Ru Chih C. Huang Professor Department of Biology CMDB Graduate Program Faculty B.S. National Taiwan University Ph.D. Ohio State University Postdoctoral California Institute of Technology

[Research Interests] [Representative Publications] [Lab Members]

RESEARCH INTERESTS

Development of a Novel Family of Antitumor Agents Targeting Cdc2 Kinase, Survivin and MDR₁

Our laboratory has extensively investigated the use of derivatives of the plant lignan nordihydroguaiaretic acid (NDGA) for blocking viral replication through the inhibition of viral transcription. We have shown that NDGA derivatives, originally isolated from the creosote bush, Larrea tridentata, and now synthesized chemically, can inhibit the production of HIV, HSV and HPV transcripts by the deactivation of their Sp1-dependent promoters. An unexpectant outgrowth of this research has been the finding that one of the derivatives, tetra-O-methyl NDGA (M₄N), also induces cell cycle arrest in mammalian tumor cell lines. When treated with M₄N, tumor cells in culture arrest in the G₂ phase of the cell cycle. Arrest appears to be the direct result of inhibition of transcription of the cdc2 gene, whose expression is dependent on Sp1 and whose gene product is a cyclin kinase required for formation of the Maturation Promoting Factor complex. The discovery of the antiproliferative activity of M₄N led us to investigate whether it could be an effective antitumor agent. Injection of the water insoluble M₄N compound into experimentally induced mouse tumors resulted in arrest of tumor growth with minimal toxicity to the surrounding normal tissues. Since these initial observations we have been able to determine that M4N induces apoptosis in these tumors by inhibiting the expression and stability of the inhibitor of apoptosis protein, survivin.  In addition we have developed several solvent systems for administering M4N systemically and  through their use have demonstrated M₄N antitumor activity in at least eight different mouse tumor model systems, including human carcinoma cell explants in nude mice, spontaneous tumors in transgenic mice and experimentally induced metastatic tumors.  Our lab continues to investigate the mechanism of M4N action and test newly synthesized NDGA derivatives for improved solubility and effectiveness. In this regard, we have found that M4N and Maltose M3N, a water soluble derivative of NDGA can effectively inhibit Sp1-regulated multidrug resistant gene expression and synthesis of PgP protein in NCI/ADR-RES cells in culture and in a xenograft model system.  Combination therapy with M4N or Maltose- M3N with Paclitaxel was synergistic in inhibiting growth of these tumors in nude mice.

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 M4N and other NDGA derivatives as effective inhibitors of HIV replication has encouraged us to pursue an anti-HIV therapeutic approach that involves a cocktail of three compounds that inhibit different steps in the HIV life cycle. In addition to the NDGA derived transcription inhibitors, our lab has also isolated two saponins from the African rain forest tree Tieghemela heckelii that inhibit viral entry. Tieghemelin and arganine C are potent inhibitors of HIV mediated cell-cell fusion and inhibit HIV replication in infected cells in culture.  We have also targeted viral integration and 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 the NDGA compounds targeting transcription factors 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 three classes of 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 intensely involved in studying the molecular mechanisms by which these compounds exert their pharmacological activity.

Inhibition of Herpes Simplex Virus Infection by Nordihydroguaiaretic Acid Plant Lignan Derivatives

Herpes simplex virus type 1 (HSV-1) is a DNA virus afflicting greater than 70% of the human population.  During an HSV-1 lytic infection, viral genes are expressed in a tightly regulated cascade.  In this cascade, several of the HSV-1 genes are dependent on the Sp1 protein for their expression, and may serve as targets for viral inhibition.  The immediate early ICP4 gene is an attractive target as it is among the first genes to be expressed in the lytic cascade, and its expression is absolutely essential for the expression of subsequent viral genes and HSV-1 replication.  Our lab has demonstrated that NDGA plant lignan derivatives inhibit replication of HSV-1 in infected Vero cells and prevent the binding of Sp1 to cognate sites on the HSV-1 ICP4 promoter.  We are interested in developing these compounds as effective treatment modalities for herpes labialis and genital herpes.    

 
A Self-Inducing Runaway-Replication Plasmid Protein Expression System

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.  The success of the Human Genome Project has opened the door to a myriad of new proteins and potential therapeutic entities. We are continuing to refine this protein expression system so that it can be a universally effective tool for investigators in the burgeoning field of proteomics.
 

Regulation of Zebrafish Egg Envelope Gene Expression

Zebrafish eggs, like the eggs of all vertebrates, are surrounded by an extracellular membrane or envelope that is composed of a few conserved protein components featuring common domains that have been preserved over millions of years of evolution.  These proteins are coordinately expressed in oocytes at the earliest stages of follicular growth.  We have cloned a cluster of genes encoding the major egg envelope protein in zebrafish and are interested in what factors regulate their expression.  These zpb genes are arranged in a tandem array of six to eight copies that evolved through a process of duplication and homogenization by gene conversion presumably in response to the zebrafish’s need to produce large quantities of eggs at frequent intervals.  The extent of homogenization among the zpb genes in their promoter regions has provided us with clues to key elements that may be important in regulating their transcription.  Upstream sequences of zpb have been coupled to the luciferase reporter gene in an expression plasmid to characterize the strength of the gene’s promoter and identify oocyte-specific enhancers.  Deletion mutants of the promoter have been analyzed and several candidate enhancers have been identified.

REPRESENTATIVE PUBLICATIONS

Yeh, H-C., Puleo, C., Lim, T.C., Ho, Y-P., Giza, P., Huang, R.C.C., Wang, T-H. 2006. A microfluidic-FCS platform for investigation on the dissociation of Sp1-DNA complex by doxorubicin. Nuc Acids Res, Accepted for publication.

Chang, C.C., Liang, Y.C., Klutz, A., Hsu, C.I., Lin, C.F., Mold, D.E., Chou, T.C., Lee, Y.C., Huang R.C.C. 2006. Reversal of multidrug resistance by two nordihydroguaiaretic acid derivatives, M4N and Maltose- M3N, and their use in combination with Doxorubicin or Paclitaxel. Cancer Chemother Pharmacol. 58:640-653.

Huang, R.C.C., Chang, C-C., and Mold, D.E. 2006. Survivin Dependent and Independent Pathways and the Induction of Cancer Cell Death by Tetra-O-methyl Nordihydroguaiaretic Acid. Eds. Daniel Van Hoff and Philip J. Gray, Jr. Invited Article, Seminars in Oncology 33:479-485.

Dohm, J.A., Hsu, M-H., Hwu, J-R., Huang ,R.C.C., Moudrianakis, E.N., Lattman, E.E., Gittis, A.G. 2005. Influence of polyamines, ions, hydration, and the transcriptional inhibitor P4N on the conformations of the Sp1 binding site. J. Molec. Bio. 349:731-744.

Park, R., Chang, C-C., Liang, Y-C., Chung, Y., Henry, R.A., Lin, E., Mold, D.E., Huang, R.C.C. 2005. Systemic treatment with tetra-o-methyl nordihydroguaiaretic acid suppresses the growth of human xenograft tumors. Clinical Cancer Res. 11:4601-4609.

Hansel, D.E., Dhara, S., Huang, R.C.C., Ashfaq, R., Deasel, M., Shimada, Y., Bernstein, H.S., Harmon, J., Brock, M., Forastiere, A., Washington, M.K., Maitra, A., Montgomery, E. 2005. CDC2/CDK1 expression in esophageal adenocarcinoma and precursor lesions serves as a diagnostic and cancer progression marker and potential novel drug target. Am J Surg Pathol. 29:390-399.

Chang, C-C., Heller, J.D., Kuo, J., Huang, R.C.C.2004. Tetra-O-methyl nordihydroguaiaretic acid induces growth arrest and cellular apoptosis of C3 transformed cells by suppressing cdc2 and survivin expression. Proc Natl Acad Sci USA. 101:13239-13244.

Gosse, B.K., Ito, Y., Huang, R.C.C. 2004.  Optimization of active saponin, arganine C, for microbicidal external use. J Liq Chromatogr Rel Technol. 27:1947-1953.

Huang, R.C.C, Li, Y., Giza, P.E., Gnabre, J.N., Abd-Elazem, I.S., King, K.Y., Hwu, J.R. 2003. Novel antiviral agent tetraglycylated nordihydroguaiaretic acid hydrochloride as a host-dependent viral inhibitor. Antiviral Res. 58:57-64.

Park, R., Giza, P.E., Mold, D.E., Huang, R.C.C. 2003  Inhibition of HSV-1 replication and reactivation by the mutation-insensitive transcription inhibitor tetra-O-glycyl-nordihydroguaiaretic acid. Antiviral Res. 58:35-45.

Gosse, B.K., Gnabre, J.N., Bates, R.B., Nakkiew, P., Huang, R.C.C. 2002.   Antiviral saponins from Tieghemella heckelii. J Nat Products. 65:1942-1944.

Gosse, B.K., Gnabre, J.N., Ito, Y., Huang, R.C. 2002 Isolation of saponins with viral entry inhibitory activity by combined chromatographic methods. J Liq Chromatogr Rel Technol. 25:3197-3209.

Abd-Elazem, I.S., Chen, H.S., Bates, R.B., Huang, R.C.C. 2002   Isolation of two highly potent and non-toxic inhibitors of human immunodeficiency virus type 1 (HIV-1) integrase from Salvia miltiorrhiza. Antiviral Res. 55:91-106.

Heller, J.D., Kuo, J., Wu, T.C., Kast, W.M., Huang, R.C.C. 2001 Tetra-O-methyl nordihydroguaiaretic acid induces G₂ arrest in mammalian cells. Cancer Res. 61:5499-5504.

Mold, D.E., Kim, I.F., Tsai, C-M., Lee, D., Chang, C-Y., Huang, R.C.C. 2001  Cluster of genes encoding the major egg envelope protein of zebrafish. Mol Reprod Dev. 58:4-14.

Craigo, J., Callahan, M., Huang, R.C., Delucia, A.L. 2000.  Inhibition of human papilloma virus type 16 gene expression by nordihydroguaiaretic acid plant lignan derivatives. Antiviral Res. 47:19-28.

Lab Members

Research Scientist:
Apostolos Gittis, PhD

Associate Research Scientists:
Ibrahim Abd-Elazem, PhD
Rossitza Gitti, PhD
David Mold, MD, PhD

Assistant Research Scientist:
Kotohiko Kimura, MD, PhD

Graduate Students:
Devin Rosenthal

Master's Degree Students:
John LeGall
Leah Liu
Omar Butt

Postdoctoral Fellow:
Hsin-Chih (Tim) Yeh

Visiting Predoctoral Fellow:
Jong Ho Chun, MD

Research Technician II:
Paul Giza

Undergraduate Students:
Mikhail Borbounov
Vincent Devlin
Amy Dinitz
John Harrington
Brian Nickel
Divya Sambandan