Glycyrrhetic acid (GA)-modified pullulan nanoparticles (GAP NPs) were synthesized as a novel carrier of curcumin (CUR) with a degree of substitution (DS) of GA moieties within the range of 1.2–6.2 groups per hundred glucose units. In the present study, we investigated the physicochemical characteristics, release behavior, in vitro cytotoxicity and cellular uptake of the particles. Self-assembled GAP NPs with spherical shapes could readily improve the water solubility and stability of CUR. The CUR release was sustained and pH-dependent. The cellular uptake of CUR-GAP NPs was confirmed by green fluorescence in the cells. An MTT study showed CUR-GAP NPs with higher cytotoxicity in HepG2 cells than free CUR, but GAP NPs had no significant cytotoxicity. GAP is thus an excellent carrier for the solubilization, stabilization, and controlled delivery of CUR.

The significance of Brf1 overexpression in human hepatocellular carcinoma

The association of alcohol consumption and breast cancer is more pronounced in cases that are positive for estrogen receptor (ER+) than in cases that are negative (ER−). Its mechanism remains to be determined. Deregulation of RNA polymerase III (Pol III) transcription enhances cellular tRNAs and 5S rRNA production, increasing translational capacity to promote cell transformation and tumor formation. Here, we report that alcohol increases Pol III gene transcription in both normal and cancer breast cell lines. The induction in ER+ breast cancer cells (MCF-7) is significantly higher than in ER− normal breast cells (MCF-10A, MCF-10F and MCF-12A) and is correlated with ER expression. E2 causes <2-fold increase in Pol III gene transcription. The addition of ethanol to this system now produces a 10–15-fold increase. Ethanol increases ERα expression, resulting in an increase in Brf1 protein and mRNA levels. In addition, ethanol markedly stimulates phosphorylation of JNK1, but not JNK2. Inhibition of JNK1 decreases ERE-Luc reporter activity and represses expression of ERα, Brf1 and Pol III genes. Reduction of ERα by its small interfering RNA represses Brf1 and Pol III gene transcription. Ethanol with E2 produces larger and more numerous colonies. Repression of ERα or Brf1 inhibits alcohol-induced cell transformation. Together, these results support the idea that alcohol increases ERα expression through JNK1 to elevate Brf1 expression and Pol III gene transcription to bring about greater phenotypic changes. These studies demonstrate that ERα mediates Pol III gene transcription through Brf1, suggesting that ERα may play a critical role in alcohol-induced deregulation of Pol III genes in ER+ breast cancer development.

Deregulation of RNA polymerase III (Pol III) transcription enhances cellular tRNAs and 5S rRNA production, leading to an increase in translational capacity to promote cell proliferation, transformation and tumor formation. Phosphorylation of histone H3 (H3ph) is induced by tumor promoters (EGF, UV and TPA) and immediate early genes, such as c-myc, c-jun and c-fos. However, it remains to be determined whether H3ph is involved in RNA Pol III transcription. Here, we report that EGF strongly induced H3ph at serine 28 (H3S28ph). EGF significantly increased transcription of RNA Pol III-dependent genes (Pol III genes), tRNA^Leu, tRNA^Tyr, 5S rRNA and 7SL RNA. Inhibition of EGFR, but not PI3K, reduced both H3S28ph and tRNA^Leu and 5S rRNA transcription. EGF enhanced occupancy of H3S28ph in the promoters of tRNA^Leu and 5S rRNA. Further analysis indicates that EGF augmented cellular levels of protein and mRNA of TFIIIB subunits, Brf1 and TATA box-binding protein (TBP). Brf1 is a specific transcription factor for RNA Pol III genes. EGF enhanced occupancy of H3S28ph in the Brf1 and TBP promoters. Inhibition of H3S28ph by mutant H3S28A repressed Brf1, TBP and tRNA^Leu and 5S rRNA expression and decreased occupancy of H3S28ph in their promoters. Reduction of Brf1 significantly decreased tRNA^Leu and 5S rRNA transcription and repressed EGF-induced anchorage-independent growth. Blocking H3S28ph signaling by using mutant H3S28A reduced EGF-induced cell transformation. Together, these results indicate that EGF activates EGFR signaling to induce H3S28ph, which, in turn, upregulates tRNA^Leu and 5S rRNA transcription through Brf1 and TBP and promotes cell transformation. The studies demonstrate that epigenetic modification of H3S28ph has a critical role in the activity of Pol III genes.

Chronic alcohol consumption is associated with steatohepatitis and cirrhosis, enhancing the risk for hepatocellular carcinoma. RNA polymerase (pol) III transcribes a variety of small, untranslated RNAs, including tRNAs and 5S rRNAs, which determine the biosynthetic capacity of cells. Increased RNA pol III-dependent transcription, observed in transformed cells and human tumors, is required for oncogenic transformation. Given that alcohol consumption increases risk for liver cancer, we examined whether alcohol regulates this class of genes. Ethanol induces RNA pol III-dependent transcription in both HepG2 cells and primary mouse hepatocytes in a manner that requires ethanol metabolism and the activation of JNK1. This regulatory event is mediated, at least in part, through the ability of ethanol to induce expression of the TFIIIB components, Brf1, and the TATA-binding protein (TBP). Induction of TBP, Brf1, and RNA pol III-dependent gene expression is driven by enhanced c-Jun expression. Ethanol promotes a marked increase in the direct recruitment of c-Jun to TBP, Brf1, and tRNA gene promoters. Chronic alcohol administration in mice leads to enhanced expression of TBP, Brf1, tRNA, and 5S rRNA gene transcription in the liver. These alcohol-dependent increases are more pronounced in transgenic animals that express the HCV NS5A protein that display increased incidence of liver tumors. Together, these results identify a new class of genes that are regulated by alcohol through the co-regulation of TFIIIB components and define a central role for c-Jun in this process.

The products of Pol III genes (RNA polymerase III-dependent genes), such as tRNAs and 5S rRNA, are elevated in both transformed and tumor cells suggesting that they play a crucial role in tumorigenesis. An increase in Brf1 (TFIIIB-related factor 1), a subunit of TFIIIB, augments Pol III gene transcription and is sufficient for cell transformation and tumor formation. We have demonstrated that enhancement of Brf1 and Pol III gene expression is associated with the occurrences of hepatocellular carcinoma (HCC) in mice. This suggests that Brf1 may be a key molecule during HCC development. Diethylnitrosamine (DEN), a chemical carcinogen, has been used to induce HCC in rodents. To determine the role of Brf1 and the epigenetic-regulating events in cell proliferation and transformation, hepatocytes were treated with DEN. The results indicate that DEN increases proliferation and transformation of AML-12 cells. DEN enhanced Brf1 expression and tRNA ^Leu and 5S rRNA transcription, as well as H3S10ph (phosphorylation of histone H3 serine 10). Interestingly, DEN-induced Pol III gene transcription and H3S10ph in tumor cells of liver are significantly higher than in non-tumor cells. Inhibition of H3S10ph by H3S10A attenuates the induction of Brf1 and Pol III genes. Further analysis indicates that H3S10ph occupies the promoters of Brf1 and Pol III genes to modulate their expression. Blocking H3S10ph represses cell proliferation and transformation. These results demonstrate that DEN induces H3S10ph, which mediate Brf1 expression, including but not limited Brf1-dependent genes, to upregulate Pol III gene transcription, resulting in an increase in cell proliferation and transformation.