Jui-Hsiang Hung

Genome-based deletion analysis reveals the prenyl xanthone biosynthesis pathway in Aspergillus nidulans.

Xanthones are a class of molecules that bind to a number of drug targets and possess a myriad of biological properties. An understanding of xanthone biosynthesis at the genetic level should facilitate engineering of second-generation molecules and increasing production of first-generation compounds. The filamentous fungus Aspergillus nidulans has been found to produce two prenylated xanthones, shamixanthone and emericellin, and we report the discovery of two more, variecoxanthone A and epishamixanthone. Using targeted deletions that we created, we determined that a cluster of 10 genes including a polyketide synthase gene, mdpG, is required for prenyl xanthone biosynthesis. mdpG was shown to be required for the synthesis of the anthraquinone emodin, monodictyphenone, and related compounds, and our data indicate that emodin and monodictyphenone are precursors of prenyl xanthones. Isolation of intermediate compounds from the deletion strains provided valuable clues as to the biosynthetic pathway, but no genes accounting for the prenylations were located within the cluster. To find the genes responsible for prenylation, we identified and deleted seven putative prenyltransferases in the A. nidulans genome. We found that two prenyltransferase genes, distant from the cluster, were necessary for prenyl xanthone synthesis. These genes belong to the fungal indole prenyltransferase family that had previously been shown to be responsible for the prenylation of amino acid derivatives. In addition, another prenyl xanthone biosynthesis gene is proximal to one of the prenyltransferase genes. Our data, in aggregate, allow us to propose a complete biosynthetic pathway for the A. nidulans xanthones.

Induction of Bcl-2 Expression by Hepatitis B Virus Pre-S2 Mutant Large Surface Protein Resistance to 5- Fluorouracil Treatment in Huh-7 Cells

Background Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide with poor prognosis due to resistance to conventional chemotherapy and limited efficacy of radiotherapy. Our previous studies have indicated that expression of Hepatitis B virus pre-S2 large mutant surface antigen (HBV pre-S2Δ) is associated with a significant risk of developing HCC. However, the relationship between HBV pre-S2Δ protein and the resistance of chemotherapeutic drug treatment is still unclear. Methodology/Principal Findings Here, we show that the expression of HBV pre-S2Δ mutant surface protein in Huh-7 cell significantly promoted cell growth and colony formation. Furthermore, HBV pre-S2Δ protein increased both mRNA (2.7±0.5-fold vs. vehicle, p = 0.05) and protein (3.2±0.3-fold vs. vehicle, p = 0.01) levels of Bcl-2 in Huh-7 cells. HBV pre-S2Δ protein also enhances Bcl-2 family, Bcl-xL and Mcl-1, expression in Huh-7 cells. Meanwhile, induction of NF-κB p65, ERK, and Akt phosphorylation, and GRP78 expression, an unfolded protein response chaperone, were observed in HBV pre-S2Δ and HBV pre-S-expressing cells. Induction of Bcl-2 expression by HBV pre-S2Δ protein resulted in resistance to 5-fluorouracil treatment in colony formation, caspase-3 assay, and cell apoptosis, and can enhance cell death by co-incubation with Bcl-2 inhibitor. Similarly, transgenic mice showed higher expression of Bcl-2 in liver tissue expressing HBV pre-S2Δ large surface protein in vivo. Conclusion/Significance Our result demonstrates that HBV pre-S2Δ increased Bcl-2 expression which plays an important role in resistance to 5-fluorouracil-caused cell death. Therefore, these data provide an important chemotherapeutic strategy in HBV pre-S2Δ-associated tumor.

Reactive oxygen species mediate Terbufos-induced apoptosis in mouse testicular cell lines via the modulation of cell cycle and pro-apoptotic proteins

Terbufos (S‐t‐butylthiomethyl‐O,O‐diethyl phosphorodithioate) is a highly toxic organophosphate which is extensively used as an insecticide and nematicide. Chronic exposure to terbufos causes neuronal injury and predisposes to neurodegenerative diseases. Accumulating evidence has shown that the exposure to terbufos, as an occupational risk factor, may also cause reproductive disorders. However, the exact mechanisms of reproductive toxicity remain unclear. The present study aimed to investigate the toxic effect of terbufos on testicular cells and to explore the mechanism of toxicity on a cellular level. The cytotoxic effects of terbufos on mouse immortalized spermatogonia (GC‐1), spermatocytes (GC‐2), Leydig (TM3), and Sertoli (TM4) cell lines were assessed by MTT assays, caspase activation, flow cytometry, TUNEL assay, Western blot, and cell cycle analysis. The exposure to different concentrations of terbufos ranging from 50 to 800 μM for 6 h caused significant death in all the used testicular cell lines. Terbufos increased reactive oxygen species (ROS) production, reduced mitochondrial membrane potential, and initiated apoptosis, which was confirmed by a dose‐dependent increase in the number of TUNEL‐positive apoptotic cells. Blocking ROS production by N‐acetyl cysteine (NAC) protected GC‐1 cells from terbufos‐induced cell death. The results demonstrated that terbufos induces ROS, apoptosis, and DNA damage in testicular cell lines and it should be considered potentially hazardous to testis. Together, this study provided potential molecular mechanisms of terbufos‐induced toxicity in testicular cells and suggests a possible protective measure.

OSU-2S/Sorafenib Synergistic Antitumor Combination against Hepatocellular Carcinoma: The Role of PKCδ/p53

Background: Sorafenib (Nexavar®) is an FDA-approved systemic therapy for advanced hepatocellular carcinoma (HCC). However, the low efficacy and adverse effects at high doses limit the clinical application of sorafenib and strongly recommend its combination with other agents aiming at ameliorating its drawbacks. OSU-2S, a PKCδ activator, was selected as a potential candidate anticancer agent to be combined with sorafenib to promote the anti-cancer activity through synergistic interaction. Methods: The antitumor effects of sorafenib, OSU-2S and their combination were assessed by MTT assay, caspase activation, Western blotting, migration/invasion assays in four different HCC cell lines. The synergistic interactions were determined by Calcusyn analysis. PKCδ knockdown was used to elucidate the role of PKCδ activation as a mechanism for the synergy. The knockdown/over-expression of p53 was used to explain the differential sensitivity of HCC cell lines to sorafenib and/or OSU-2S. Results: OSU-2S synergistically enhanced the anti-proliferative effects of sorafenib in the four used HCC cell lines with combination indices <1. This effect was accompanied by parallel increases in caspase 3/7 activity, PARP cleavage, PKCδ activation and inhibition of HCC cell migration/invasion. In addition, PKCδ knockdown abolished the synergy between sorafenib and OSU-2S. Furthermore, p53 restoration in Hep3B cells through the over-expression rendered them more sensitive to both agents while p53 knockdown from HepG2 cells increased their resistance to both agents. Conclusion: OSU-2S augments the anti-proliferative effect of sorafenib in HCC cell lines, in part, through the activation of PKCδ. The p53 status in HCC cells predicts their sensitivity toward both sorafenib and OSU-2S. The proposed combination represents a therapeutically relevant approach that can lead to a new HCC therapeutic protocol.

Modulation of Cyclins, p53 and Mitogen-Activated Protein Kinases Signaling in Breast Cancer Cell Lines by 4-(3,4,5-Trimethoxyphenoxy)benzoic Acid

Despite the advances in cancer therapy and early detection, breast cancer remains a leading cause of cancer-related deaths among females worldwide. The aim of the current study was to investigate the antitumor activity of a novel compound, 4-(3,4,5-trimethoxyphenoxy)benzoic acid (TMPBA) and its mechanism of action, in breast cancer. Results indicated the relatively high sensitivity of human breast cancer cell-7 and MDA-468 cells towards TMPBA with IC50 values of 5.9 and 7.9 μM, respectively compared to hepatocarcinoma cell line Huh-7, hepatocarcinoma cell line HepG2, and cervical cancer cell line Hela cells. Mechanistically, TMPBA induced apoptotic cell death in MCF-7 cells as indicated by 4′,6-diamidino-2-phenylindole (DAPI) nuclear staining, cell cycle analysis and the activation of caspase-3. Western blot analysis revealed the ability of TMPBA to target pathways mediated by mitogen-activated protein (MAP) kinases, 5′ adenosine monophosphate-activated protein kinase (AMPK), and p53, of which the concerted action underlined its antitumor efficacy. In addition, TMPBA induced alteration of cyclin proteins’ expression and consequently modulated the cell cycle. Taken together, the current study underscores evidence that TMPBA induces apoptosis in breast cancer cells via the modulation of cyclins and p53 expression as well as the modulation of AMPK and mitogen-activated protein kinases (MAPK) signaling. These findings support TMPBA’s clinical promise as a potential candidate for breast cancer therapy.

Induction of Apurinic Endonuclease 1 Overexpression by Endoplasmic Reticulum Stress in Hepatoma Cells

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide with poor prognosis due to resistance to conventional chemotherapy and limited efficacy of radiotherapy. Previous studies have noted the induction of endoplasmic reticulum stress or apurinic endonuclease 1 (APE1) expression in many tumors. Therefore, the aim of this study was to investigate the relationship between endoplasmic reticulum (ER stress) and APE1 in hepatocellular carcinoma. Here we investigate the expression of APE1 during ER stress in HepG2 and Huh-7 cell lines. Tunicamycin or brefeldin A, two ER stress inducers, increased APE1 and GRP78, an ER stress marker, expression in HepG2 and Huh-7 cells. Induction of APE1 expression was observed through transcription level in response to ER stress. APE1 nuclear localization during ER stress was determined using immunofluorescence assays in HepG2 cells. Furthermore, expression of Hepatitis B virus pre-S2∆ large mutant surface protein (pre-S2∆), an ER stress-induced protein, also increased GRP78 and APE1 expression in the normal hepatocyte NeHepLxHT cell line. Similarly, tumor samples showed higher expression of APE1 in ER stress-correlated liver cancer tissue in vivo. Our results demonstrate that ER stress and HBV pre-S2∆ increased APE1 expression, which may play an important role in resistance to chemotherapeutic agents or tumor development. Therefore, these data provide an important chemotherapeutic strategy in ER stress and HBV pre-S2∆-associated tumors.

Prevention of chronic HBV infection induced hepatocellular carcinoma development by using antiplatelet drugs

Hepatocellular carcinoma (HCC) is a common malignancy affecting approximately one million people worldwide annually. It is one of the most common causes of cancer morbidity and mortality in Asia and Africa. The successful control of HCC in children after HBV vaccination further supports a role of chronic HBV infection in the development of HCC. Known risk factors for HCC include chronic viral infection, cirrhosis, extensive alcohol intake, non-alcoholic fatty liver disease, certain inherited metabolic conditions, environmental exposure, and transgenic oncogenes (1-4).

Enhancing the Anticancer Activity of Antrodia cinnamomea in Hepatocellular Carcinoma Cells via Cocultivation With Ginger: The Impact on Cancer Cell Survival Pathways

Antrodia cinnamomea (AC) is a medicinal fungal species that has been widely used traditionally in Taiwan for the treatment of diverse health-related conditions including cancer. It possesses potent anti-inflammatory and antioxidant properties in addition to its ability to promote cancer cell death in several human tumors. Our aim was to improve the anticancer activity of AC in hepatocellular carcinoma (HCC) through its cocultivation with ginger aiming at tuning the active ingredients. HCC cell lines, Huh-7 and HepG2 were used to study the in vitro anticancer activity of the ethanolic extracts of AC (EAC) alone or after the cocultivation in presence of ginger (EACG). The results indicated that the cocultivation of AC with ginger significantly induced the production of important triterpenoids and EACG was significantly more potent than EAC in targeting HCC cell lines. EACG effectively inhibited cancer cells growth via the induction of cell cycle arrest at G2/M phase and induction of apoptosis in Huh-7 and HepG2 cells as indicated by MTT assay, cell cycle analysis, Annexin V assay, and the activation of caspase-3. In addition, EACG modulated cyclin proteins expression and mitogen-activated protein kinase (MAPK) signaling pathways in favor of the inhibition of cancer cell survival. Taken together, the current study highlights an evidence that EACG is superior to EAC in targeting cancer cell survival and inducing apoptotic cell death in HCC. These findings support that EACG formula can serve as a potential candidate for HCC adjuvant therapy.

Nuclear factor erythroid-2-related factor regulates LRWD1 expression and cellular adaptation to oxidative stress in human embryonal carcinoma cells

Leucine-rich repeats and WD repeat domain-containing protein 1 (LRWD1) is implicated in the regulation of signal transduction, transcription, RNA processing and tumor development. However, LRWD1 transcriptional regulation is not fully understood. This study aimed to investigate the relationship between LRWD1 expression and reactive oxygen species (ROS) level in human embryonal carcinoma cell line, NT2/D1 cells, which will help in understanding the transcriptional regulatory role of ROS in cells. Results showed that the exposure of NT2/D1 cells to various concentrations of hydrogen peroxide (H2O2) and the nitric oxide (NO) donor sodium nitroprusside (SNP) caused a significant increase in the mRNA and protein expression of LRWD1. In addition, LRWD1 promoter luciferase reporter assay, and Chromatin Immunoprecipitation assay (CHIP assay) showed that nuclear factor erythroid-2-related factor (Nrf2) was involved in the regulation of LRWD1 expression in response to oxidative stress. The involvement of Nrf2 was confirmed by shRNA-mediated knockdown of Nrf2 in NT2/D1 cells, which caused a significant decrease in LRWD1 expression in response to oxidative stress. Similarly, LRWD1 knockdown resulted in the accumulation of H2O2 and superoxide anion radical (O2-). Blocking ROS production by N-acetyl cysteine (NAC) protected NT2/D1 shLRWD1cells from H2O2-induced cell death. Collectively, oxidative stress increased LRWD1 expression through a Nrf2-dependent mechanism, which plays an important role in cellular adaptation to oxidative stress. These results highlight an evidence, on the molecular level, about LRWD1 transcriptional regulation under oxidative stress.

Antrodia cinnamomea boosts the anti-tumor activity of sorafenib in xenograft models of human hepatocellular carcinoma

Hepatocellular carcinoma (HCC) has been recognized worldwide as one of the major causes of cancer death. The medicinal fungus Antrodia cinnamomea (A. cinnamomea) has been served as a functional food for liver protection. The aim of the present study was to investigate the potential activity of A. cinnamomea extracts as a safe booster for the anticancer activity of sorafenib, a multi-kinase inhibitor approved for the treatment of HCC. The biologically active triterpenoids in the ethanolic extracts of A. cinnamomea (EAC) were initially identified by HPLC/LC/MS then the different extracts and sorafenib were assessed in vitro and in vivo. EAC could effectively sensitize HCC cells to low doses of sorafenib, which was perceived via the ability of the combination to repress cell viability and to induce cell cycle arrest and apoptosis in HCC cells. The ability of EAC to enhance sorafenib activity was mediated through targeting mitogen-activated protein (MAP) kinases, modulating cyclin proteins expression and inhibiting cancer cell invasion. Moreover, the proposed combination significantly suppressed ectopic tumor growth in mice with high safety margins compared to single-agent treatment. Thus, this study highlights the advantage of combining EAC with sorafenib as a potential adjuvant therapeutic strategy against HCC.