Hypoxia Targeting of Hepatocellular Carcinoma with Nitroimidazoles Conjugated to Trimethyl Nordihydroguaiaretic Acid
Hepatocellular carcinoma (HCC) develops through cirrhosis brought on by chronic liver injury. This chronic injury results in fibrogenesis that damages the normal liver circulatory system and leads to a shortage of blood perfusion and oxygen delivery. Moreover, in tumor tissues, a high rate of cell proliferation in the tumor cells and abnormalities of structure and function of tumor vessels increases the need for oxygen. We are interested in developing therapies that target hypoxic HCC cells and have been experimenting with nitroimadazoles conjugated to the methylated nordihydroguaiaretic acid (NDGA) anticancer agents developed in our lab.
In hypoxic tumor cells, nitroimidazoles undergo a series of enzymic reductions, mediated by nitroreductase enzymes, and followed by ring fragmentation. Reactive radicals are thus generated, which then irreversibly bind to the cellular components. After the compounds enter the cell, reduction enables more drugs to accumulate in the cell by a favorable concentration gradient. In normoxic cells, the presence of oxygen prevents the enzymic reduction of nitroimidazole, and hence no binding occurs. In addition, nitroimidazole derivatives show preferential toxicity to hypoxic cells as hypoxic cytotoxins. Their cytotoxicity toward hypoxic cells is a result of abstraction of hydrogen from target molecules by free radicals formed in the reduction of the nitro group.
To enhance the effectiveness of tetra-O-methyl NDGA (M4N, terameprocol), a semi-synthetic small molecule anticancer agent developed in our lab, we have designed molecules which incorporate trimethyl NDGA with a nitroimidazole unit. To improve the water solubility, we have also tethered the nitroimidazole unit to NDGA with poly(ethylene glycol). So far we have successfully synthesized eight regioisomeric NDGA–nitroimidazole conjugates in pure form. All of them exhibit anti-hepatocellular carcinoma activities with potency in the range of 10–25 µM and our preliminary results indicate they are even more effective against HCC cells when the cells are grown under hypoxic conditions. Future studies of these compounds in mouse models of HCC are planned.
Rapid Cell Death Induction and Growth and Metastasis Suppression of Human Cancer Cells by Tetra-O-methyl Nordihydroguaiaretic Acid in Combination with Protein Kinase Inhibitors
Transcription factor Sp1 regulates the expression of a variety of cellular genes, including Sp1 gene itself in eukaryotic cells. High levels of Sp1 have been found to correlate with human cancer development and progression. The anticancer drug M4N (terameprocol), by blocking the binding of Sp1 protein to its recognition site represses the overexpression of a series of growth related genes in all cancer types tested. Tumor growth arrest is observed in cancer cell cultures, in a variety of tumor xenografts, in animal models and in 30 – 40% patients who received terameprocol in clinical trials. However, rapid cancer cell death in vivo has not often been achieved, especially when the amount of terameprocol in the tumor is below the therapeutic level, and this has limited its effectiveness in suppressing more aggressive cancers. Using human cancer xenografts in mouse model, we have been able to raise the amount of M4N in plasma and in tumors of the mice to a level of >1mM with a single cycle treatment of daily IV injections of M4N for 14 – 21 days. In addition, M4N in combination with either rapamycin or with etoposide is able to synergistically induce rapid cell death in prostate, colon, pancreas, breast, brain cancer cells in culture and eliminate LNCaP, the orthotopic prostate tumors and metastasis in nude mice. Treated animals have survived in good health (currently beyond 240 days) while all non treated mice , or mice subjected to only single drug treatments died in less than 35 days due to tumor burden. We are currently studying how M4N and inhibitors of protein kinases in the PI3/AKT pathway act synergistically to induce rapid apoptosis in cancer cells.
A Series of Antiviral Agents Derived from Natural Products for Use in Combination Therapy of HIV-1
The AIDS epidemic continues to be a devastating problem for millions of people in the world. Our success with tetra-O-methyl NDGA (M4N, terameprocol) and other NDGA derivatives as effective inhibitors of Sp1-dependent HIV replication has encouraged us to pursue an anti-HIV therapeutic approach that involves a cocktail of compounds that inhibit different steps in the HIV life cycle. In addition to the NDGA derived transcription inhibitors, we have purified two potent inhibitors of HIV integrase, lithospermic acid and lithospermic acid B, from the roots of Salvia miltiorrhiza. They are also potent inhibitors of HIV replication. All of these compounds have been found to be mutation insensitive and should therefore increase the effectiveness and reduce the toxicity of the therapy. We are currently testing these compounds in combination for their effectiveness at blocking HIV production in cell culture and in peripheral blood mononuclear cells (PBMCs). As a therapeutic regimen is developed, the lab will be involved in studying in more detail the molecular mechanisms by which these compounds exert their pharmacological activity.
A Self-Inducing Runaway-Replication Plasmid Protein Expression System Engineered for Twenty First Century Proteomics
A unique, highly efficient prokaryotic expression system has been developed in our laboratory that produces unparalleled amounts of protein. The system consists of a temperature-sensitive-copy-number plasmid that carries the rop gene and promoter downstream from the trp promoter. Any sequence cloned into the rop gene alters Rop protein activity and causes lethal runaway plasmid DNA replication. This plasmid replication can be suppressed in trans by complementation with a similar wild-type plasmid. Cells harboring both plasmids are quite stable, and induction of plasmid DNA synthesis occurs only after cells are grown for several generations under conditions that lead to the loss of the trans-acting repressor. Large amounts of Rop fusion proteins accumulate in the cell as the trp operon is gradually induced via repressor titration. The expression plasmid has been engineered with an affinity tag for easy purification of the fusion protein and a thrombin cleavage site for cleavage of the protein of interest from the Rop fusion. We have begun to explore ways to adapt this system to the generation of proteome libraries amenable to high throughput screening.