Rodent models for the preclinical evaluation of drugs suitable for pharmacological intervention in aging.

INTRODUCTION: There is a growing scientific and public interest in the development of new antiaging drugs for the purposes of extending mean and/or maximum life span, maintaining normal physiological function, and alleviating the onset and severity of age-associated diseases. This review looks at the current screening approaches used to evaluate the efficacy of such compounds, with a particular focus on those that extend life span. AREAS COVERED: This article reviews the current preclinical approaches for assessing longevity therapy including the assessment of antiaging drugs (aging reversal) and geroprotectors (drugs that prevent premature aging and/or slowdown or postpone aging). This article also discusses the methods and the importance in evaluating the anticarcinogenic potential and safety of antitumor drugs. EXPERT OPINION: Based on more than 30 years of experience in the field, the authors believe that the standard testing protocols for antiaging drugs should include the simultaneous evaluation of the drug's safety, as well as its antitumor and anticarcinogenic activity potential. The authors also believe that the principles of international programs for the expert critical evaluation of pharmacological interventions should be created to improve the range of antiaging interventions available for human studies.

Loss of CAK phosphorylation of RAR{alpha} mediates transcriptional control of retinoid-induced cancer cell differentiation.

Although the role of the classic retinoic acid (RA)-induced genomic pathway in cancer cell differentiation is well recognized, the underlying mechanisms remain to be dissected. Retinoic acid receptor alpha (RARalpha) is a transcription factor activated by RA, and its serine 77 (RARalphaS77) is the main residue phosphorylated by the cyclin-dependent kinase (CDK)-activating kinase (CAK) complex. We report here that in both human myeloid leukemia and mouse embryonic teratocarcinoma stem cells, either RA-suppressed CAK phosphorylation of RARalpha or mutation of RARalphaS77 to alanine (RARalphaS77A) coordinates CAK-dependent G(1) arrest with cancer cell differentiation by transactivating RA-target genes. Both hypophosphorylated RARalpha and RARalphaS77A reduce binding to retinoic acid-responsive elements (RARE) in the promoters of RA-target genes while stimulating gene transcription. The enhanced transactivation and reduced RARalpha-chromatin interaction are accompanied by RARalpha dissociation from the transcriptional repressor N-CoR and are association with the coactivator NCoA-3. Such effects of decreased CAK phosphorylation of RARalphaS77 on mediating RA-dependent transcriptional control of cancer cell differentiation are examined correspondingly in both RA-resistant myeloid leukemia and embryonic teratocarcinoma stem RARalpha(-/-) cells. These studies demonstrate, for the first time, that RA couples G(1) arrest to transcriptional control of cancer cell differentiation by suppressing CAK phosphorylation of RARalpha to release transcriptional repression.-Wang, A., Alimova, I. N., Luo, P. Jong, A., Triche, T. J., Wu, L. Loss of CAK phosphorylation of RARalpha mediates transcriptional control of retinoid-induced cancer cell differentiation.

Metformin induces unique biological and molecular responses in triple negative breast cancer cells.

Triple negative (TN) breast cancer is more frequent in women who are obese or have type II diabetes, as well as young women of color. These cancers do not express receptors for the steroid hormones estrogen or progesterone, or the type II receptor tyrosine kinase (RTK) Her-2 but do have upregulation of basal cytokeratins and the epidermal growth factor receptor (EGFR). These data suggest that aberrations of glucose and fatty acid metabolism, signaling through EGFR and genetic factors may promote the development of TN cancers. The anti-type II diabetes drug metformin has been associated with a decreased incidence of breast cancer, although the specific molecular subtypes that may be reduced by metformin have not been reported. Our data indicates that metformin has unique anti-TN breast cancer effects both in vitro and in vivo. It inhibits cell proliferation (with partial S phase arrest), colony formation and induces apoptosis via activation of the intrinsic and extrinsic signaling pathways only in TN breast cancer cell lines. At the molecular level, metformin increases P-AMPK, reduces P-EGFR, EGFR, P-MAPK, P-Src, cyclin D1 and cyclin E (but not cyclin A or B, p27 or p21), and induces PARP cleavage in a dose- and time-dependent manner. These data are in stark contrast to our previously published biological and molecular effects of metformin on luminal A and B, or Her-2 type breast cancer cells. Nude mice bearing tumor xenografts of the TN line MDA-MB-231, treated with metformin, show significant reductions in tumor growth (p = 0.0066) and cell proliferation (p = 0.0021) as compared to untreated controls. Metformin pre-treatment, before injection of MDA-MB-231 cells, results in a significant decrease in tumor outgrowth and incidence. Given the unique anti-cancer activity of metformin against TN disease, both in vitro and in vivo, it should be explored as a therapeutic agent against this aggressive form of breast cancer.

Metformin inhibits breast cancer cell growth, colony formation and induces cell cycle arrest in vitro.

The anti-diabetic drug metformin reduces human cancer incidence and improves the survival of cancer patients, including those with breast cancer. We studied the activity of metformin against diverse molecular subtypes of breast cancer cell lines in vitro. Metformin showed biological activity against all estrogen receptor (ER) positive and negative, erbB2 normal and abnormal breast cancer cell lines tested. It inhibited cellular proliferation, reduced colony formation and caused partial cell cycle arrest at the G(1) checkpoint. Metformin did not induce apoptosis (as measured by DNA fragmentation and PARP cleavage) in luminal A, B or erbB2 subtype breast cancer cell lines. At the molecular level, metformin treatment was associated with a reduction of cyclin D1 and E2F1 expression with no changes in p27(kip1) or p21(waf1). It inhibited mitogen activated protein kinase (MAPK) and Akt activity, as well as the mammalian target of rapamycin (mTOR) in both ER positive and negative, erbB2-overexpressing and erbB2-normal expressing breast cancer cells. In erbB2-overexpressing breast cancer cell lines, metformin reduced erbB2 expression at higher concentrations, and at lower concentrations within the therapeutic range, it inhibited erbB2 tyrosine kinase activity evidenced by a reduction of phosphorylated erbB2 (P-erbB2) at both auto- and Src- phosphorylation sites. These data suggest that metformin may have potential therapeutic utility against ER positive and negative, erbB2-overexpressing and erbB2-normal expressing breast cancer cells.

Effect of Epitalon on biomarkers of aging, life span and spontaneous tumor incidence in female Swiss-derived SHR mice.

From the age of 3 months until their natural deaths, female outbred Swiss-derived SHR mice were subcutaneously injected on 5 consecutive days every month with 0.1 ml of normal saline (control) or with 1.0 microg/mouse (approximately 30-40 microg/kg) of tetrapeptide Epitalon (Ala-Glu-Asp-Gly) dissolved in 0.1 ml saline. There were 54 mice in each group. The results of this study show that treatment with Epitalon did not influence food consumption, body weight or mean life span of mice. However, it slowed down the age-related switching-off of estrous function and decreased the frequency of chromosome aberrations in bone marrow cells (by 17.1%, P<0.05). It also increased by 13.3% the life span of the last 10% of the survivors (P<0.01) and by 12.3% the maximum life span in comparison with the control group. We also found that treatment with Epitalon did not influence total spontaneous tumor incidence, but inhibited the development of leukemia (6.0-fold), as compared with the control group. The data obtained suggest a geroprotector activity of Epitalon and the safety of its long-term administration in mice.

Effect of delta-sleep inducing peptide-containing preparation Deltaran on biomarkers of aging, life span and spontaneous tumor incidence in female SHR mice.

From the age of 3 months until their natural deaths, female Swiss-derived SHR mice were subcutaneously injected 5 consecutive days every month with 0.1 ml of normal saline (control) or with 2.5 microg/mouse (approximately 100 microg/kg) of delta-sleep inducing peptide (DSIP, Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu) as the preparation Deltaran solved in 0.1 ml of saline. There were 54 mice in each group. The results of this study show that the treatment with Deltaran did not influence food consumption, but decreased the body weight of mice; it slowed down the age-related switching-off of estrous function; it decreased by 22.6% the frequency of chromosome aberrations in bone marrow cells; it did not influence mean life span; and it increased by 17.1% life span of the last 10% of the survivors and by 24.1% maximum life span in comparison with the control group. We also found that treatment with Deltaran significantly decreased total spontaneous tumor incidence (by 2.6-fold), mainly mammary carcinomas and leukemias in mice as compared with the control group. This is the first report on geroprotector and anticarcinogenic effect of DSIP-containing preparation Deltaran.

Dose-dependent effect of melatonin on life span and spontaneous tumor incidence in female SHR mice.

From the age of 3 months until their natural death, female Swiss-derived SHR mice were given melatonin with their drinking water (2 or 20mg/l) for 5 consecutive days every month. Intact mice served as controls. There were 54 mice in each group. The results of this study show that the treatment of melatonin did not significantly influence food consumption, but its administration at lower doses did decrease the body weight of mice; it slowed down the age-related switching-off of estrous function; it did not influence the frequency of chromosome aberrations in bone marrow cells; it did not influence mean life span; and it increased life span of the last 10% of the survivors in comparison to controls. We also found that treatment with low dose melatonin (2mg/l) significantly decreased spontaneous tumor incidence (by 1,9-fold), mainly mammary carcinomas, in mice whereas higher doses (20mg/l) failed to influence tumor incidence as compared to controls. For this reason, we conclude that the effect of melatonin as a geroprotector is dose-dependent.

Number of pregnancies and ovariectomy modify mammary carcinoma development in transgenic HER-2/neu female mice.

We studied effect of pregnancy and ovariectomy on the development of mammary tumors in homozygous female HER-2/neutransgenic mice. The mean life span of uniparous mice was decreased by 16% in comparison to the control (P<0.05) and of mice which have two pregnancies decreased by 11% (P<0.05). Ovariectomy at the age of 2 months was followed by 32.7% increase in mean life span of mice. The incidence or multiplicity of mammary adenocarcinomas did not change in uniparous mice, whereas the size of the tumors and metastatic potential were decreased as compared to the virgins. When mice have two full-time pregnancies, there was an increase in multiplicity of mammary carcinomas and significant (2.1-fold) decrease in the survival time of tumor-bearing mice. Ovariectomy significantly decreased the total incidence of mammary carcinomas, the number of tumors per tumor-bearing animal, and inhibited metastasizing into lungs. Our results indicate that pregnancy accelerated the development of mammary adenocarcinomas in transgenic HER-2/neu mice whereas ovariectomy inhibits their development.

The effect of melatonin treatment regimen on mammary adenocarcinoma development in HER-2/neu transgenic mice.

The effect of various regimens of treatment with melatonin on the development of mammary tumors in HER2/neu transgenic mice was investigated. Female HER-2/neu mice starting from the age of 2 months were kept under standard light/dark regimen and as given melatonin with tap water (20 mg/l) during the night time 5 times monthly (interrupted treatments) or constantly to natural death. Intact mice served as controls. Treatment with melatonin slowed down age-related disturbances in estrous function most in the group exposed to interrupted treatment with the hormone. Constant treatment with melatonin decreased incidence and size of mammary adenocarcinomas, and incidence of lung metastases, compared to controls. The number of mice bearing 4 and more tumors was reduced in the group with constant melatonin treatment. Interrupted treatment with melatonin promote mammary carcinogenesis in HER-2/neu transgenic mice. The data demonstrate the regimen-dependent inhibitory effect of melatonin on the development of spontaneous mammary tumors in HER-2/neu mice but not on overall survival with implication about the likely cause of the effect. Polycystic kidney disease is common in this transgenic line. Adverse effect of melatonin on the life span in our study may be unique to the transgenic model used and may not be relevant to the suppressive effect of melatonin in delay of mammary cancer.

Inhibitory effect of the peptide epitalon on the development of spontaneous mammary tumors in HER-2/neu transgenic mice.

Female FVB/N HER-2/neu transgenic mice from the age of 2 months were subcutaneously injected with saline, the peptide Epitalon(R) (Ala-Glu-Asp-Gly) or with the peptide Vilon(R) (Lys-Glu) in a single dose of 1 microg/mouse for 5 consecutive days every month. Epitalon treatment reduced the cumulative number and the maximum size of tumors (p < 0.05). Furthermore, the number of mice bearing 1 mammary tumor was increased, whereas the number of mice bearing 2 or more mammary tumors was reduced in Epitalon-treated in comparison to saline-treated animals (p < 0.05). The size but not the number of lung metastases was reduced in Epitalon-treated compared to saline-treated mice (p < 0.05). The treatment with Vilon produced significant negative effects when compared to the control group, with an increased incidence of mammary cancer development (p < 0.05), a shorter mean latent period of tumors (p < 0.05) and an increased cumulative number of tumors (p < 0.05). A 3.7-fold reduction in the expression of HER-2/neu mRNA was found in mammary tumors from HER-2/neu transgenic mice treated with Epitalon compared to control animals. The expression of mRNA for HER-2/neu was also partially reduced in Vilon-treated mice, but it remained significantly higher in Vilon- than in Epitalon-treated animals (1.9-fold increase). The data demonstrate the inhibitory effect of Epitalon in the development of spontaneous mammary tumors in HER-2/neu mice, suggesting that a downregulation of HER-2/neu gene expression in mammary adenocarcinoma may be responsible, at least in part, for the antitumor effect of the peptide.