Cycloastragenol is a potent telomerase activator in neuronal cells: implications for depression management.

Cycloastragenol (CAG) is an aglycone of astragaloside IV. It was first identified when screening Astragalus membranaceus extracts for active ingredients with antiaging properties. The present study demonstrates that CAG stimulates telomerase activity and cell proliferation in human neonatal keratinocytes. In particular, CAG promotes scratch wound closure of human neonatal keratinocyte monolayers in vitro. The distinct telomerase-activating property of CAG prompted evaluation of its potential application in the treatment of neurological disorders. Accordingly, CAG induced telomerase activity and cAMP response element binding (CREB) activation in PC12 cells and primary neurons. Blockade of CREB expression in neuronal cells by RNA interference reduced basal telomerase activity, and CAG was no longer efficacious in increasing telomerase activity. CAG treatment not only induced the expression of bcl2, a CREB-regulated gene, but also the expression of telomerase reverse transcriptase in primary cortical neurons. Interestingly, oral administration of CAG for 7 days attenuated depression-like behavior in experimental mice. In conclusion, CAG stimulates telomerase activity in human neonatal keratinocytes and rat neuronal cells, and induces CREB activation followed by tert and bcl2 expression. Furthermore, CAG may have a novel therapeutic role in depression.

Astragaloside IV and cycloastragenol stimulate the phosphorylation of extracellular signal-regulated protein kinase in multiple cell types.

Two Chinese herb-derived small molecule telomerase activators, astragaloside IV (AG-IV) and cycloastragenol (CAG), have recently been shown to improve the proliferative response of CD8+ T lymphocytes from HIV-infected patients by upregulating telomerase activity. Here, we examined the signaling mechanism of AG-IV and CAG. Telomerase activity in human embryonic kidney HEK293 fibroblasts was increased upon treatment with increasing concentrations of AG-IV or CAG. Both compounds induced the phosphorylation of extracellular signal-regulated protein kinase (ERK) in a time- and dose-dependent manner in HEK293 cells and HEK-neo keratinocytes. AG-IV and CAG also stimulated ERK phosphorylation in other cell lines of lung, brain, mammary, endothelial, and hematopoietic origins. Use of selective inhibitors and dominant negative mutants revealed the involvement of c-Src, MEK (ERK kinase), and epidermal growth factor receptor in CAG-induced ERK phosphorylation. Our data indicate that AG-IV and CAG may exert their cellular effects through the activation of the Src/MEK/ERK pathway.

In vitro intestinal absorption and first-pass intestinal and hepatic metabolism of cycloastragenol, a potent small molecule telomerase activator.

Cycloastragenol (CAG) is the aglycone derivative of astragaloside IV which has recently been demonstrated to activate telomerase and represents a potential drug candidate for the treatment of degenerative diseases. In the present study, intestinal absorption and metabolism of CAG were examined using the Caco-2 model and liver microsomes, respectively. The results showed that CAG rapidly passes through the Caco-2 cell monolayer by passive diffusion. Four different glucuronide conjugates and two oxidized CAG metabolites were found in the apical and basolateral sides of Caco-2 monolayer, suggesting that first-pass intestinal metabolism of CAG might occur upon passage through the intestinal epithelium. CAG underwent extensive metabolism in rat and human liver microsomes with only 17.4% and 8.2%, respectively, of the starting amount of CAG remaining after 30 min of incubation. Monohydroxylation of the parent and oxidization of the hydroxylated CAG were found in the liver samples. The present study indicates that CAG is efficiently absorbed through intestinal epithelium. However, extensive first-pass hepatic metabolism would limit the oral bioavailability of this compound.

Telomerase-based pharmacologic enhancement of antiviral function of human CD8+ T lymphocytes.

Telomerase reverse transcribes telomere DNA onto the ends of linear chromosomes and retards cellular aging. In contrast to most normal somatic cells, which show little or no telomerase activity, immune cells up-regulate telomerase in concert with activation. Nevertheless, during aging and chronic HIV-1 infection, there are high proportions of dysfunctional CD8(+) CTL with short telomeres, suggesting that telomerase is limiting. The present study shows that exposure of CD8(+) T lymphocytes from HIV-infected human donors to a small molecule telomerase activator (TAT2) modestly retards telomere shortening, increases proliferative potential, and, importantly, enhances cytokine/chemokine production and antiviral activity. The enhanced antiviral effects were abrogated in the presence of a potent and specific telomerase inhibitor, suggesting that TAT2 acts primarily through telomerase activation. Our study is the first to use a pharmacological telomerase-based approach to enhance immune function, thus directly addressing the telomere loss immunopathologic facet of chronic viral infection.

Telomerase antagonists GRN163 and GRN163L inhibit tumor growth and increase chemosensitivity of human hepatoma.

Most cancer cells have an immortal growth capacity as a consequence of telomerase reactivation. Inhibition of this enzyme leads to increased telomere dysfunction, which limits the proliferative capacity of tumor cells; thus, telomerase inhibition represents a potentially safe and universal target for cancer treatment. We evaluated the potential of two thio-phosphoramidate oligonucleotide inhibitors of telomerase, GRN163 and GRN163L, as drug candidates for the treatment of human hepatoma. GRN163 and GRN163L were tested in preclinical studies using systemic administration to treat flank xenografts of different human hepatoma cell lines (Hep3B and Huh7) in nude mice. The studies showed that both GRN163 and GRN163L inhibited telomerase activity and tumor cell growth in a dose-dependent manner in vitro and in vivo. The potency and efficacy of the lipid-conjugated antagonist, GRN163L, was superior to the nonlipidated parent compound, GRN163. Impaired tumor growth in vivo was associated with critical telomere shortening, induction of telomere dysfunction, reduced rate of cell proliferation, and increased apoptosis in the treatment groups. In vitro, GRN163L administration led to higher prevalence of chromosomal telomere-free ends and DNA damage foci in both hepatoma cell lines. In addition, in vitro chemosensitivity assay showed that pretreatment with GRN163L increased doxorubicin sensitivity of Hep3B. In conclusion, our data support the development of GRN163L, a novel lipidated conjugate of the telomerase inhibitor GRN163, for systemic treatment of human hepatoma. In addition to limiting the proliferative capacity of hepatoma, GRN163L might also increase the sensitivity of this tumor type to conventional chemotherapy.

Lipid modification of GRN163, an N3'-->P5' thio-phosphoramidate oligonucleotide, enhances the potency of telomerase inhibition.

The vast majority of human cancers express telomerase activity, while most human somatic cells do not have detectable telomerase activity. Since telomerase plays a critical role in cell immortality, it is an attractive target for a selective cancer therapy. Oligonucleotides complementary to the RNA template region of human telomerase (hTR) have been shown to be effective inhibitors of telomerase and, subsequently, cancer cell growth in vitro. We show here that a lipid-modified N3'-->P5' thio-phosphoramidate oligonucleotide (GRN163L) inhibits telomerase more potently than its parental nonconjugated thio-phosphoramidate sequence (GRN163). Cells were treated with both the first- (GRN163) and second-generation (GRN163L) oligonucleotides, including a mismatch control, with or without a transfection enhancer reagent. GRN163L inhibited telomerase activity effectively in a dose-dependent manner, even without the use of a transfection reagent. The IC50 values for GRN163 in various cell lines were on average sevenfold higher than for GRN163L. GRN163L inhibition of telomerase activity resulted in a more rapid loss of telomeres and cell growth than GRN163. This report is the first to show that lipid modification enhanced the potency of the novel GRN163 telomerase inhibitor. These results suggest that the lipid-conjugated thio-phosphoramidates could be important for improved pharmacodynamics of telomerase inhibitors in cancer therapy.

Telomerase inhibition with an oligonucleotide telomerase template antagonist: in vitro and in vivo studies in multiple myeloma and lymphoma.

The effects of telomerase inhibition with an oligonucleotide N3' --> P5' thiophosphoramidate (GRN163) complementary to the telomerase template region were examined on human multiple myeloma (MM) and non-Hodgkin lymphoma (NHL) cell lines, primary MM cells, and tumor xenografts. GRN163 treatment reduced telomerase levels in all cells and induced more rapid telomeric shortening. Continuous GRN163 treatment for 7 to 14 days resulted in proliferative arrest, morphologic changes, and apoptosis characteristic of cell crisis in tumor cell lines with short (1.7-5.4 kb) but not long (9-11 kb) telomeres. Intratumoral administration of GRN163 also inhibited the growth of MM and NHL xenografts established from cell lines with short telomeres (Hs602 lymphoma, 2.7 kb; CAG myeloma, 2.7 kb) and increased tumor apoptosis. However, GRN163 therapy of NHL xenografts established from cells with long telomeres (11.0 kb) had equivocal effects on tumor growth and did not induce apoptosis during this time frame. Systemic daily intraperitoneal administration of GRN163 in myeloma xenografts with short telomere lengths also decreased tumor telomerase levels and reduced tumor volumes. These data demonstrate that telomerase is important for the replication of mature B-cell neoplasia by stabilizing short telomeres, and they suggest that telomerase inhibition represents a novel therapeutic approach to MM and NHL.

A novel telomerase template antagonist (GRN163) as a potential anticancer agent.

Telomerase, the enzyme responsible for proliferative immortality, is expressed in essentially all cancer cells, but not in most normal human cells. Thus, specific telomerase inhibition is potentially a universal anticancer therapy with few side effects. We designed N3'-->P5' thio-phosphoramidate (NPS) oligonucleotides as telomerase template antagonists and found that their ability to form stable duplexes with the telomerase RNA subunit was the key factor for antitelomerase activity. In biochemical assays 11-13-mer NPS oligonucleotides demonstrated sequence- and dose-dependent inhibition of telomerase with IC(50) values <1 nM. Optimization of the sequence, length, and bioavailability resulted in the selection of a 13-mer NPS oligonucleotide, GRN163, as a drug development candidate. GRN163 inhibited telomerase in a cell-free assay at 45 +/- 7 pM, and in various tumor cell lines at approximately 1 nM and approximately 0.3-1.0 micro M in the presence and absence of carriers, respectively. GRN163 was competitive with telomeric primer binding, primarily because of hybridization to human telomerase RNA (hTR) component. Tumor cells treated with GRN163 in culture underwent telomere shortening, followed by cellular senescence or apoptosis after a period of time that generally correlated with initial telomere length. In a flank DU145 (prostate cancer) xenograft model, parenterally administered GRN163 caused suppression of tumor growth in the absence of gross toxicity. These data demonstrate that GRN163 has significant potential for additional development as an anticancer agent.