98 was significantly downregulated in activated HSCs. miR-98 overexpression inhibited HSCs activation. Furthermore, we hypothesized that miR-98 regulated hepatic leukemia factor (HLF) expression by binding to the 3' UTR of its mRNA directly, as evidenced by luciferase reporter assay. HLF overexpression increased HSCs activation by inducing hypoxia inducible factor-1 alpha (HIF-1?) expression, resulting in the activation of TGF-?/Smad2/3 signaling pathway. Besides, low expression of miR-98 was also found in liver tissues from various fibrotic murine models, including carbon tetrachloride (CCl4), bile duct ligation (BDL), and high-fat diet (HFD)-induced liver fibrosis. miR-98 overexpression in vivo by ago-miR-98 injection could attenuate CCl4-, BDL-, and HFD-induced murine hepatic fibrosis. Meanwhile, miR-98 overexpression suppressed HLF expression and reduced fibrosis marker expression. Collectively, our study demonstrates that miR-98 suppress HSCs activation by targeting HLF directly and interacting with HIF-1?/TGF-?/Smad2/3 signaling pathway, which may be an effective therapeutic target for liver fibrosis.
Project description:Cadmium, a heavy metal pollutant, causes cancer. The existence of cancer stem cells (CSCs) in tumors is widely considered to be the reason for the recurrence and treatment failure of cancer. Increasing evidence has confirmed that under certain conditions non-CSCs could be converted into CSCs. The impact of cadmium on the development of CSC lineage in the bulk tumor cell population is not yet studied. The aim of this study was to evaluate the effect of cadmium on the conversion of non-CSCs to CSCs and the identification of CSCs based on the concurrent monitoring of multiple CSC markers. High-content monitoring of molecular markers was performed using quantum dot (QD) nanoprobes and an acousto-optical tunable filter (AOTF)-based imaging device. Cadmium treatment significantly increased the CSC population in MCF-7 and HepG2 cell lines. The cadmium-induced CSCs were identified by a concurrent analysis of stem-cell markers, namely, CD44, CD24, CD133, and ALDH1. Moreover, increased m-RNA expression of CD44, ALDH1, and CD133 and protein expression of p-Ras, p-Raf-1, p-MEK-1, and p-ERK-1 were observed in the cadmium-treated MCF-7 and HepG2 cells. This study demonstrates that cadmium induces the gene expression of CSC markers in the breast and liver cancer cell lineage and promotes the conversion of non-CSCs to CSCs.
Project description:Although non?alcoholic fatty liver disease (NAFLD) is considered a benign disorder, hepatic steatosis has been proposed to be involved in the tumorigenesis of liver cancer. However, the underlying mechanism for carcinogenesis in fatty liver diseases remains unclear. Cancer stem cells (CSCs) have been hypothesized to serve a key role in tumorigenesis. Tumor formation begins with a subset of heterogeneous cells that share properties with stem cells, such as self?renewal and undifferentiated properties. Our previous study reported that the saturated fatty acid palmitate (PA) significantly enhanced the CSC properties of the HepG2 human liver cancer cell line; however, its underlying mechanisms are unknown. In the present study, a proteomic approach was used to investigate the palmitoylation of proteins in HepG2 CSCs. CSC behavior was induced in HepG2 cells via 200 µM PA. Proteomic analysis was performed to identify post?transcriptional modifications of proteins in HepG2 CSCs in response to PA treatment. The present study identified proteins modified by palmitoylation in HepG2 CSC spheres formed following PA treatment. It was therefore hypothesized that palmitoylation may be crucial for CSC sphere formation. Furthermore, the present study demonstrated that two palmitoylation inhibitors, tunicamycin (5, 10 and 25 µg/ml) and 2?bromohexadecanoic acid (25, 50 and 150 µM), significantly decreased CSC sphere formation without affecting cell viability. An association was identified between sphere formation capacity and tumor?initiating capacity of CSCs. The results of the present study demonstrated that protein palmitoylation may influence the PA?induced CSC tumor?initiating capacity, and that the inhibition of palmitoylation may be a suitable chemopreventive strategy for treating patients with NAFLD.
Project description:PURPOSE:Overexpression of epithelial cell adhesion molecule (EpCAM) correlates with poor prognosis, therapeutic failure and early tumor recurrence in hepatocellular carcinoma (HCC) patients. The tumor microenvironment dictates the fate of tumor-initiating cancer stem cells (CSCs); however, very limited studies were attempted to evaluate CSC tumorigenesis in the liver microenvironment. Here, we have systemically investigated the role of EpCAM+ cancer cells in tumor initiation in orthotopic HCC models. RESULTS:Control mice and the mice with bland steatosis failed to develop tumors. In the mice with steatohepatitis, EpCAM+ CSCs have shown significantly increased ability in terms of tumor initiation and growth, compared to that with EpCAM- non-CSCs inoculation (p < 0.005). For Hep3B inoculation, EpCAM-High group has shown significantly higher tumor growth compared with EpCAM-Low (p < 0.005). For HepG2 inoculation, both EpCAM-High and EpCAM-Low groups confirmed similar tumor incidence and growth. METHODS:Diet-induced compromised microenvironments were established to mimic clinical fatty liver and non-alcoholic steatohepatitis (NASH) patients and the tumorigenic capabilities of Hepa1-6 cells were evaluated. CSCs were enriched by spheroid culture and labeled with copGFP for EpCAM+ CSCs and with mCherry for non-CSCs. FACS-sorted cells were inoculated into left liver lobes, and tumor growth was monitored by high-frequency ultrasound. The subpopulations of Hep3B and HepG2 cells in terms of EpCAM-Low and EpCAM-High were evaluated in the orthotopic model of athymic mice. CONCLUSIONS:NASH microenvironment promotes the EpCAM+ CSCs initiated tumorigenesis in immunocompetent mouse model. Differential EpCAM expression demonstrates distinct tumor biology in athymic mouse models.
Project description:Low expression levels of the programmed cell death 5 (PDCD5) gene have been reported in numerous human cancers, however, PDCD5 expression has not been investigated in hepatic cancer. The present study aims to investigate the biological behavior of PDCD5 overexpression in hepatocellular carcinoma (HCC) cells. The PDCD5 gene was stably transfected into the HepG2 HCC cell line (HepG2-PDCD5), and the expression levels of PDCD5 were examined by quantitative polymerase chain reaction and western blotting. An MTT assay was used to assess the cellular proliferating ability, and propidium iodide (PI) staining was used to evaluate the cell cycle by flow cytometry. The cells were incubated with 2 ng/ml transforming growth factor (TGF)-? for 7 days in order to induce invasion and epithelial-mesenchymal transition (EMT). Apoptosis was measured by Annexin V-fluorescein isothiocyanate and PI double labeling. A Boyden chamber invasion assay was carried out to detect tumor invasion. Western blotting was performed to detect the protein expression levels of PDCD5, insulin-like growth factor (IGF)-1 and the EMT marker, Snail. The results showed that the HepG2-PDCD5 cells exhibited slower proliferation rates and high G2/M cell numbers compared with those of the HepG2 and HepG2-Neo controls (P<0.05). The PDCD5 transfected cells showed higher sensitivity to cisplatin treatment than the HepG2-Neo cells, with a higher p53 protein expression level. PDCD5 overexpression can attenuate tumor invasion, EMT and the level of IGF-1 protein induced by TGF-? treatment. In conclusion, stable transfection of the PDCD5 gene can inhibit growth and induce cell cycle arrest in HepG2 cells, and its also notably improves the apoptosis-inducing effects of cisplatin, and reverses invasion and EMT induced by TGF-?. The use of PDCD5 is a novel strategy for improving the chemotherapeutic effects on HCC.
Project description:We report a preclinical study of a pyrrole-imidazole (PI) polyamide that targets the human transforming growth factor (hTGF)-?1 gene as a novel transcriptional gene silencer in a common marmoset primate model. We designed and then synthesized PI polyamides to target the hTGF-?1 promoter. We examined effects of seven PI polyamides (GB1101-1107) on the expression of hTGF-?1 mRNA stimulated with phorbol 12-myristate 13-acetate (PMA) in human vascular smooth muscle cells. GB1101, GB1105 and GB1106 significantly inhibited hTGF-?1 mRNA expression. We examined GB1101 as a PI polyamide to hTGF-?1 for hypertrophic scars in marmosets in vivo. Injection of GB1101 completely inhibited hypertrophic scar formation at 35 days post-incision and inhibited cellular infiltration, TGF-?1 and vimentin staining, and epidermal thickness. Mismatch polyamide did not affect hypertrophic scarring or histological changes. Epidermis was significantly thinner with GB1101 than with water and mismatch PI polyamides. We developed the PI polyamides for practical ointment medicines for the treatment of hypertrophic scars. FITC-labeled GB1101 with solbase most efficiently distributed in the nuclei of epidermal keratinocytes, completely suppressed hypertropic scarring at 42 days after incision, and considerably inhibited epidermal thickness and vimentin-positive fibroblasts. PI polyamides targeting hTGF-?1 promoter with solbase ointment will be practical medicines for treating hypertrophic scars after surgical operations and skin burns.
Project description:Aberrant overexpression of ERG induced by the TMPRSS2-ERG gene fusion is likely involved in the development of prostate cancer. Synthetic pyrrole-imidazole (PI) polyamides recognize and attach to the minor groove of DNA with high affinity and specificity. In the present study, we designed a PI polyamide targeting TMPRSS2-ERG translocation breakpoints and assessed its effect on human prostate cancer cells. Our study identified that this PI polyamide repressed the cell and tumor growth of androgen-sensitive LNCaP prostate cancer cells. Targeting of these breakpoint sequences by PI polyamides could be a novel approach for the treatment of prostate cancer.