Tetra-<i>O</i>-Methyl Nordihydroguaiaretic Acid Broadly Suppresses Cancer Metabolism and Synergistically Induces Strong Anticancer Activity in Combination with Etoposide, Rapamycin and UCN-01

<div><p>The ability of Tetra-<i>O</i>-methyl nordihydroguaiaretic acid (M₄N) to induce rapid cell death in combination with Etoposide, Rapamycin, or UCN-01 was examined in LNCaP cells, both in cell culture and animal experiments. Mice treated with M₄N drug combinations with either Etoposide or Rapamycin showed no evidence of tumor and had a 100% survival rate 100 days after tumor implantation. By comparison all other vehicles or single drug treated mice failed to survive longer than 30 days after implantation. This synergistic improvement of anticancer effect was also confirmed in more than 20 cancer cell lines. In LNCaP cells, M₄N was found to reduce cellular ATP content, and suppress NDUFS1 expression while inducing hyperpolarization of mitochondrial membrane potential. M₄N-treated cells lacked autophagy with reduced expression of BNIP3 and ATG5. To understand the mechanisms of this anticancer activity of M₄N, the effect of this drug on three cancer cell lines (LNCaP, AsPC-1, and L428 cells) was further examined via transcriptome and metabolomics analyses. Metabolomic results showed that there were reductions of 26 metabolites essential for energy generation and/or production of cellular components in common with these three cell lines following 8 hours of M₄N treatment. Deep RNA sequencing analysis demonstrated that there were sixteen genes whose expressions were found to be modulated following 6 hours of M₄N treatment similarly in these three cell lines. Six out of these 16 genes were functionally related to the 26 metabolites described above. One of these up-regulated genes encodes for CHAC1, a key enzyme affecting the stress pathways through its degradation of glutathione. In fact M₄N was found to suppress glutathione content and induce reactive oxygen species production. The data overall indicate that M₄N has profound specific negative impacts on a wide range of cancer metabolisms supporting the use of M₄N combination for cancer treatments.</p></div

Analysis of the effect of M₄N on metabolism.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148685#pone.0148685.g004" target="_blank">Fig 4</a> shows the metabolites whose contents were suppressed by M₄N treatment in common with three cell lines (LNCaP, AsPC-1, and L428). The shortage of metabolites induced by M₄N should negatively affect the metabolic pathways in which these metabolites were involved. Based on this premise, the data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148685#pone.0148685.g004" target="_blank">Fig 4</a> was analyzed and this figure was drawn. On the right side, the names of metabolites from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148685#pone.0148685.g004" target="_blank">Fig 4</a> were shown in square boxes. In the middle, the metabolic pathways related to these metabolites were shown in elliptical circles. The relationships between metabolites and their related metabolic pathways were depicted by thin arrows. On the left side, the three major functions of these metabolic pathways were shown in circles. The relationships between metabolic pathways and their related major functions were depicted by thick arrows. An asterisk indicates that ATP concentration was measured by luminescent-based enzymatic assay (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148685#pone.0148685.g006" target="_blank">Fig 6A</a>). The analysis showed that M₄N suppressed various metabolic pathways in both energy generation and cellular component production. Additionally the analysis showed that M₄N suppressed ROS scavenging mechanisms as well.</p

Effect of combination treatments on cell death-related cellular functions.

<p><b>A-B:</b> Analysis of caspase-dependent cell death mechanisms induced by combination treatments of M₄N with Etoposide, Rapamycin, or UCN-01. <b>Aa & Ab:</b> Cleavage of caspase-3, -4, -7, and -9 along with poly-ADP ribose polymerase (PARP) in LNCaP cells treated by the combination treatments for 17 h. β-Actin was used as a control. <b>B:</b> Caspase-7 enzymatic activity in LNCaP cells treated by the combination treatments for 13 h. Data are presented as mean±SD in triplicate. <b>A & B:</b> C: control, E: Etoposide (10 μM), Ra: Rapamycin (10 μM), Ra30μM: Rapamycin (30 μM), U: UCN-01 (2 μM), M4N: M₄N (80 μM), and Casp: Caspase. <b>C:</b> Effect of combination treatments on mitochondrial membrane potential (ΔΨ_m). LNCaP cells were treated with M₄N (80 μM) in combination with Etoposide (10 μM), Rapamycin (10 μM), or UCN-01 (2 μM) for 4 h. The ΔΨ_m was measured using JC-1 dye. The image shows the ratios obtained by dividing the intensity at 568 nm-excitation light (J-aggregates) by that at 488 nm-excitation light (J-aggregates + monomer). The color bar is shown on the right side of the figure (H: high, L: low ratio).</p

A schema depicting the mechanisms about how M₄N combination treatments work.

<p>Processes depicted by thick arrows (in the combination treatment) operate better than those by thin arrows (in M₄N treatment only).</p

Metabolism-related data from combined results of DNA microarray analysis about the effects of M₄N treatment in three different cell lines (LNCaP, AsPc-1, and L428).

<p>Metabolism-related data from combined results of DNA microarray analysis about the effects of M₄N treatment in three different cell lines (LNCaP, AsPc-1, and L428).</p

Suppressive effect of M₄N on autophagy.

<p><b>A:</b> Effect of M₄N on autophagy in LNCaP cells. The expression of LC3B-I and II was examined by the western blotting in LNCaP cells treated with combination treatments of M₄N with etoposide, rapamycin, or UCN-01 for 18 h in the presence of bafilomycin A₁ (100 nM), an autophagosome degradation inhibitor. Bafilomycin A₁ was added to measure the net activity of autophagy. The concentrations of M₄N, etoposide, rapamycin, and UCN-01 were 80, 20, 20, and 5 μM, respectively. C: Control, E: etoposide, Ra: rapamycin, U: UCN-01. <b>B:</b> The mRNA expression of <i>BNIP3</i> gene, examined by northern blotting, in LNCaP cells treated with M₄N (80 μM) under normoxic or hypoxic condition for 2 or 6 h. Hyp: hypoxic condition. <b>C:</b> The expression of BNIP3 and BNIP3L, examined by the western blotting, in LNCaP cells treated with M₄N (80 μM) under normoxic or hypoxic condition for 6 or 18 h. <b>D:</b> The expression of ATG5, examined by the western blotting, in LNCaP cells treated with M₄N (80 μM) for 18 h. β-Actin was used as a control.</p

Effect of M₄N on nucleic acid metabolism in LNCaP, AsPC-1, and L428 cells.

<p>Schemas for purine (<b>A</b>) and pyrimidine (<b>B</b>)-related metabolic pathways. The raw data of the metabolite assay is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148685#pone.0148685.s003" target="_blank">S1 Table</a>. The selection of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148685#pone.0148685.s003" target="_blank">S1 Table</a> can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148685#pone.0148685.g004" target="_blank">Fig 4</a>. The upward pointing arrows indicate that the contents of the metabolites associated with these arrows were significantly (p≤0.05) induced by M₄N in at least two out of three cell lines (LNCaP, AsPC-1, and L428 cells) under the additional condition that the effect of M₄N on the contents of these metabolites in the third cell line was an induction without statistical significant difference, no significant change, or not examined. Meanwhile the downward pointing arrows indicate that the contents of the metabolites associated with these arrows were significantly (p≤0.05) suppressed by M₄N in at least two out of three cell lines (LNCaP, AsPC-1, and L428 cells) under the additional condition that the effect of M₄N on the contents of these metabolites in the third cell line was a suppression without statistical significant difference, no significant change, or not examined. The exception to these rules was xanthine (whose content was induced by M₄N in all three cell lines but the difference was statistically significant only in L428 cells). The effect of M₄N on ATP content in the whole cells is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148685#pone.0148685.g006" target="_blank">Fig 6A</a>. PRPP: phosphoribosyl pyrophosphate, PPi: pyrophosphate, Pi: phosphate.</p

Interaction and Collaboration of SP1, HIF-1, and MYC in Regulating the Expression of Cancer-Related Genes to Further Enhance Anticancer Drug Development

Specificity protein 1 (SP1), hypoxia-inducible factor 1 (HIF-1), and MYC are important transcription factors (TFs). SP1, a constitutively expressed housekeeping gene, regulates diverse yet distinct biological activities; MYC is a master regulator of all key cellular activities including cell metabolism and proliferation; and HIF-1, whose protein level is rapidly increased when the local tissue oxygen concentration decreases, functions as a mediator of hypoxic signals. Systems analyses of the regulatory networks in cancer have shown that SP1, HIF-1, and MYC belong to a group of TFs that function as master regulators of cancer. Therefore, the contributions of these TFs are crucial to the development of cancer. SP1, HIF-1, and MYC are often overexpressed in tumors, which indicates the importance of their roles in the development of cancer. Thus, proper manipulation of SP1, HIF-1, and MYC by appropriate agents could have a strong negative impact on cancer development. Under these circumstances, these TFs have naturally become major targets for anticancer drug development. Accordingly, there are currently many SP1 or HIF-1 inhibitors available; however, designing efficient MYC inhibitors has been extremely difficult. Studies have shown that SP1, HIF-1, and MYC modulate the expression of each other and collaborate to regulate the expression of numerous genes. In this review, we provide an overview of the interactions and collaborations of SP1, HIF1A, and MYC in the regulation of various cancer-related genes, and their potential implications in the development of anticancer therapy

Metabolites whose cellular contents were suppressed by M₄N treatment in common with three cell lines (LNCaP, AsPC-1, and L428).

<p>LNCaP, AsPC-1 and L428 cells were treated with M₄N (80μM) for 8 h and metabolite contents of the samples were measured by LC/GC mass spectroscopy by Metabolon (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148685#pone.0148685.s003" target="_blank">S1 Table</a>). Among the metabolites examined, only the metabolites whose contents were significantly (p≤0.05) suppressed by M₄N in at least two out of three cell lines were selected and listed here under the additional condition that the effect of M₄N on the contents of metabolites in the third cell line was a suppression, no significant change, or not examined. 'M/C' indicates the ratio of metabolite contents for the samples treated with M₄N vs. control. N.A. indicates 'data not available'. The exceptions for these rules were ADP and UDP-glucose (whose contents were both suppressed by M₄N in two of the three cell lines but the difference was statistically significant only in one of the two cell lines. In the third cell line the data was not available). The numbers for the ratio shaded in green indicate that metabolite contents were statistically smaller in the treated samples than the control while those without shades indicate that there was not a statistical difference between the control and the treated samples.</p