Abstract
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.
Highlights
We have previously reported that tetra-O-methyl nordihydroguaiaretic acid (M4N), known as EM1421 and terameprocol, possessed antiviral and anti-cancer activities [1,2,3,4] and that M4N could be potentially useful as an anticancer drug
As a pilot experiment to determine the most appropriate drugs to be used in combination with M4N for clinical applications, we selected three anticancer drugs, namely Etoposide [17], Rapamycin [18], and UCN-01 [19] and evaluated anticancer efficacy of combination treatments with these drugs in LNCaP cells using both the transferase dUTP nick end labeling (TUNEL) assay and Trypan blue exclusion assay
The amount of cell death detected by Trypan blue exclusion assay far exceeded that detected by TUNEL assay (Fig 1A), indicating that TUNELnegative cell death played a major role in the synergistic cell death, especially in M4N combination treatment with Rapamycin
Summary
We have previously reported that tetra-O-methyl nordihydroguaiaretic acid (M4N), known as EM1421 and terameprocol, possessed antiviral and anti-cancer activities [1,2,3,4] and that M4N could be potentially useful as an anticancer drug. In addition Wang et al showed that the overexpression of SP1 facilitated development and progression of gastric cancer [5], further suggesting that suppression of SP1-regulated transcription could induce anticancer activities. Other than these studies on transcription inhibition, there have been but a few studies related to the pharmacological activities of M4N. Because of the similarity in molecular structure between M4N and NDGA (Fig 1A), these data regarding NDGA are relevant to our studies on M4N Overall, these previous findings of M4N seem to indicate that M4N might have multiple pharmacological activities
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