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.

Tetra-O-methyl-nordihydroguaiaretic acid inhibits energy metabolism and synergistically induces anticancer effects with temozolomide on LN229 glioblastoma tumors implanted in mice while preventing obesity in normal mice that consume high-fat diets.

Tetra-O-methyl-nordihydroguaiaretic acid (terameprocol; M4N), a global transcription inhibitor, in combination with a second anticancer drug induces strong tumoricidal activity and has the ability to suppress energy metabolism in cultured cancer cells. In this study, we showed that after continuous oral consumption of high-fat (HF) diets containing M4N, the M4N concentration in most of the organs in mice reached ~1 µM (the M4N concentration in intestines and fat pads was as high as 20-40 µM) and treatment with the combination of M4N with temozolomide (TMZ) suppressed glycolysis and the tricarboxylic acid cycle in LN229 human glioblastoma implanted in xenograft mice. Combination treatment of M4N with TMZ also reduced the levels of lactate dehydrogenase A (LDHA), a key enzyme for glycolysis; lactate, a product of LDHA-mediated enzymatic activity; nicotinamide phosphoribosyltransferase, a rate-limiting enzyme for nicotinamide adenine dinucleotide plus hydrogen (NADH)/NAD+ salvage pathway; and NAD+, a redox electron carrier essential for energy metabolism. It was also shown that M4N suppressed oxygen consumption in cultured LN229 cells, indicating that M4N inhibited oxidative phosphorylation. Treatment with M4N and TMZ also decreased the level of hypoxia-inducible factor 1A, a major regulator of LDHA, under hypoxic conditions. The ability of M4N to suppress energy metabolism resulted in induction of the stress-related proteins activating transcription factor 4 and cation transport regulator-like protein 1, and an increase in reactive oxygen species production. In addition, the combination treatment of M4N with TMZ reduced the levels of oncometabolites such as 2-hydroxyglutarate as well as the aforementioned lactate. M4N also induced methylidenesuccinic acid (itaconate), a macrophage-specific metabolite with anti-inflammatory activity, in tumor microenvironments. Meanwhile, the ability of M4N to suppress energy metabolism prevented obesity in mice consuming HF diets, indicating that M4N has beneficial effects on normal tissues. The dual ability of combination treatment with M4N to suppress both energy metabolism and oncometabolites shows that it is potentially an effective therapy for cancer.

In vivo amelioration of endogenous antitumor autoantibodies via low-dose P4N through the LTA4H/activin A/BAFF pathway.

Cancer progression is associated with the development of antitumor autoantibodies in patients' sera. Although passive treatment with antitumor antibodies has exhibited remarkable therapeutic efficacy, inhibitory effects on tumor progression by endogenous antitumor autoantibodies (EAAs) have been limited. In this study, we show that P4N, a derivative of the plant lignan nordihydroguaiaretic acid (NDGA), enhanced the production of EAAs and inhibited tumor growth at low noncytotoxic concentrations via its immunoregulatory activity. Intratumoral injection of P4N improved the quantity and quality of EAAs, and passive transfer of P4N-induced EAAs dramatically suppressed lung metastasis formation and prolonged the survival of mice inoculated with metastatic CT26 tumor cells. P4N-induced EAAs specifically recognized two surface antigens, 78-kDa glucose-regulated protein (GRP78) and F1F0 ATP synthase, on the plasma membrane of cancer cells. Additionally, P4N treatment led to B-cell proliferation, differentiation to plasma cells, and high titers of autoantibody production. By serial induction of autocrine and paracrine signals in monocytes, P4N increased B-cell proliferation and antibody production via the leukotriene A4 hydrolase (LTA4H)/activin A/B-cell activating factor (BAFF) pathway. This mechanism provides a useful platform for studying and seeking a novel immunomodulator that can be applied in targeting therapy by improving the quantity and quality of the EAAs.

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

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.

Hepatocellular carcinoma targeting agents: conjugates of nitroimidazoles with trimethyl nordihydroguaiaretic acid.

Cancer has been a primary global health issue for decades, with hepatocellular carcinoma (HCC) resulting in more than half a million new cases annually. With survival rates as low as <5% after five years, it remains a poorly treated cancer. Nordihydroguaiaretic acid (NDGA), an antioxidant, was previously proven effective against cancer cells. Nitroimidazole derivatives convert into reactive compounds under hypoxic conditions. In this study, eight methylated NDGAs containing a 2- or 4-nitroimidazole moiety were synthesized as leads against HCC. Four of these conjugates, possessing a poly(ethylene glycol) tether, had superior aqueous solubility. These four NDGA-nitroimidazole conjugates were found to inhibit the proliferation HCC Hep3B cells with IC50 values between 10 and 15 µM. Furthermore, nitroimidazole-conjugated NDGA derivatives exhibit better antiproliferative activity under hypoxic conditions.

Glycosylated nordihydroguaiaretic acids as anti-cancer agents.

Three perglycosylated nordihydroguaiaretic acids (NDGA) were synthesized through the Huiseng 1,3-dipolar cycloaddition reaction. These sugar-NDGA conjugates containing triazole-linkages possessed good solubility in water. NDGA-(triazol-galactose)(4) (12b) and NDGA-(triazol-glucose)(4) (12c) were found to act as inhibitors against human hepatocellular carcinoma Hep3B cells in culture.

Immobilization of growth factors on collagen scaffolds mediated by polyanionic collagen mimetic peptides and its effect on endothelial cell morphogenesis.

Angiogenesis, a morphogenic event endothelial cells (ECs) undergo in response to 3-D environmental triggers, is critical to the survival and ultimate functional capacity of engineered tissue constructs. Here we present a new collagen mimetic peptide (CMP) architecture consisting of multiple anionic charges at the peptide's N-terminus designed to attract growth factors by charge-charge interactions and bind to collagen by CMP-collagen interaction. The anionic CMPs exhibited specific binding affinity to type I collagen substrates while attracting vascular endothelial growth factors (VEGFs), which led to enhanced morphological features of ECs, indicative of tubulogenesis. The results show that these new CMPs could be used to direct proliferation and differentiation of cells in collagen scaffolds by localization and sustained delivery of growth factors and other morphogens.

New nordihydroguaiaretic acid derivatives as anti-HIV agents.

Reaction of nordihydroguaiaretic acid with various alkyl chloride, 1-piperidinecarbonyl chloride, methyl chloroformate, or 1,1'-carbonyldiimidazole under alkaline conditions produced the corresponding phenol ethers, carbamates and carbonates, respectively, in 67-83% yields. Among these derivatives, the nitrogen-containing compounds were converted to the corresponding hydrochloride salts. Having good solubility, these NDGA derivatives were found stable in aqueous solution. These new compounds exerted appealing activity against HIV Tat-regulated transactivation in human epithelial cells. The most potent compound meso-2,3-dimethyl-1,4-bis(3,4-[2-(piperdino)ethoxyphenyl])butane tetrakishydrochloride salt (5b) showed IC(50) value of 0.88 microM.

A microfluidic-FCS platform for investigation on the dissociation of Sp1-DNA complex by doxorubicin.

The transcription factor (TF) Sp1 is a well-known RNA polymerase II transcription activator that binds to GC-rich recognition sites in a number of essential cellular and viral promoters. In addition, direct interference of Sp1 binding to DNA cognate sites using DNA-interacting compounds may provide promising therapies for suppression of cancer progression and viral replication. In this study, we present a rapid, sensitive and cost-effective evaluation of a GC intercalative drug, doxorubicin (DOX), in dissociating the Sp1-DNA complex using fluorescence correlation spectroscopy (FCS) in a microfluidic system. FCS allows assay miniaturization without compromising sensitivity, making it an ideal analytical method for integration of binding assays into high-throughput, microfluidic platforms. A polydimethylsiloxane (PDMS)-based microfluidic chip with a mixing network is used to achieve specific drug concentrations for drug titration experiments. Using FCS measurements, the IC50 of DOX on the dissociation of Sp1-DNA complex is estimated to be 0.55 microM, which is comparable to that measured by the electrophoretic mobility shift assay (EMSA). However, completion of one drug titration experiment on the proposed microfluidic-FCS platform is accomplished using only picograms of protein and DNA samples and less than 1 h total assay time, demonstrating vast improvements over traditional ensemble techniques.

Survivin-dependent and -independent pathways and the induction of cancer cell death by tetra-O-methyl nordihydroguaiaretic acid.

The inhibitor of apoptosis protein (IAP) family encodes a group of Baculovirus IAP repeat domain (BIR)-containing proteins that suppress apoptosis. Some of the IAPs, survivin and XIAP in particular, are differentially overexpressed in many types of human cancer and are deemed attractive anticancer targets. Here we review the regulation of survivin expression and survivin's functions in both normal and cancerous cells, and some of the current survivin-targeted cancer therapy. We further discuss the possible mechanisms of tetra-O-methyl nordihydroguaretic acid (M(4)N), a global transcription inhibitor, in the induction of cancer cell death in tumors via survivin-dependent and -independent pathways.

Reversal of multidrug resistance by two nordihydroguaiaretic acid derivatives, M4N and maltose-M3N, and their use in combination with doxorubicin or paclitaxel.

PURPOSE: Multidrug resistance (MDR) continues to be a major obstacle for successful anticancer therapy. One of the principal factors implicated in MDR is the over expression of P-glycoprotein (Pgp), the product of the MDR1 gene. METHODS: Here we explore the possibility of using the transcription inhibitor tetra-O-methyl nordihydroguaiaretic acid (M4N) to inhibit Sp1-regulated MDR1 gene expression and restore doxorubicin and paclitaxel sensitivity to multidrug resistant human cancer cells in vitro and in vivo. RESULTS: We found that M4N acted synergistically with doxorubicin and paclitaxel in inhibiting the growth of the cells in culture allowing significant dose reductions of both drugs. We observed no such synergism when M4N was used in combination with cisplatin, another chemotherapeutic agent, but not a Pgp substrate, as analyzed by the combination index and isobologram methods. Analysis of MDR1 mRNA and Pgp levels revealed that at sublethal doses, M4N inhibited MDR1 gene expression in the multidrug resistant NCI/ADR-RES cells and reversed the MDR phenotype as measured by Rhodamine-123 retention. In addition, M4N was found to inhibit doxorubicin-induced MDR1 gene expression in drug sensitive MCF-7 breast cancer cells. CONCLUSIONS: M4N and maltose-tri-O-methyl nordihydroguaiaretic acid (maltose-M3N), a water-soluble derivative of NDGA, were also able to reverse the MDR phenotype of the tumor cells in a xenograft model system and combination therapy with M4N or maltose-M3N and paclitaxel was effective at inhibiting growth of these tumors in nude mice.

Systemic treatment with tetra-O-methyl nordihydroguaiaretic acid suppresses the growth of human xenograft tumors.

PURPOSE: We have previously shown that the transcriptional inhibitor tetra-O-methyl nordihydroguaiaretic acid (M4N) induces growth arrest in tumor cells and exhibits tumoricidal activity when injected intratumorally into tumor cell explants in mice. The experiments reported here were designed to determine whether M(4)N can be given systemically and inhibit the growth of five different human xenograft tumors. EXPERIMENTAL DESIGN: Nude (nu/nu) mice bearing xenografts of each of five human tumor types (i.e., hepatocellular carcinoma, Hep 3B; prostate carcinoma, LNCaP; colorectal carcinoma, HT-29; breast carcinoma, MCF7; and erythroleukemia, K-562) were treated with M4N given i.v. or i.p. in a Cremophor EL-based solvent system or orally in a corn oil based diet. Tumors from the treated animals were measured weekly and analyzed for the expression of the Cdc2 and survivin genes, both previously shown to be down-regulated by M4N. RESULTS: Systemic M4N treatment suppressed the in vivo growth of xenografts in each of the five human tumor types. Four of the five tumor models were particularly sensitive to M4N with tumor growth inhibitions (T/C values) of < or = 42%, whereas the fifth, HT-29, responded to a lesser extent (48.3%). Growth arrest and apoptosis in both the xenograft tumors and in the tumor cells grown in culture were accompanied by reductions in both Cdc2 and tumor-specific survivin gene expression. Pharmacokinetic analysis following oral and i.v. administration to ICR mice indicated an absolute bioavailability for oral M4N of approximately 88%. Minimal drug-related toxicity was observed. CONCLUSION: These preclinical studies establish that when given systemically, M4N can safely and effectively inhibit the growth of human tumors in nude mice.

Influence of ions, hydration, and the transcriptional inhibitor P4N on the conformations of the Sp1 binding site.

Three crystal structures containing the entire Sp1 consensus sequence d(GGGGCGGGG) with two or three additional base-pairs on either the 5' or 3' ends and overhangs have been determined. Despite the different lengths of DNA in the pseudo-dodecamers and pseudo-tridecamer, all three structures form A-DNA duplexes that share a common set of crystal contacts, including a T*(G.C) base triplet and a 5'-overhang that flips out and away from the helical axes to form a Hoogsteen base-pair with the 3'-overhang of a symmetry mate. The global conformations of the three structures differ, however, in the widths of their respective major grooves, the lengths of the molecules, and the extent of crystal packing. The structures were determined from crystals grown in an unusual precipitant for A-DNA, polyethylene glycol (PEG) 400, in combination with polyamines or ions; cobalt hexamine for the pseudo-tridecamer, and spermidine for the pseudo-dodecamers. As the Sp1 binding site is a target for antiviral and anticancer drugs, pseudo-dodecamer crystals were soaked with one such antiviral and anticancer compound, P4N. Although P4N was not visualized unambiguously in the electron density maps, the effect of the drug is evident from significant differences in the lattice constants, crystal packing, and overall conformation of the structure.

Tetra-O-methyl nordihydroguaiaretic acid induces growth arrest and cellular apoptosis by inhibiting Cdc2 and survivin expression.

We previously reported that Sp1-dependent Cdc2 gene expression is inhibited by tetra-O-methyl nordihydroguaiaretic acid (M(4)N) and that M(4)N is likely responsible for causing growth arrest in M(4)N-treated transformed C3 cells. Here, we show that after M(4)N treatment and cell-cycle arrest, expression of the Sp1-dependent survivin gene, a member of the inhibitor of apoptosis family, is also suppressed, and the mitochondrial apoptotic pathway is activated. To confirm that inhibition of Cdc2 and survivin gene expression is necessary for M(4)N-induced growth arrest and apoptosis, we tested the effect of adding Cdc2 and survivin back to M(4)N-treated cells. Cell division was transiently restored in the presence of M(4)N after transfection of an exogenous Cdc2 gene copy under the control of the Sp1-independent cytomegalovirus promoter. Caspase-3 activation was also reduced by 50% and 75% in transiently and stably survivin-transfected C3 cells, respectively. The results suggest that M(4)N induces growth arrest and apoptosis by suppressing Cdc2 and survivin expression, which constitutes the cellular basis of its antitumoric action.

Inhibition of HSV-1 replication and reactivation by the mutation-insensitive transcription inhibitor tetra-O-glycyl-nordihydroguaiaretic acid.

Methylated derivatives of nordihydroguaiaretic acid (NDGA)were previously shown to be potent mutation-resistant inhibitors of herpes simplex virus type 1 (HSV-1) which target Sp1 protein binding to critical viral promoters. The hydrophobic nature of these agents, however, renders them relatively water-insoluble and, therefore, limits their applicability. We report here on the anti-HSV-1 properties of a related but water-soluble glycylated derivative of NDGA, tetra-O-glycyl-NDGA (G(4)N). In yield reduction assays, G(4)N inhibited replication of laboratory and clinical strains of wild type HSV-1 and ACV-resistant (HSV-1(R)) strains of HSV-1 in a dose-dependent manner, with average IC(50) values of 4.7 and 3.2 microM against wild-type and HSV-1(R) strains, respectively. An MTT-based cytotoxicity assay revealed a TC(50) value of 73.2 microM for G(4)N on Vero cells, with no reduction in viability detected at concentrations below 30 microM. Similar to its methylated counterparts, G(4)N was found to inhibit transcription of the HSV-1 ICP4 gene, a major immediate early viral regulator, and gel mobility shift assays showed it can block Sp1 protein binding to cognate sites on the ICP4 promoter. In anticipation of its potential use as a systemic anti-HSV-1 agent, we tested G(4)N in a murine trigeminal ganglia (TG) explant model system, and found G(4)N was able to prevent HSV-1 reactivation from explanted and cultured latently infected TG.

Novel antiviral agent tetraglycylated nordihydroguaiaretic acid hydrochloride as a host-dependent viral inhibitor.

A water soluble derivative of nordihydroguaiaretic acid (NDGA), G(4)N (2), synthesized by reaction of NDGA (1) with N,N-dimethylglycine in the presence of dicyclohexylcarbodiimide and dimethylaminopyridine and then with HCl(g) (Scheme 1), competes effectively with the DNA binding domain of recombinant Sp1 protein for binding to the human immunodeficiency virus (HIV) LTR as demonstrated by an electrophoretic mobility-shift assay (EMSA). By blocking Sp1 binding to the HIV LTR, G(4)N suppresses Sp1-regulated HIV Tat transactivation and replication in cultured cells with an IC(50) of 12 microM similar to that of 3'-O-methyl-NDGA as we have previously reported. In addition simian immunodeficiency virus (SIV) replication was completely inhibited by G(4)N at 5.0 microM. G(4)N showed no toxic effect to 174 x CEM cells and H9 cells at 100 microM.

Antiviral saponins from Tieghemella heckelii.

Arganine C (1) and a new saponin, tieghemelin (2), were isolated from Tieghemella heckelii fruits. Arganine C (1) strongly inhibited HIV entry into cells in a cell fusion assay. The less potent tieghemelin (2) was converted into arganine C (1) by reduction of its ethyl ester with sodium borohydride. The removal of the four-sugar chains from arganine C (1) and tieghemelin (2) to give 16alpha-hydroxyprotobassic acid 3-O-beta-D-glucopyranoside (3) and 16alpha-hydroxyprotobassic acid 3-O-beta-D-glucuronopyranoside (4), respectively, caused total loss of activity in both cases. Arganine C (1) was not significantly cytotoxic to HeLa-CD4(+) cells at the level required to reduce the syncytium count to zero, suggesting it to be a promising candidate for further study as an antiviral drug.

Isolation of two highly potent and non-toxic inhibitors of human immunodeficiency virus type 1 (HIV-1) integrase from Salvia miltiorrhiza.

Water soluble extracts of the herbal plant, Salvia miltiorrhiza (Danshen) exhibited potent effect against HIV-1 integrase activity in vitro and viral replication in vivo. We have developed an extensive purification scheme to isolate effective, non-toxic inhibitors against human immunodeficiency virus type 1 (HIV-1) using the 3'-processing activity of integrase as a purification guide and assay. Two water soluble compounds, M(5)22 and M(5)32, have been discovered by isolating them from S. miltiorrhiza roots in purities of >99.5% as shown by NMR spectral analysis with yields of 0.018 and 0.038%, respectively. Structural determination revealed that M(5)22 is lithospermic acid and M(5)32 is lithospermic acid B. These two structurally related compounds are potent anti-HIV inhibitors and showed no cytotoxicity to H9 cells at high concentrations (CC(100)>297 microM for M(5)22 and >223 microM for M(5)32). The IC50 for inhibition of 3'-processing by HIV-1 integrase was found to be 0.83 microM for M(5)22 and 0.48 microM for M(5)32. In addition, M(5)22 and M(5)32 inhibited HIV-1 integrase catalytic activities of 3'-joining to the target DNA with IC50 of 0.48 microM for M(5)22 and 0.37 microM for M(5)32. Furthermore, kinetic and mechanistic studies suggested that drug binding to HIV-1 integrase and inhibition of enzymatic activity occur at a fast rate. Both M(5)22 and M(5)32 do not prevent HIV entry in H9 cells. They also show no inhibition of reverse transcriptase activity in infected cells. The levels of intracellular strong stop and full-length viral DNA remained unchanged following drug treatment. However, both inhibitors strongly suppressed the acute HIV-1 infection of H9 cells with IC50 values of 2 and 6.9 microM for M(5)22 and M(5)32, respectively. Thus these two selective integrase inhibitors hold promise as a novel class of therapeutic drugs for AIDS based on their high potencies and absence of cytotoxicity.