PLoS One. 2016 Feb 17;11(2):e0148685. doi: 10.1371/journal.pone.0148685. eCollection 2016.
Tetra-O-Methyl Nordihydroguaiaretic Acid Broadly Suppresses Cancer Metabolism and Synergistically Induces Strong Anticancer Activity in Combination with Etoposide, Rapamycin and UCN-01.
Kotohiko Kimura1, Ru Chih C. Huang1*.
1 Department of Biology, Johns Hopkins University, Baltimore, Maryland, United States of America
*Corresponding author (email@example.com)
The ability of Tetra-O-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.
What is tetra-O-methyl nordihydroguaiaretic acid (M4N)?
M4N, also known as EM1421 and terameprocol, is a novel, semi-synthetic derivative of a naturally occurring plant lignan. Our laboratory previously reported that M4N possessed antiviral and anti-cancer activities [1-4]. We also showed that one of principal pharmacological activities of M4N was to inhibit the activity of SP1 transcription factor by binding to GC-rich regions (SP1 consensus sequences) of gene promoters competitively with SP1 . Phase I trials data so far indicated that M4N possesses potential anticancer properties [5, 6]. For non-aggressive cancers, when in situ drug concentrations can be kept high, M4N is an effective anticancer agent. However, when the amount of M4N in the tumor is below the therapeutic level, rapid cancer cell death in vivo is often not achieved, and this has limited its effectiveness in suppressing more aggressive cancers. One of the most frequently attempted strategies to increase anticancer efficacy of chemotherapy drugs is combination treatment with one or more appropriately selected drugs. We has examined if it was possible to increase anticancer activity of M4N by using the drug in combination with other anticancer drugs commercially available. An important finding from the clinical trials with M4N was that patients were able to tolerate high doses of M4N with minimal side-effects [5, 6], which made this drug very suitable for being use in multidrug treatments.
The animal experiments proved that M4N combination treatments really worked great!
For the reason described above, our laboratory explored a possible usage of M4N in combination with different anticancer drugs [7, 8, 9]. To our amazement, as described in our PLoS One e0148685 article, it was found that M4N had unusually strong activity to help other anticancer drugs (Etoposide, Rapamycin, UCN-01, and Ly294002+Rottlerin) to work much better for prostatic cancers in tissue culture experiments and mouse xenograft experiments  (Fig. 1A and B). This effect of synergistic improvement of anticancer efficacy by M4N was also found in M4N combination treatment with Sorafenib for treating various cancers from different origins: mammary, pancreatic, and hepatic cancers [8, 9] (Fig. 1C-E).
In all our xenograft experiments we administered M4N and another anticancer drug in combination to mice at a moderate dosage each daily for a few weeks since this method was shown to be effective to maintain a reasonable blood concentration of the anticancer drugs. Recently it was shown that the prolonged treatment of hepatic cancer patients with Sorafenib after radiation therapy was effective , which supported our methodology. Using such M4N combinations [7, 8, 9], our laboratory has been experimentally successful in eliminating the entire population of heterogeneous cancer cells in solid tumors and stopping the growth of metastasis without side effects to the host. Xenograft mice were able to live beyond a hundred days while non-treated, or single drug treated mice all died around 30 days. Plans for phase II human clinical trials using a short term, continuous intravenous and oral drug treatment study design are in progress.
The importance of M4N activity to suppress cancer metabolism in facilitating anticancer effect by combination treatments.
Since Otto Warburg (a Nobel prize winner for discovering glycolysis mechanisms) proposed in 1956 that cancer cells used metabolic biochemical reactions differently from normal cells , there have been many efforts to utilize the difference between cancer and normal cells in metabolism to preferentially eliminate cancer cells. We showed by gas chromatography/mass spectroscopy assay (performed by Metabolon Inc.) that M4N could suppress cancer metabolisms in various biochemical reactions including glycolysis, TCA cycle, amino acid and lipid metabolism, and autophagy (a cellular mechanism to recycle unwanted cellular components for necessary cellular activities) (Fig. 2A).
Since cancer cells require enormous amounts of metabolic energy and nutrients to grow rapidly, the suppressive effect of M4N on multiple biochemical mechanisms related to cellular metabolisms have profound negative effects on biological activities of cancer cells. In fact we found that M4N combination treatment induced strong cellular stress reactions and reactive oxygen generation in cancer cells. Thus the negative effect on metabolism induced by M4N is among the main reasons why M4N combination treatment is so effective.
Based on RNA deep sequence analysis, we examined the genes whose expressions were modulated by M4N in common with two cell lines, LNCaP (prostatic) and AsPC-1 (pancreatic). We found that 67 genes were commonly modulated by M4N and did the cluster analysis by GoMiner  for these genes to see if there were any particular cellular functions associated with the genes. The cluster analysis picked up three clusters which represented ‘cell death-related genes’, ‘metabolism-related genes’, and ‘stress-related genes’ (Fig. 3). Thus it was concluded that M4N modulated the expressions of genes which were related to the aforementioned activity of M4N to suppress cancer metabolism as well as to the additional activities of the drug to induce stress and cell death stimulus.
Mitochondria, the epicenter of cellular metabolic mechanisms, is a major target of M4N.
We also showed in our PLoS One e0148685 article that the mitochondria, the very important organelle for all sorts of metabolic activity, was a major target of pharmacological activity of M4N. For instance we found that ATP cellular contents were suppressed by M4N (Fig. 4A; A majority of ATP is generated by mitochondrial electron transport system (METS) which is constituted from complex I/II/III/IV). We also found that M4N induced hyperpolarization of mitochondrial membrane potential (DY) (Fig 4B; When DY is hyperpolarized, METS does not work well and ATP generation is reduced) . We also found that the expression of NDFUS1 , a major component of complex I in METS, was suppressed by M4N. Other than that we found that M4N suppressed the expression of PTEN, a protein known to be involved in mitochondrial metabolism through complex IV (Fig. 4C)  and that M4N also induced the expression of ATF4 and SESN2, which were known to be activated by mitochondrial dysfunction . The data overall clearly showed that M4N induced mitochondrial dysfunction and suppressed the function of METS.
One of the interesting findings from our study on cancer metabolism was that M4N had an activity to preferentially suppress the cellular contents of aspartate among all the amino acids (Fig. 2B). Recently it was shown that even when METS was inhibited (thus cellular respiration was suppressed), the cells could survive and grow as far as a plenty of aspartate was supplied to the cells [17, 18]. As described above, M4N has an activity to suppress METS. Therefore under the influence of M4N, the cells need to rely on aspartate to survive. However, since M4N has an activity to reduce cellular aspartate contents as well as suppress activity of METS, the cells treated with M4N lack much capability to survive and grow (Fig. 4D). Since cancer cells demand far more materials and energy for their strong appetites than normal cells, this double whammy on the mitochondrial metabolism induced by M4N thus causes much stronger adverse effects against cancer cells than normal cells.
A future of M4N.
M4N is potentially a game changer in multi-drug anticancer chemotherapy by its activity to strongly facilitate anticancer efficacy of various drugs which are either already in commercially use or in developmental stages. Multiple clinical trials to study anticancer efficacy of M4N combination treatments are very imminent considering the vast amount of cancer cases that are in need of effective treatment.
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