Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2012 Nov 1.
Published in final edited form as: Cancer Res. 2011 Sep 12;71(21):6807–6816. doi: 10.1158/0008-5472.CAN-11-1575

Verticillin A Overcomes Apoptosis Resistance in Human Colon Carcinoma through DNA methylation-dependent Upregulation of BNIP3

Feiyan Liu 1,3,*, Qianqian Liu 2, Dafeng Yang 3, Wendy B Bollag 4, Keith Robertson 5, Ping Wu 1, Kebin Liu 3,5,*
PMCID: PMC3206150  NIHMSID: NIHMS324813  PMID: 21911457

Abstract

Drug resistance is a major cause of failure in cancer chemotherapy. Therefore, identification and combined use of adjuvant compounds that can overcome drug resistance may improve the efficacy of cancer therapy. We screened extracts of Verticillium sp-infected mushrooms for anti-tumor compounds and identified the compound Verticillin A as an inducer of hepatoma cell apoptosis in vitro and an inhibitor of tumor xenograft growth in vivo. Verticillin A exhibited a potent apoptosis sensitizing activity in human colon carcinoma cells exposed to TRAIL or Fas in vitro. Furthermore, Verticillin A effectively sensitized metastatic human colon carcinoma xenograft to TRAIL-mediated growth inhibition in vivo. At the molecular level, we observed that Verticillin A induces cell cycle arrest in the G2 phase of the cell cycle in human colon carcinoma cells, markedly upregulating BNIP3 in both hepatoma and colon carcinoma cells. Notably, silencing BNIP3 decreased the sensitivity of tumor cells to Verticillin A-induced apoptosis in the absence or presence of TRAIL. We found that the BNIP3 promoter are methylated in both human hepatoma and colon carcinoma cells and tumor specimens. Verticillin A upregulated the expression of a panel of genes known to be regulated at the level of DNA methylation, in support of the concept that Verticillin A may act by demethylating the BNIP3 promoter to upregulate BNIP3 expression. Taken together, our findings identify Verticillin A as a potent apoptosis sensitizer with great promise for further development as an adjuvant agent to overcome drug resistance in human cancer therapy.

Keywords: Verticillin A, Drug Resistance, TRAIL, Fas, BNIP3

Introduction

The ideal cancer therapy should meet two criteria: First, the therapeutic agent has to be effective in killing cancer cells; and second, the agent needs to exhibit low toxicity by showing selectivity for the cancer cells and avoiding systemic or off-target toxicity. Currently, cancer cell resistance to chemotherapeutic drugs and the high cytotoxicity of chemotherapeutic agents are the two major problems in human cancer therapy (1). Cancer cells may be intrinsically resistant to chemotherapeutic drugs, especially to cytotoxic agents, prior to treatment. Tumors can also acquire resistance during treatment. Drug resistance, whether intrinsic or acquired, is believed to account for treatment failure in over 90% of patients with metastatic cancer (1). Therefore, finding ways to overcome drug resistance may greatly improve the survival of patients with cancer. Multiple mechanisms confer cancer cell resistance to chemotherapeutic drugs, however, when it comes to effective eradication of cancer cells by chemotherapies, all roads lead to apoptosis. Essentially most cytotoxic anticancer drugs currently in clinical use or in clinical trials kill cancer cells through inducing apoptosis. Thus, tumor cell resistance to apoptosis, whether intrinsic or acquired, represents a major challenge in chemotherapeutic intervention of cancer, especially metastatic cancer.

TNF-related apoptosis-inducing ligand (TRAIL, also known as TNFSF10 or APO2L) has been under intense study for its obvious potential as a selective anticancer agent in cancer therapy since it preferentially induces apoptosis in tumor cells but not in normal cells (2–3). TRAIL-based cancer therapies are now in multiple Phase I and Phase II clinical trials to treat human cancer, including metastatic human colorectal cancer. However, although human patients exhibit excellent tolerance to humanized TRAIL receptor agonist mAbs, the efficacy of these TRAIL receptor agonist mAbs so far is disappointing. This poor efficacy is obviously expected since most cancer cells, especially metastatic cancer cells, often exhibit a TRAIL-resistance phenotype (4–8).

To increase the efficacy of TRAIL-based therapy, various therapeutic agents have been tested for their effectiveness in enhancing TRAIL-induced apoptosis in cancer cell lines and in human cancer patients (4, 9–17). These therapeutic agents have shown great promise in enhancing TRAIL efficacy. However, because the most attractive feature of TRAIL therapy is its tumor selectivity-conferred low toxicity, combining cytotoxic agents with TRAIL may bring back toxicity associated with the therapeutic agents. Therefore, identifying novel TRAIL sensitizers with low toxicity and high sensitization activity is urgently needed for TRAIL-based cancer therapy. We report here the identification and characterization of Verticillin A, a natural compound from pathogen-infected mushroom, as a potent TRAIL sensitizer. Our data suggest that Verticillin A hold great promise for further development as a potent sensitizer to enhance the efficacy of TRAIL- and potentially other cytotoxic agent-based therapy against human colorectal cancer.

Materials and Methods

Purification and identification of Verticillin A

The fresh fruiting bodies of Verticillin sp-infected mushroom (Amanita flavorubescens Alk) were lyophilized and then extracted successively with light petroleum and ethyl acetate. The ethyl acetate extract was fractionated by countercurrent chromatography using a two-phase solvent system composed of light petroleum, chloroform and acetonitrile with a volume ratio of 6:1:3. Fractions that exhibited significant cytotoxicity was subjected to semi-preparative chromatography on a reverse-phase C8 column (Hypersil ODS 20 ×250 mm), followed by elution with a mixture of acetonitrile and water with a gradient from 10 to 100%. The structures of purified cytotoxic compounds were determined by electro-spray ionization mass spectrometry (ESI-MS) and one- and two-dimensional nuclear magnetic resonance (NMR) spectra.

Cell Lines

All cell lines except MPNST-724 used in this study were obtained from American Type Culture Collection(ATCC) (Mannassas, VA). ATCC characterizes these cells by morphology, immunology, DNA fingerprint, and cytogenetics. MPNST-724 has been previously characterized (18).

Reagents

Recombinant TRAIL protein was expressed and purified as previously described (19). TRAIL receptor DR5 agonist mAb, CD3 mAb (OKT3), and CD28 mAb (CD28.2) were obtained from Biolegend (San Diego, CA). Etoposide and cisplatin were obtained from Sigma (St. Louis, MO). Mega-Fas Ligand® (FasL) (Kindly provided by Drs. Steven Butcher and Lars Damstrup, Topotarget A/S, Denmark) is a recombinant fusion protein that consists of three human FasL extracellular domains linked to a protein backbone comprising the dimmer-forming collagen domain of human adiponectin. The Mega-Fas Ligand was produced as a glycoprotein in mammalian cells in Topotarget A/S (Copenhagen, Denmark).

Mice

Athymic mice were obtained from the NCI Frederick mouse facility. Six to eight weeks old female mice were used. Experiments and care/welfare were in agreement with federal regulations and an approved protocol by the GHSU/IACUC committee.

Cell viability and apoptosis assays

Cell viability assay was carried out using the MTT cell proliferation assay kit (ATCC, Manassas, VA). For the DNA fragmentation assay, genomic DNA was isolated from cells and analyzed by agarose gel electrophoresis. For the quantitative apoptosis assay, cells were cultured in the absence or presence of recombinant TRAIL protein with or without Verticillin A (20), followed by staining with propidine iodide (PI) (Trevigen, Gaithersburg, MD) or PI plus Annexin V-Alex Fluor 647 (Biolegend) and analyzed by flow cytometry.

Cell surface marker analysis

Tumor cells were stained with anti-TRAIL receptor DR4, DR5, T-R3 and T-R4 mAbs or an isotype-matched control IgG (Alexis Biochemicals, San Diego, CA) as previously described (20). For Fas receptor analysis, tumor cells were stained with FITC-conjugated anti-human Fas mAb (BD Biosciences). The stained cells were analyzed by flow cytometry.

RT-PCR analysis

Total RNA was isolated from cells or tissues using Trizol (Invitrogen, San Diego, CA) and used for semi quantitative and real-time RT-PCR analysis of gene expression as described (21–22). The PCR primer sequences are listed in supplemental table 1.

Western analysis

Western analysis was performed as previously described (20). The following primary antibodies were obtained from Cell Signaling Biotech (Danvers, MA): anti-FLIP (1:250 dilution), anti-cIAP1 (1:250), anti-xIAP (1:500), anti-Bad (1:1000), anti-Bok (1:1000), anti-p53 (1:500), anti-PUMA (1:2000), and anti-cleaved PARP (1:500). The following primary antibodies were obtained from Santa Cruz Biotech (Santa Cruz, CA): anti-Bax (1:2000), anti-survivin (1:100), anti-Mcl-1 (1:100), anti-BNIP3 (1:100). Anti-β-actin was obtained from Sigma (St Louis, MO) and used at 1:8000.

In Vivo tumor growth inhibition

For HepG2 tumor, athymic mice were subcutaneously (s.c.) inoculated with the tumor cells. The control mice were given saline. The treatment group was intravenously injected with Verticillin A at doses of 1 and 2 mg/kg body weight, respectively. Seven mice were used in each group. For SW620 tumors, SW620 cells (3×106 cells/mouse) were injected s.c. into athymic mice at the right flank. Three days later, the tumor-bearing mice were treated with Verticillin A (0.125mg/kg body weight, n=6), TRAIL (100 mg/mouse, n=5) and Verticillin A plus TRAIL (n=5) every 2 days for 14 days. Tumor size was measured in 2 dimensions with a digital micrometer caliper at the indicated time points. Tumor volume was calculated by the formula (tumor length × tumor width2)/2.

Cell cycle analysis

Cell cycle was analyzed as previously described (19).

MS-PCR analysis

Genomic DNA was isolated using a DNeasy Tissue Kit (Qiagea). Sodium bisulfite treatment of genomic DNA was carried out using CpGenome™ Universal DNA Modification Kit (Chemicon, Temecula, CA). Methylation sensitive (MS)-PCR was carried out as previously described (23). The PCR primers are listed in supplemental Table 1.

Gene silencing

Scramble siRNA (Dharmacon, Lafayette, CO) and human BNIP3-specific siRNA (Santa Cruz, Cat# sc-37451) were used. For HepG2 cells, tumor cells were transiently transfected with the siRNAs using Lipofectamine 2000 (Invitrogen) for approximately 24 h. Cells were then harvested and reseeded in 24-well plates in the absence or presence of 200 nM Verticillin A for approximately 24 h before analysis for apoptosis. For SW620 cells, cells were transfected with scramble siRNA or BNIP3-specific siRNAs. Verticillin A was added to the transfection culture 6 h later to a final concentration of 10 nM and the cells were cultured overnight. Cells were then harvested and reseeded in the presence of 10 nM Verticillin A with or without TRAIL (10 ng/ml) for another 24 h and analyzed for apoptosis.

Statistical analysis

Where indicated, data were represented as the means ± SD. Statistical analysis was performed using two-sided t test, with p-values<0.05 considered statistically significant.

Results

Purification and identification of Verticillin Aas an anti-tumor cytotoxic agent

The fresh bodies of mushroom (Amanita flavorubescens Alk) infected by fungus Verticillium sp were extracted, fractionated and screened for anti-tumor cytotoxicity. From approximately 1500 g fresh mushrooms, we purified a compound (approximately 10 mg) with 99% purity and potent inhibitory activity against HepG2 cells. This compound has a formula of C30H28N6O6S4 and a molecular weight of 696.3. Analysis with electro-spray ionization mass spectrometry and nuclear magnetic resonance (NMR) spectrometry, in combination with comparing the crystal structure with the database (24) identified this compound as Verticillin A (Supplemental Fig. 1).

Verticillin Ainhibits the growth of hepatoma cells in vitro

Verticillin A exhibited a growth inhibitory effect on HepG2 cells in a dose-dependent manner with an IC50 of approximately 62 nM based on MTT assays (Fig. 1A). Analysis of apoptosis markers, including Annexin V plus PI, PARP cleavage and genomic DNA fragmentation, revealed that Verticillin A increased PI and Annexin V-double positive cells, induced PARP cleavage and genomic DNA fragmentation in Hepg2 cells at a dose-dependent manner (Fig. 1B). Our data thus suggest that Verticillin A inhibits HepG2 cell growth at least partially through inducing apoptosis.

Figure 1. Verticillin A induces human hepatoma cell apoptosis in vitro and growth inhibition in vivo.

Figure 1

Open in a new tab

A. Verticillin A exhibits a potent growth inhibitory effect against hepatoma cells. HepG2 cells were seeded in 96-well plates and cultured in the presence of various concentrations of Verticillin A for 24 and 72 h, respectively. The tumor cell growth rate was measured using the MTT assay. B. Verticillin A induces apoptosis in hepatoma cells. HepG2 cells were treated with Verticillin A at the indicated concentrations and analyzed for apoptosis by staining with PI and Annexin V (top left panel), PARP cleavage by Western blotting (bottom left panel) and DNA fragmentation by agarose gel electrophoresis (bottom right panel). % PI and Annexin V-double positive cells are indicated in each plot (top left panel). % cell death was calculated as PI and Annexin V-double positive cells of the treated cells - % PI and Annexin V-double positive cells of untreated cells, and presented in the top right panel. C. Verticillin A suppresses hepatoma xenograft growth in vivo. HepG2 cells were injected s.c. into athymic mice (n=7 per group). The tumor-bearing mice were treated with Verticillin A at the indicated doses, and tumor growth was measured over time after Verticillin A treatment. * p<0.05.

To determine whether the growth inhibitory effect of Verticillin A can be extended to in vivo tumor growth inhibition, HepG2 cells were injected s.c. into athymic mice. Tumor-bearing mice were then treated with Verticillin A by i.v. injection. Verticillin A inhibited tumor growth in a dose-dependent manner, with significant inhibition of HepG2 tumor growth at a dose of 2 mg/kg body weight (Fig. 1C).

Verticillin A is a potent suppressor of multiple types of tumor cells

To determine whether Verticillin A inhibits the growth of other types of tumor cells, six types of tumor cells were cultured in the presence of different concentrations of Verticillin A and examined for the their growth in vitro. Verticillin A significantly inhibited the growth of all of these tumor cells. More importantly, Verticillin A inhibited the growth of these tumor cells with concentrations in the nanomolar range with IC50 from 30 to 122 nM (Supplemental Table 2), To test the toxicity of Verticillin A to normal human cells, we cultured normal human colon epithelial cell line CCD-841 in the presence of Verticillin A and determined the IC50 is 666.7 nM. As expected, CCD-841 is not sensitive to TRAIL, and Verticillin A exhibited no sensitization effect on CCD-841 cell sensitivity to TRAIL (Supplemental Figure 2). We then obtained human white blood cells from two normal donors and stimulated the T cells in anti-CD3/anti-CD28-coated 96-well plates for 2 days. Verticillin A was then added to the culture for another 24 h. The IC50s were 65.1 and 78.8 nM, respectively, for donors 1 and 2. As expected, the activated human normal T cells are not sensitive to TRAIL, and Verticillin A exhibited a small degree of effect on activated human T cell sensitivity to TRAIL (Supplemental Figure 2).

Verticillin A is a potent apoptosis sensitizer that overcomes TRAIL resistance in the metastatic human colon carcinoma cells

Combinational therapy has been shown often to be effective than single agent therapy in suppression of tumor cell growth (25). SW620 is a metastatic human colon carcinoma cell line that is highly resistant to therapeutic agents, including TRAIL (Fig. 2A)(26). We observed that in addition to its ability to inhibit SW620 cell growth, Verticillin A also effectively sensitized SW620 cells to TRAIL, and TRAIL agonist mAb-induced cell death at a concentration as low as 10 nM (Fig. 2A). The sensitization effect of Verticillin A was also observed in 6 other human colon carcinoma cells (Fig. 2B). Next, we examined the sensitization effects of Verticillin A in other types of tumor cells. Pre-treatment of sarcoma (MPNST724), lung adenocarcinoma (A549) and mammary carcinoma (MCF-7) with Verticillin A also significantly increased these tumor cells sensitivity to TRAIL-induced cell death (Fig. 2C). Analysis of tumor cell death using PI and Annexin V double staining and PARP cleavage indicated that combination treatment of Verticillin A and TRAIL induces apoptosis in SW620 cells (Fig 2D).

Figure 2. Verticillin A is a potent sensitizer of the TRAIL-induced cell death in human colon carcinoma cells.

Figure 2

Open in a new tab

A. Verticillin A overcomes TRAIL resistance of metastatic human colon carcinoma cells. SW620 cells were incubated with various concentrations of Verticillin A for 24 h and analyzed for cell death by PI staining (left panel). SW620 cells were also cultured in the absence or presence of Verticillin A (10 nM) overnight, followed by incubation with various concentrations of TRAIL protein (middle panel), or DR5 agonist mAb (right panel) for 24 h. The tumor cells were then stained using PI and analyzed for cell death by flow cytometry. % cell death was calculated by the formula: % PI+ cells of the TRAIL or DR5-treated cells − % PI+ cells of untreated cells. B–C. Verticillin A sensitizes human colon carcinoma (B), as well as sarcoma, lung and mammary carcinoma (C) cells, to TRAIL-induced cell death. Tumor cells were treated with Verticillin A (20 nM) overnight, followed by incubation with TRAIL protein (50 ng/ml) and then analyzed for cell death by PI staining as in A. ** p<0.01 as compared to TRAIL or Verticillin A treatment alone. Column: mean; Bar: SD. D. Verticillin A induces tumor cell apoptosis. SW620 cells were cultured in the absence or presence of Verticillin A (10 nM) overnight, followed by incubation with TRAIL protein (10 ng/ml) for another 24 h as in A and stained with Annexin V and PI. The percentage of apoptotic cells (Annexin V+ and PI+ cells) is indicated in the top right. The tumor cells were also analyzed for PARP cleavage by Western blotting analysis (right panel).

Verticillin A is also a potent apoptosis sensitizer that overcomes resistance to FasL-induced cell death

Because Fas-mediated and TRAIL-induced apoptosis share similar signaling pathways, we next tested whether Verticillin A also sensitizes tumor cells to FasL-induced cell death. Verticillin A pre-treatment significantly increased SW620 cells to FasL-induced cell death (Supplemental Figure 3). We next extended our study to the chemotherapeutic drugs etoposide and cisplatin, two anti-cancer drugs that kill tumor cells by inducing apoptosis. SW620 cells were essentially resistant to both etoposide and cisplatin. Verticillin A dramatically sensitized SW620 cells to both etoposide and cisplatin-induced cell death (Supplemental Figure 4).

Verticillin A overcomes metastatic human colon carcinoma TRAIL resistance in vivo

To determine whether the observation that Verticillin A effectively sensitizes metastatic colon carcinoma cells to TRAIL-induced cell death in vitro can be extended to enhance TRAIL-mediated tumor suppression in vivo, SW620 cells were injected s.c. into athymic mice. Verticillin A and TRAIL, either used as single agents or in combination, were then injected into tumor-bearing mice. To differentiate the function of Verticillin A in TRAIL sensitization from its direct tumor growth inhibitory activity, a low dose (0.125mg/kg body weight) of Verticillin A was used. At this low dose, Verticillin A did not exhibit significant tumor suppression activity (Fig. 3). As expected, SW620 tumors were resistant to TRAIL (Fig. 3) (26). However, combined treatment with low dose of Verticillin A and TRAIL significantly inhibited tumor cell xenograft growth (Fig. 3). Taken together, our data suggest that Verticillin A is an effective sensitizer in TRAIL-mediated suppression of metastatic colon carcinoma in vivo.

Figure 3. Verticillin A overcomes TRAIL resistance in vivo.

Figure 3

Open in a new tab

SW620 cells were injected s.c. into athymic mice. Three days later, the tumor-bearing mice were treated with Verticillin A, recombinant TRAIL protein, and Verticillin A plus recombinant TRAIL protein, respectively, every 2 days for 14 days. Tumor growth was measured over time. Column: mean; Bar: SD. * p<0.05.

Verticillin A induces cell cycle arrest

In the literature, enhanced cell cycle arrest has been suggested as a possible mechanism for the synergistic effect of natural compounds combined with therapeutic agents on tumor cell apoptosis (27). To determine whether Verticillin A alters cell cycle progression, we treated HepG2 and SW620 cells with Verticillin A and analyzed cell cycle in the treated cells. Verticillin A altered cell cycle progression in SW620 cells (Fig. 4A), but not in HepG2 cells (Fig. 4B). It is apparent that Verticillin A induced a dramatic arrest at the G2 phase of the cell cycle in SW620 cells (Fig. 4C).

Figure 4. Effects of Verticillin A on cell cycle progression.

Figure 4

Open in a new tab

SW620 (A) and HepG2 (B) cells were treated with Verticillin A at the indicated concentrations for 24 h. Cells were then fixed, stained with PI and analyzed by flow cytometry. C: Quantification of percentages of cells in G1, S and G2 phase of the cell cycle as shown in A & B. Column: mean; Bar: SD.

Verticillin A upregulates BNIP3 expression

The above observation that Verticillin A induces cell cycle arrest in SW620 cells but not in HepG2 cells (Fig. 4) suggests that enhanced cell cycle arrest is unlikely the sole mechanism of Verticillin A function. Our data also indicate that Verticillin A does not significantly alter TRAIL or Fas receptor expression (Supplemental Fig. 5). Because Verticillin A induced tumor cells apoptosis (Figs. 1 and 2), we analyzed the protein levels of genes with known functions in the mitochondrion-dependent apoptosis pathway. As shown in Figure 5A, Verticillin A did not alter the expression level of those anti-apoptotic genes examined in SW620 cells. However, among the pro-apoptotic protein examined, Verticillin A increased BNIP3 protein levels in a dose-dependent manner in SW620 cells (Fig. 5B). Analysis of BNIP3 protein level revealed that Verticillin A also increased BNIP3 protein level in HepG2 cells (Fig. 5C).

Figure 5. Verticillin A upregulates BNIP3 expression in human colon carcinoma and hepatoma cells.

Figure 5

Open in a new tab

Tumor cells were treated with Verticillin A at the indicated concentrations for 24 h. Cells were then analyzed by Western blotting analysis for anti-apoptotic (A) and pro-apoptotic (B & C) proteins as indicated.

We next silenced Verticillin A-induced BNIP3 in both HepG2 and SW620 cells and analyzed the effects of loss of BNIP3 on Verticillin A-enhanced apoptosis. Verticillin A up-regulated BNIP3 expression and BNIP3 siRNA blocked Verticillin A-induced BNIP3 expression in HepG2 cells (Fig. 6A). Silencing BNIP3 significantly decreased Verticillin A-induced apoptosis in HepG2 cells (Fig. 6B). Verticillin A induced BNIP3 expression in SW620 cells and BNIP3 siRNA blocked BNIP3 upregulation by Verticillin A (Fig. 6C). Incubation of BNIP3-transfected and Verticillin A-treated cells with TRAIL showed that silencing BNIP3 significantly reduces Verticillin A-sensitized and TRAIL-induced apoptosis in SW620 cells (Fig. 6D).

Figure 6. Verticillin A mediates apoptosis partially through regulating BNIP3 expression.

Figure 6

Open in a new tab

A. Silencing BNIP3 decreases Verticillin A-induced apoptosis in HepG2 cells. HepG2 cells were transfected with scramble and BNIP3-specific siRNAs, respectively, for approximately 24 h. Transfected cells were then treated with Verticillin A (200 nM) for approximately 24 h and analyzed for BNIP3 mRNA level by semi-quantitative (top panel) and real-time (bottom panel) RT-PCR. B. The cells were also stained with PI and Annexin V and analyzed by flow cytometry for apoptosis. Number at the upper right corner indicates % of PI and Annexin V-positive cells. The cell death was quantified as in Fig. 1B and presented at the right panel. Column: mean; Bar: SD. ** p<0.01. C. Silencing BNIP3 expression decreases Verticillin A-sensitized and TRAIL-induced apoptosis in SW620 cells. SW620 cells were transfected with scramble and BNIP3-specific siRNAs, respectively. Verticillin A was added to the transfected cultures 6 h later. Cells were harvested approximately 16 h later and analyzed for BNIP3 mRNA level by semi-quantitative (top panel) and real-time (right panel) RT-PCR. D. The transfected cells were re-cultured in the presence of Verticillin A (10nM) with or without TRAIL protein (10 ng/ml) for another 24 h and stained with PI and Annexin V. Cell death was analyzed and quantified as in B (right panel). Column: mean; Bar: SD. ** p<0.01.

Verticillin A upregulates BNIP3 expression potentially through inducing DNA demethylation

Analysis of the human BNIP3 promoter region revealed that the BNIP3 promoter is GC-rich and contains CpG islands (Fig. 7A). We then used MS-PCR to analyze the methylation status of the BNIP3 in 3 human colon carcinoma cell lines, HepG2 cells and tumor tissues dissected from 5 paraffin-embedded human colorectal carcinoma specimens (4 liver metastases and 1 primary adenocarcinoma). BNIP3 promoter is methylated in all the cell lines and the tumor specimens examined (Fig. 7B).

Figure 7. Verticillin A regulates BNIP3 expression potentially by inhibiting DNA methylation.

Figure 7

Open in a new tab

A. The human BNIP3 gene promoter, showing CpG islands (grey area). B. MS-PCR analysis of the human BNIP3 gene promoter DNA methylation status. U: unmethylated, M: methylated. P: primary colon carcinoma, LM: liver metastases. C. RT-PCR analysis of a panel of DNA-methylation-regulated genes. SW620 cells were treated with various concentrations of azacytidine for 3 days or Verticillin A for 24 h and analyzed by RT-PCR for the expression levels of the indicated genes.

The above observations suggest that Verticillin A might activate BNIP3 expression in human cancer cells through inhibiting DNA methylation. To determine whether Verticillin A-inhibited DNA methylation is a general phenomenon, we tested the effects of Verticillin A on the expression of a panel of 4 genes known to be regulated by DNA methylation (23, 28–29). As expected, azacytidine treatment increased the expression level of BNIP3 and these 4 genes (Fig. 7C). At the same time, Verticillin A treatment also increased the expression of BNIP3 and these 4 genes. Thus, our data suggest that Verticillin A might function at least partially through upregulating BNIP3 in a DNA demethylation-dependent manner.

Discussion

Verticillin A is a compound of the epidithiodioxopiprazine structural class. In a screening for anti-tumor cytotoxic natural compounds, we purified a compound from Verticillium-infected mushrooms Amanita flavorubescens Alk and identified this compound as Verticillin A. Neither uninfected Amanita flavorubescens Alk nor the pathogen fungus Verticillium contains Verticillin A, suggesting that Verticillin A is synthesized during the host and pathogen interaction. Here, we demonstrated that Verticillin A is an effective tumor suppressor that induces tumor cell apoptosis at nanomolar concentrations. More importantly, Verticillin A exhibited potent activity as an apoptosis sensitizer that effectively overcame metastatic human colon carcinoma cell resistance to TRAIL-, Fas- and other cytotoxic agent-induced apoptosis in vitro at a concentration as low as 10 nM. Furthermore, Verticillin A also overcame human colon carcinoma xenograft resistance to TRAIL therapy in vivo. Therefore, Verticillin A is a potent apoptosis sensitizer.

TRAIL is considered a selective anticancer drug (2, 30), and TRAIL-based cancer therapy is currently in Phase I and II clinical trials. However, most cancer cells, especially cancer cells in advanced stages, are resistant to TRAIL (12). Overcoming TRAIL resistance is thereby of urgent significance (3). Current approaches to overcome TRAIL resistance largely focus on combination treatment with conventional chemotherapeutic agents (4, 10, 16–17, 31–34). Combinations of TRAIL receptor mAb with conventional chemotherapeutic drugs are currently tested in clinical trials against metastatic human colorectal cancer. However, although proven effective, these chemotherapeutic agents’ toxicity may offset the advantage of tumor-selectivity and low toxicity of TRAIL therapy. Natural compounds have been shown to possess TRAIL sensitization activity (35–39). One example of such a compound is sulforaphane, a dietary isothiocyanate found in broccoli and cauliflower (35–37). Here, we identified Verticillin A from fungus-infected mushrooms as another natural compound that possesses biological activity as a TRAIL sensitizer. Compared to sulforaphane, which sensitizes tumor cells to TRAIL-induced apoptosis in micromolar concentrations (35–37), Verticillin A sensitizes multiple types of tumor cells to TRAIL-induced apoptosis at nanomolar concentrations, suggesting that Verticillin is potentially a more potent TRAIL sensitizer that warrants clinical testing for its effectiveness in enhancing the efficacy of TRAIL therapy in human cancer patients.

Structurally related Verticillin compounds have been shown to possess biological activities to inhibit induction of several oncogenes (40–41). 11,11′-dideoxy-verticillin, a natural compound isolated from herb Shiraia bambusicola, has also been shown to inhibit epidermal growth factor receptor tyrosine kinase activity and to suppress tumor growth (40). Although Verticillin A shares some structure similarity with 11,11′-dideoxy-verticillin, Verticillin A apparently possesses very different biological activity. We demonstrated here that Verticillin A induces the expression of BNIP3 in both hepatoma and colon carcinoma cells. We also demonstrated that this Verticillin A-elicited increase in BNIP3 expression either directly induces apoptosis in HepG2 cells, or sensitizes the metastatic colon carcinoma cells to TRAIL-induced apoptosis (Fig. 6). BNIP3 is a proapoptotic member of the Bcl-2 family (42) that mediates tumor cell apoptosis (43–47). However, it has also been reported that BNIP3 up-regulation is not associated with arsenic trioxide-mediated TRAIL sensitization in human glioma (48), suggesting that the function of BNIP3 in apoptosis might be tumor type- or cellular context-dependent. Nevertheless, we demonstrated here that Verticillin A upregulates BNIP3 in both human hepatoma and colon carcinoma cells, and BNIP3 upregulation is at least partially responsible for the increased apoptosis in hepatoma and colon carcinoma cells. It should be pointed out that although Verticillin A induces BNIP3 in both colon carcinoma and hepatoma cells, Verticillin A sensitized the human colon carcinoma to TRAIL-induced apoptosis at a low dose (10 nM). In contrast, lower dose (10 nM) of Verticillin A did not overcome HepG2 cell resistance to TRAIL-induced cell death (Supplemental Fig. 6). This difference might be due, at least in part, to the different cell cycle arrest induction in these two types of tumors (Fig. 4). However, whether higher doses of Verticillin A sensitize hepatoma cells to TRAIL-induced apoptosis remains to be determined.

BNIP3 expression has been shown to be regulated by DNA methylation in tumor cells (49). The promoter region of the human BNIP3 gene contains CpG islands (Fig. 7) and inhibition of DNA methylation increases BNIP3 expression in the human colon carcinoma cells (Fig. 7C). We demonstrated here that the BNIP3 promoter is methylated in both human hepatoma and colon carcinoma cells, as well as in human colon carcinoma specimens (Fig. 7B). Furthermore, inhibition of DNA methylation with azacytidine increased BNIP3 expression in both human hepatoma and colon carcinoma cells (Fig. 7C). Thus, our data suggest that Verticillin A increases BNIP3 expression possibly by inhibiting DNA methylation or inducing DNA demethylation. However, how Verticillin A alters DNA methylation to mediate BNIP3 expression remains to be determined.

In conclusion, we have identified the natural compound Verticillin A as a potent cytotoxic agent that has the potential to be developed as a low toxicity anticancer drug. More significantly, we demonstrated that Verticillin A is also a potent apoptosis sensitizer that has great potential to be developed as an effective, yet potentially less toxic, adjuvant agent to overcome drug resistance in cancer chemotherapy against metastatic human colorectal cancer.

Supplementary Material

1
2
3
4
5
6
7
8
9

Acknowledgments

We thank Dr. Yongchang Zhao for assistance in collecting mushroom, Ms. Sally McCarty for assistance in collecting human white blood cells and Dr. Jeanene Pihkala for assistance in flow cytometry analysis.

Grant support

National Institutes of Health (CA133085 to K.L), the American Cancer Society (RSG-09-209-01-TBG to K.L.), National High Technology Research and Development Program of China (2007AA021504 to FL and PW), and Siyuan Foundation (to FL and PW).

Footnotes

Conflict of interest: None

References

  • 1.Longley DB, Johnston PG. Molecular mechanisms of drug resistance. J Pathol. 2005;205:275–92. doi: 10.1002/path.1706. [DOI] [PubMed] [Google Scholar]
  • 2.Holoch PA, Griffith TS. TNF-related apoptosis-inducing ligand (TRAIL): a new path to anti-cancer therapies. Eur J Pharmacol. 2009;625:63–72. doi: 10.1016/j.ejphar.2009.06.066. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Rowinsky EK. Targeted induction of apoptosis in cancer management: the emerging role of tumor necrosis factor-related apoptosis-inducing ligand receptor activating agents. J Clin Oncol. 2005;23:9394–407. doi: 10.1200/JCO.2005.02.2889. [DOI] [PubMed] [Google Scholar]
  • 4.Galligan L, Longley DB, McEwan M, Wilson TR, McLaughlin K, Johnston PG. Chemotherapy and TRAIL-mediated colon cancer cell death: the roles of p53, TRAIL receptors, and c-FLIP. Mol Cancer Ther. 2005;4:2026–36. doi: 10.1158/1535-7163.MCT-05-0262. [DOI] [PubMed] [Google Scholar]
  • 5.White-Gilbertson S, Mullen T, Senkal C, Lu P, Ogretmen B, Obeid L, et al. Ceramide synthase 6 modulates TRAIL sensitivity and nuclear translocation of active caspase-3 in colon cancer cells. Oncogene. 2009;28:1132–41. doi: 10.1038/onc.2008.468. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Garofalo M, Di Leva G, Romano G, Nuovo G, Suh SS, Ngankeu A, et al. miR-221&222 regulate TRAIL resistance and enhance tumorigenicity through PTEN and TIMP3 downregulation. Cancer Cell. 2009;16:498–509. doi: 10.1016/j.ccr.2009.10.014. [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]
  • 7.Kim SH, Ricci MS, El-Deiry WS. Mcl-1: a gateway to TRAIL sensitization. Cancer Res. 2008;68:2062–4. doi: 10.1158/0008-5472.CAN-07-6278. [DOI] [PubMed] [Google Scholar]
  • 8.Menke C, Goncharov T, Qamar L, Korch C, Ford HL, Behbakht K, et al. TRAIL receptor signaling regulation of chemosensitivity in vivo but not in vitro. PLoS One. 2011;6:e14527. doi: 10.1371/journal.pone.0014527. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Rosato RR, Almenara JA, Coe S, Grant S. The multikinase inhibitor sorafenib potentiates TRAIL lethality in human leukemia cells in association with Mcl-1 and cFLIPL down-regulation. Cancer Res. 2007;67:9490–500. doi: 10.1158/0008-5472.CAN-07-0598. [DOI] [PubMed] [Google Scholar]
  • 10.Dhandapani L, Yue P, Ramalingam SS, Khuri FR, Sun SY. Retinoic acid enhances TRAIL induced apoptosis in cancer cells by upregulating TRAIL receptor 1 expression. Cancer Res. 2011 doi: 10.1158/0008-5472.CAN-10-4180. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Lagneaux L, Gillet N, Stamatopoulos B, Delforge A, Dejeneffe M, Massy M, et al. Valproic acid induces apoptosis in chronic lymphocytic leukemia cells through activation of the death receptor pathway and potentiates TRAIL response. Exp Hematol. 2007;35:1527–37. doi: 10.1016/j.exphem.2007.06.014. [DOI] [PubMed] [Google Scholar]
  • 12.Ricci MS, Kim SH, Ogi K, Plastaras JP, Ling J, Wang W, et al. Reduction of TRAIL-induced Mcl-1 and cIAP2 by c-Myc or sorafenib sensitizes resistant human cancer cells to TRAIL-induced death. Cancer Cell. 2007;12:66–80. doi: 10.1016/j.ccr.2007.05.006. [DOI] [PubMed] [Google Scholar]
  • 13.Nawrocki ST, Carew JS, Douglas L, Cleveland JL, Humphreys R, Houghton JA. Histone deacetylase inhibitors enhance lexatumumab-induced apoptosis via a p21Cip1-dependent decrease in survivin levels. Cancer Res. 2007;67:6987–94. doi: 10.1158/0008-5472.CAN-07-0812. [DOI] [PubMed] [Google Scholar]
  • 14.Shankar S, Davis R, Singh KP, Kurzrock R, Ross DD, Srivastava RK. Suberoylanilide hydroxamic acid (Zolinza/vorinostat) sensitizes TRAIL-resistant breast cancer cells orthotopically implanted in BALB/c nude mice. Mol Cancer Ther. 2009;8:1596–605. doi: 10.1158/1535-7163.MCT-08-1004. [DOI] [PubMed] [Google Scholar]
  • 15.Jani TS, DeVecchio J, Mazumdar T, Agyeman A, Houghton JA. Inhibition of NF-kappaB signaling by quinacrine is cytotoxic to human colon carcinoma cell lines and is synergistic in combination with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or oxaliplatin. J Biol Chem. 2010;285:19162–72. doi: 10.1074/jbc.M109.091645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Fu L, Lin YD, Elrod HA, Yue P, Oh Y, Li B, et al. c-Jun NH2-terminal kinase-dependent upregulation of DR5 mediates cooperative induction of apoptosis by perifosine and TRAIL. Mol Cancer. 2010;9:315. doi: 10.1186/1476-4598-9-315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Menke C, Bin L, Thorburn J, Behbakht K, Ford HL, Thorburn A. Distinct TRAIL resistance mechanisms can be overcome by proteasome inhibition but not generally by synergizing agents. Cancer Res. 2011;71:1883–92. doi: 10.1158/0008-5472.CAN-10-2252. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Subramanian S, Thayanithy V, West RB, Lee CH, Beck AH, Zhu S, et al. Genome-wide transcriptome analyses reveal p53 inactivation mediated loss of miR-34a expression in malignant peripheral nerve sheath tumours. J Pathol. 2010;220:58–70. doi: 10.1002/path.2633. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Yang D, Wang S, Brooks C, Dong Z, Schoenlein PV, Kumar V, et al. IFN regulatory factor 8 sensitizes soft tissue sarcoma cells to death receptor-initiated apoptosis via repression of FLICE-like protein expression. Cancer Res. 2009;69:1080–8. doi: 10.1158/0008-5472.CAN-08-2520. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Liu F, Hu X, Zimmerman M, Waller J, Wu P, Hayes-Jordan A, et al. TNFα Cooperates with IFN-γ to Repress Bcl-xL Expression to Sensitize Metastatic Colon Carcinoma Cells to TRAIL-mediated Apoptosis. PLoS ONE. 2011;6:e16241. doi: 10.1371/journal.pone.0016241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Zimmerman M, Yang D, Hu X, Liu F, Singh N, Browning D, et al. IFN-γ Upregulates Survivin and Ifi202 Expression to Induce Survival and Proliferation of Tumor-Specific T Cells. PLoS One. 2010;5:e14076. doi: 10.1371/journal.pone.0014076. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Hu X, Yang D, Zimmerman M, Liu F, Yang J, Kannan S, et al. IRF8 regulates acid ceramidase expression to mediate apoptosis and suppresses myelogeneous leukemia. Cancer Res. 2011;71:2882–91. doi: 10.1158/0008-5472.CAN-10-2493. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.McGough JM, Yang D, Huang S, Georgi D, Hewitt SM, Rocken C, et al. DNA methylation represses IFN-gamma-induced and signal transducer and activator of transcription 1-mediated IFN regulatory factor 8 activation in colon carcinoma cells. Mol Cancer Res. 2008;6:1841–51. doi: 10.1158/1541-7786.MCR-08-0280. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Minato H, Matsumoto M, Katayama T. Studies on the metabolites of Verticillium sp. structures of Verticillins A, B, and C. J Chem Soc Perkin 1. 1973;17:1819–25. doi: 10.1039/p19730001819. [DOI] [PubMed] [Google Scholar]
  • 25.Bareford MD, Park MA, Yacoub A, Hamed HA, Tang Y, Cruickshanks N, et al. Sorafenib enhances pemetrexed cytotoxicity through an autophagy -dependent mechanism in cancer cells. Cancer Res. 2011 doi: 10.1158/0008-5472.CAN-11-0898. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Voelkel-Johnson C, Hannun YA, El-Zawahry A. Resistance to TRAIL is associated with defects in ceramide signaling that can be overcome by exogenous C6-ceramide without requiring down-regulation of cellular FLICE inhibitory protein. Mol Cancer Ther. 2005;4:1320–7. doi: 10.1158/1535-7163.MCT-05-0086. [DOI] [PubMed] [Google Scholar]
  • 27.DiPaola RS. To arrest or not to G(2)-M Cell-cycle arrest: commentary re: A. K. Tyagi et al., Silibinin strongly synergizes human prostate carcinoma DU145 cells to doxorubicin-induced growth inhibition, G(2)-M arrest, and apoptosis. Clin. cancer res. 8:3512–3519, 2002. Clin Cancer Res. 2002;8:3311–4. [PubMed] [Google Scholar]
  • 28.Weisenberger DJ, Siegmund KD, Campan M, Young J, Long TI, Faasse MA, et al. CpG island methylator phenotype underlies sporadic microsatellite instability and is tightly associated with BRAF mutation in colorectal cancer. Nat Genet. 2006;38:787–93. doi: 10.1038/ng1834. [DOI] [PubMed] [Google Scholar]
  • 29.Yang D, Thangaraju M, Greeneltch K, Browning DD, Schoenlein PV, Tamura T, et al. Repression of IFN regulatory factor 8 by DNA methylation is a molecular determinant of apoptotic resistance and metastatic phenotype in metastatic tumor cells. Cancer Res. 2007;67:3301–9. doi: 10.1158/0008-5472.CAN-06-4068. [DOI] [PubMed] [Google Scholar]
  • 30.Wu GS. TRAIL as a target in anti-cancer therapy. Cancer Lett. 2009;285:1–5. doi: 10.1016/j.canlet.2009.02.029. [DOI] [PubMed] [Google Scholar]
  • 31.Xu J, Zhou JY, Wei WZ, Wu GS. Activation of the Akt survival pathway contributes to TRAIL resistance in cancer cells. PLoS One. 2010;5:e10226. doi: 10.1371/journal.pone.0010226. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Kauh J, Fan S, Xia M, Yue P, Yang L, Khuri FR, et al. c-FLIP degradation mediates sensitization of pancreatic cancer cells to TRAIL-induced apoptosis by the histone deacetylase inhibitor LBH589. PLoS One. 2010;5:e10376. doi: 10.1371/journal.pone.0010376. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Wang S, Ren W, Liu J, Lahat G, Torres K, Lopez G, et al. TRAIL and doxorubicin combination induces proapoptotic and antiangiogenic effects in soft tissue sarcoma in vivo. Clin Cancer Res. 2010;16:2591–604. doi: 10.1158/1078-0432.CCR-09-2443. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Sanchez-Perez T, Ortiz-Ferron G, Lopez-Rivas A. Mitotic arrest and JNK-induced proteasomal degradation of FLIP and Mcl-1 are key events in the sensitization of breast tumor cells to TRAIL by antimicrotubule agents. Cell Death Differ. 2010;17:883–94. doi: 10.1038/cdd.2009.176. [DOI] [PubMed] [Google Scholar]
  • 35.Shankar S, Ganapathy S, Srivastava RK. Sulforaphane enhances the therapeutic potential of TRAIL in prostate cancer orthotopic model through regulation of apoptosis, metastasis, and angiogenesis. Clin Cancer Res. 2008;14:6855–66. doi: 10.1158/1078-0432.CCR-08-0903. [DOI] [PubMed] [Google Scholar]
  • 36.Kim H, Kim EH, Eom YW, Kim WH, Kwon TK, Lee SJ, et al. Sulforaphane sensitizes tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-resistant hepatoma cells to TRAIL-induced apoptosis through reactive oxygen species-mediated up-regulation of DR5. Cancer Res. 2006;66:1740–50. doi: 10.1158/0008-5472.CAN-05-1568. [DOI] [PubMed] [Google Scholar]
  • 37.Kallifatidis G, Rausch V, Baumann B, Apel A, Beckermann BM, Groth A, et al. Sulforaphane targets pancreatic tumour-initiating cells by NF-kappaB-induced antiapoptotic signalling. Gut. 2009;58:949–63. doi: 10.1136/gut.2008.149039. [DOI] [PubMed] [Google Scholar]
  • 38.Chen Q, Ganapathy S, Singh KP, Shankar S, Srivastava RK. Resveratrol induces growth arrest and apoptosis through activation of FOXO transcription factors in prostate cancer cells. PLoS One. 2010;5:e15288. doi: 10.1371/journal.pone.0015288. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Ganapathy S, Chen Q, Singh KP, Shankar S, Srivastava RK. Resveratrol enhances antitumor activity of TRAIL in prostate cancer xenografts through activation of FOXO transcription factor. PLoS One. 2010;5:e15627. doi: 10.1371/journal.pone.0015627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Zhang YX, Chen Y, Guo XN, Zhang XW, Zhao WM, Zhong L, et al. 11,11′-dideoxy-verticillin: a natural compound possessing growth factor receptor tyrosine kinase-inhibitory effect with anti-tumor activity. Anticancer Drugs. 2005;16:515–24. doi: 10.1097/00001813-200506000-00007. [DOI] [PubMed] [Google Scholar]
  • 41.Chu M, Truumees I, Rothofsky ML, Patel MG, Gentile F, Das PR, et al. Inhibition of c-fos proto-oncogene induction by Sch 52900 and Sch 52901, novel diketopiperazine produced by Gliocladium sp. J Antibiot (Tokyo) 1995;48:1440–5. doi: 10.7164/antibiotics.48.1440. [DOI] [PubMed] [Google Scholar]
  • 42.Chen G, Ray R, Dubik D, Shi L, Cizeau J, Bleackley RC, et al. The E1B 19K/Bcl-2-binding protein Nip3 is a dimeric mitochondrial protein that activates apoptosis. J Exp Med. 1997;186:1975–83. doi: 10.1084/jem.186.12.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Bristow N, Burton TR, Henson ES, Ong-Justiniano C, Brown M, Gibson SB. Truncated forms of BNIP3 act as dominant negatives inhibiting hypoxia-induced cell death. Biochim Biophys Acta. 2011;1812:302–11. doi: 10.1016/j.bbadis.2010.11.013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Burton TR, Gibson SB. The role of Bcl-2 family member BNIP3 in cell death and disease: NIPping at the heels of cell death. Cell Death Differ. 2009;16:515–23. doi: 10.1038/cdd.2008.185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Daido S, Kanzawa T, Yamamoto A, Takeuchi H, Kondo Y, Kondo S. Pivotal role of the cell death factor BNIP3 in ceramide-induced autophagic cell death in malignant glioma cells. Cancer Res. 2004;64:4286–93. doi: 10.1158/0008-5472.CAN-03-3084. [DOI] [PubMed] [Google Scholar]
  • 46.Bandyopadhyay S, Zhan R, Wang Y, Pai SK, Hirota S, Hosobe S, et al. Mechanism of apoptosis induced by the inhibition of fatty acid synthase in breast cancer cells. Cancer Res. 2006;66:5934–40. doi: 10.1158/0008-5472.CAN-05-3197. [DOI] [PubMed] [Google Scholar]
  • 47.Mahon PC, Baril P, Bhakta V, Chelala C, Caulee K, Harada T, et al. S100A4 contributes to the suppression of BNIP3 expression, chemoresistance, and inhibition of apoptosis in pancreatic cancer. Cancer Res. 2007;67:6786–95. doi: 10.1158/0008-5472.CAN-07-0440. [DOI] [PubMed] [Google Scholar]
  • 48.Kim EH, Yoon MJ, Kim SU, Kwon TK, Sohn S, Choi KS. Arsenic trioxide sensitizes human glioma cells, but not normal astrocytes, to TRAIL-induced apoptosis via CCAAT/enhancer-binding protein homologous protein-dependent DR5 up-regulation. Cancer Res. 2008;68:266–75. doi: 10.1158/0008-5472.CAN-07-2444. [DOI] [PubMed] [Google Scholar]
  • 49.Bacon AL, Fox S, Turley H, Harris AL. Selective silencing of the hypoxia-inducible factor 1 target gene BNIP3 by histone deacetylation and methylation in colorectal cancer. Oncogene. 2007;26:132–41. doi: 10.1038/sj.onc.1209761. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS324813-supplement-1.tif (170.2KB, tif)
2
NIHMS324813-supplement-2.tif (303.4KB, tif)
3
NIHMS324813-supplement-3.doc (30.5KB, doc)
4
NIHMS324813-supplement-4.doc (39.5KB, doc)
5
NIHMS324813-supplement-5.doc (26KB, doc)
6
NIHMS324813-supplement-6.tif (177.6KB, tif)
7
NIHMS324813-supplement-7.tif (173.9KB, tif)
8
NIHMS324813-supplement-8.tif (274.7KB, tif)
9
NIHMS324813-supplement-9.tif (251KB, tif)

RESOURCES