177 - 188: Conservative Growth Hormone/IGF-1 and mTOR Signaling Pathways as a Target for Aging and Cancer Prevention: Do We Really Have an Antiaging Drug

There are nine tentative hallmarks of aging in mammals, which may represent common denominators of aging in different organisms: genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, deregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, and altered cell-to-cell communication [1]. At the same time, there is also sufficient similarity in the patterns of changes observed during normal aging and the process of carcinogenesis (table 1) [2]. As can be seen in figure 1, DNA damage induced by environmental and endogenous carcinogenic factors [reactive oxygen species, ionizing radiation, ultraviolet, constant illumination (light at night), some diets, oncogenes, etc.] may lead to cellular senescence or cellular lesions which could be deleted by apoptosis. The same agents can induce damage which is followed by neoplastic transformation, thus leading to cancer [2,3]. During the last decade, the intensive search for antiaging remedies has led to the conclusion that both insulin/IGF-1 signaling (IIS) and nutrient response pathways defined by the mTOR protein kinase pathways control aging and age-associated pathology in worms, insects and mammals [4]. In each of these organisms, genetic downregulation or interruption of this signaling pathway can lead to major extension of longevity. There are two functionally distinct mTOR complexes called mTORC1 and mTORC2. mTORC1 is activated by insulin and related growth factors through phosphatidylinositol-3-OH kinase and AKT kinase signaling and repressed by AMP-activated protein kinase, a key sensor of cellular energy status [4]. The mTORC1 is involved in promoting messenger RNA translation and protein synthesis through ribosomal protein S6 kinases (S6Ks) and 4E-BP protein, which in the hypophosphorylated form acts as a negative regulator of the cap-binding protein eIF4E. mTORC1 also stimulates lipid biosynthesis, inhibits autophagy, and through hypoxic response transcription factor HIF-1α regulates mitochondrial function and glucose metabolism. Rapamycin suppresses mTORC1 and indirectly mTORC2, which leads to metabolic lesions like glucose intolerance and abnormal lipid profile [4]. The phosphorylation of S6K1 at T389 by TORC1 is susceptible to rapamycin. The life span of S6K1-deficient female mice increased by 19% in comparison to the wild-type controls without any effect on the incidence of tumor development [5]. It is worth noting that there was no significant effect of the protein knockout on the life span of male mice. These data suggest that S6K1 is involved in mammalian life span regulation downstream of TORC1. Taking into consideration the negative effect of rapamycin on glucose tolerance and liver insulin sensitivity [5], Lamming et al. [6] studied the effect of mTORC1 and mTORC2 regulator gene modification on the life span of mice. There was no increase in life span in either female or male mtor^+/-, Raptor^+/-, mlst8^+/- or mtor^+/-Raptor^+/- mice. However, female mtor^+/-mlst8^+/- mice lived longer by 14.4% in comparison to wild-type mice. The longevity of male mtor^+/- mlst8^+/- mice was unaffected. Female mtor^+/-mlst8^+/- mice were not calorie restricted through reduced food intake or increased energy expenditure, and had normal body weights and levels of activity consistent with the phenotypic effects. mtor^+/- mlst8^+/- mice exhibited an approximately 30-60% reduction in the abundance of hepatic mTOR, Raptor, mLST8, and Rictor, whereas the expression of mTOR complex subunits was less affected in Raptor^+/- and mtor^+/-Raptor^+/- heterozygotes. The authors believe that suppression mTORC1 signaling is sufficient for life span prolongation independent of changes in glucose homeostasis.

Calorie restriction (CR) is the only known intervention in mammals that has been consistently shown to increase life span, reduce incidence and retard the onset of age-related diseases, including cancer and diabetes. CR has also been shown to increase resistance to stress and toxicity, and maintain youthful levels of function and vitality in laboratory mammals at advanced chronological age [7]. CR in rhesus monkeys have produced physiological responses strikingly similar to those observed in rodents and delayed the onset of age-related diseases, but effects on longevity were not consistent [8]. Data from these studies indicate that long-term CR reduces morbidity and mortality in primates, and thus may exert beneficial ‘antiaging' effects in humans. Although understanding the role of GH and IIS in the control of human aging is incomplete and somewhat controversial, available data indicate that dietary prevention of excessive IGF-1 and insulin secretion and using diet and exercise to enhance insulin sensitivity may represent the most hopeful approaches to cancer prevention and to extending human health span and life span [2,3,4]. Metformin, rapamycin, and some other compounds with mTOR- and IIS-inhibitory potential (resveratrol, melatonin) are able to modify both aging and carcinogenesis. This chapter focuses on the effects of biguanides and rapamycin, whereas data on resveratrol and melatonin will be discussed elsewhere.

The concept of CR mimetics is now being intensively explored [3]. The antidiabetic biguanides, phenformin, buformin and metformin were observed to reduce hyperglycemia, improve glucose utilization, reduce free fatty acid utilization, gluconeogenesis, serum lipids, insulin, and IGF-1, reduce body weight and decrease metabolic immunodepression both in humans and rodents [3]. The results of studies on the effect of antidiabetic biguanides on the life span in mice and rats are summarized in table 2.

The treatment with phenformin prolonged the mean life span of female C3H/Sn mice by 21% (p < 0.05) and the maximum life span by 26% in comparison with the controls [9]. At the time of death of the last mice in the control group, 42% of phenformin-treated mice were alive. The treatment with phenformin failed to influence the mean life span of female LIO rats; however, it increased the maximum life span by 3 months (10%) in comparison with the controls [9]. The treatment with phenformin slightly decreased the body weight of rats and delayed age-related switching off of estrous function. Similar findings were observed in female rats exposed to another biguanide, buformin [9]. Administration of metformin to female transgenic HER-2/neu mice did not change the body weight or temperature; it slowed down the age-related rise in blood glucose and triglyceride levels, decreased the serum level of cholesterol and β-lipoproteins, delayed the age-related irregularity in estrous cycle, extended the mean life span by 4-8% and the maximum life span by 1 month in comparison with the control animals [10,11]. It is well known that excess of body weight and obesity leads to development of metabolic syndrome, type 2 diabetes, premature switching off of reproductive function and risk of cancer [2,9]. The mechanism behind the geroprotective effect of metformin could reside in its ability to lower body weight.

Metformin increased the mean life span of the last 10% of survivors by 20.8% and the maximum life span by 2.8 months (10.3%) in female SHR mice in comparison with control mice [12]. The decreased body temperature and postponed age-related switching off of estrous function were observed in the group of metformin-treated mice. In another set of experiments, female SHR mice were given metformin from the age of 3, 9 or 15 months [13]. Metformin started at the age of 3 months increased the mean life span by 14% and maximum life span by 1 month, whereas the treatment started at the age of 9 months by 6%; metformin started at the age of 15 months did not affect life span.

The treatment with metformin slightly modified the food consumption but failed to influence the dynamics of body weight. Metformin decreased by 13.4% the mean life span of male 129/Sv mice and slightly increased the mean life span of females (by 4.4%). Metformin failed to influence spontaneous tumor incidence in male 129/Sv mice, decreased 3.5-fold the incidence of malignant neoplasms in female mice, while somewhat stimulated formation of benign vascular tumors [14].

Significant prolongation (by 20.1%) of the survival time was observed in male (but not female) transgenic mice with Huntington's disease without affecting fasting blood glucose levels. Increasing the dose of the drug did not improve the survival of mice [15]. In the NIA study [16], 6-month-old male F344 rats were randomized to one of four diets: control, CR, diet supplemented with metformin and standard diet pair fed to metformin. There were no significant differences in the mean life span of the last surviving 10% of each group in the CR, metformin-treated and pair fed rats as compared with control [16]. CR significantly increased life span in the 25th quantile but not the 50th, 75th, or 90th quantile. The groups of rats exposed to metformin or pair feeding were not significantly different from controls at any quantile. The reduced efficacy of CR in this study might provide a partial explanation for the lack of an increase in life span with metformin.

Male C57BL/6 mice were given ad libitum diet with supplementation of 0.1 or 1% of metformin starting from the age of 12 months until natural death [17]. The mean life span of mice given 0.1% metformin in the diet was increased by 5.83% as compared with the relevant control mice. The 1% dose was toxic and reduced the mean life span by 14.4%. Diet supplementation with 0.1% metformin increased life span by 4.15% in male B6C3F1 mice. There were no significant differences in pathologies observed in both strains of mice fed diet with 0.1% metformin. However, diet with 1% metformin reduced the incidence of liver cancers (3.3% in the metformin group vs. 26.5% in control group, p < 0.001). Male C57BL/6 mice given metformin had decreased rates of cataracts [17]. The treatment with metformin mimics some of the benefits of CR, such as improved physical performance, improved glucose-tolerance test, increased insulin sensitivity, and reduced low-density lipoprotein and cholesterol levels without a decrease in caloric intake. Metformin also increased AMP-activated protein kinase activity and increased antioxidant protection, resulting in reductions in both oxidative damage accumulation and chronic inflammation [17]. The administration of metformin to mice induced CR-like genomic and metabolic responses which were interpreted as induction of pathways associated with longevity.

Thus, available data showed that antidiabetic drugs can increase survival of rodents in some cases (table 2). This effect varied depending on the strain and species of animals. Female mice were treated in the majority of these studies. Due to gender differences in the effect of metformin [3], the experiments with males of different strains need to be performed to draw a conclusion regarding the geroprotective potential of antidiabetic biguanides. Only single studies were performed with female rats treated with buformin or phenformin and with male rats treated with metformin. Both male and female animals of different strains need to be treated in the same study for a more exact conclusion on the geroprotective potential of antidiabetic biguanides.

Routes of administration and doses of metformin were different in the majority of experiments discussed here. The NIA (National Institute of Aging, NIH USA) team used diet supplementation with 0.1 or 1% metformin in male C57BL/6 mice and a diet with 0.1% metformin in male B6C3F1 mice [17], whereas the PRIO (Petrov Research Institute of Oncology, St. Petersburg, Russia) team administered metformin with drinking water at a dose of 100 mg/kg to female HER-2/neu mice in two independent studies, to outbreed female Swiss-derived SHR mice also in two sets of experiments, and in male and female inbred 129/Sv mice in one study [10,11,12,13,14] (table 2). Calculations show that C57BL/6 mice consuming the diet with 0.1% metformin received the drug at a dose ranging from 75 to 100 mg/kg body weight, and B6C3F1 mice at a dose between 67 and 90 mg/kg. They are practically the same doses given with drinking water in our studies. The biggest dose of metformin given to C57BL/6 mice (1% in diet) reduced their mean life span by 14.4%, and at first seems toxic for the kidney as it induces its enlargement, lumpiness and decoloration [17]. The NIA team started the treatment with metformin at the age of 12 months, whereas the PRIO team started the treatment at the age of 2-3.5 months in the majority of experiments. When metformin was given to female SHR mice staring at the age of 3, 9 or 15 months, an attenuation of the effect on life span with the increase in the age at start was observed [13].

On the whole, the data in the literature and the results of our experiments suggest that antidiabetic biguanides are promising interventions for slowing down aging and life span extension. Additional studies are required to provide more information on the optimal doses, appropriate age intervals, and other conditions under which exposure to metformin could prevent premature aging in humans.

In the National Institute on Aging Intervention Testing Program, male and female genetically heterogeneous mice (UM_HET3) aged 600 days were fed with encapsulated rapamycin in diet [18]. On the basis of age at 90% mortality, rapamycin led to an increase in the mean life span by 14% for females and 9% for males. The authors claimed that disease patterns of rapamycin-treated mice did not differ from those of control mice. It was stressed that rapamycin may extend life span by postponing death from cancer, by retarding aging, or both. However, only a small part of mice was really studied pathomorphologically. In the same paper, similar results of the treatment with rapamycin started at the age of 270 days have been reported. In another set of experiments, rapamycin was given with food (14 mg/kg food; 2.24 mg/kg mouse weight per day) to UM_HET3 mice from the age of 9 months [19]. The mean life span was increased by 10% in males and 18% in females. For male mice, 3% of the control and 24% of the rapamycin-treated animals were alive at the age of 90% mortality. Practically the same survival rate was in females. The causes of death were similar in control and rapamycin-treated mice.

In our study [20], fifty-eight 2-month-old female FVB/N transgenic HER-2/neu mice were randomly divided into two groups. The first group of animals received 1.5 mg/kg rapamycin subcutaneously (s.c.) 3 times a week for a period of 2 weeks followed by 2-week intervals. In the second group, mice received solvent without rapamycin and served as controls. Treatment with rapamycin significantly inhibited age-related body weight gain. While control mice constantly gained weight during their life span, mice that received rapamycin demonstrated a very modest weight increase. Most importantly, in the control group, only 4 mice survived until the age of 11 months (14.3%) compared to 13 (43.3%) in the rapamycin-treated group (p < 0.001). Rapamycin treatment slightly increased the mean (+4.1%) and maximal life span (+12.4%). The mean life span of long-living animals (last 10% of survivors) was significantly greater in the group receiving rapamycin (+11%) compared to control. Parameter α of the Gompertz model, which is interpreted as the rate of demographic aging, was 1.8 times lower in the group subjected to rapamycin treatment than in control. A half of control mice develop mammary adenocarcinomas (MAC) by day 206, whereas in rapamycin-treated group, this period was extended to 240 days. Remarkably, rapamycin decreased the mean number of tumors per tumor-bearing mouse by 33.7% and the mean size of MAC by 23.5%.

In another our study, 66 female 129/Sv mice at the age of 2 months were randomly divided into two groups. The first group of animals received rapamycin as HER-2/neu mice, and the second group served as controls. Treatment with rapamycin significantly inhibited age-related weight gain in female mice [21]. While control mice constantly gained weight during their life span, mice that received rapamycin demonstrated a very modest weight increase. As a result, from the 5th to the 23rd month, body weight was increased by 21.9% and 12.4% in the control and rapamycin-treated groups, respectively. The body weight in the rapamycin-treated animals was significantly less compared with the control between the age of 20 and 27 months. Rapamycin slightly affected food consumption in young mice and decreased food consumption by 23% in old mice. There was no significant difference in age-related dynamics of the length of the estrous cycle and in the ratio between the estrous cycle phases in the control and rapamycin-treated groups. However, at the age of 18 months, 46 and 65% of mice had a regular estrous cycle in the control and rapamycin-treated groups, respectively. Most importantly, 35.5% of control mice survived until the age of 800 days compared to 54.3% in the rapamycin-treated group, whereas until the age of 900 days - 9.7 and 31.4%, respectively (p < 0.01). Twenty-tree percent of female mice exposed to rapamycin survived the age at death of the last mouse in the control group. Rapamycin significantly decreased the incidence of spontaneous tumors in these mice as well.

In the study of Komarova et al. [22], rapamycin was given in drinking water to 35 male mice heterozygous for a germline p53 null allele (p53^+/-) beginning at various ages. Thirty-eight intact mice served as controls. The mean life span of animals in the control group was 373 days, whereas in rapamycin-treated mice 410 days. Spontaneous carcinogenesis was significantly delayed in rapamycin-treated mice compared to control mice. Then, during analysis of the results, all mice were subdivided into two groups: ‘young' (receiving rapamycin from the age of 5 months or earlier) and ‘old' (receiving rapamycin starting at 5 months of age or older). The mean life span in rapamycin-treated ‘young' mice reached 480 days, a 3.5-month increase over the control group. Thus, the life-extending effect of rapamycin is more pronounced in the group exposed to the treatment earlier in life.

Nanoformulated micelles of rapamycin, Rapatar, were given as gavage to p53 null (p53^-/-) male mice [23]. The treatment with Rapatar extended the mean life span by 30% and delayed tumor development in highly tumor-prone p53^-/- mice. Mean tumor latencies for the control p53^-/- and Rapatar-treated p53^-/- mice were 161 and 261 days, respectively.

Beginning at 9 weeks of age until natural death, Rb1^+/- (B6.129S2(Cg)-Rb1^tm1Tyj) and Rb1^+/+ mice were fed a diet without or with enterically released formulation of rapamycin [24]. The mean life span of rapamycin-treated female Rb1^+/- mice was increased by 8.9% and by 13.8% in males as compared with the sex-matched controls. Once all Rb1^+/-mice had died, the Rb1^+/+ littermates were euthanized. Approximately 85% of rapamycin-treated versus 50% of controls survived this age in females, whereas 60 versus 25%, respectively, in males. Thus, the life span was extended more in female than in male wild-type littermates. Exposure to rapamycin was followed by inhibition of C-cell thyroid carcinomas in Rb1^+/- mice.

Data on physiological and molecular mechanisms of the beneficial effects of biguanides and rapamycin on life span and inhibitory tumorigenesis have been discussed in several recent papers [3,10,17,22] and are summarized in table 1. The effects of biguanides and rapamycin are presented in accordance with their levels of integration: molecular, cellular/tissue and systemic/organismal. There is a significant similarity in the majority of effects of these two groups of drugs and in the main patterns of their activities as antiaging and anticarcinogenic remedies. It means that the key targets as well as signaling pathways and regulatory signals are also similar. Moreover, there is also sufficient similarity in patterns of changes observed during normal aging and in the process of carcinogenesis. DNA damage response signaling seems to be a key mechanism in the establishment and maintenance of senescence as well as carcinogenesis. Some aspects of the problem have been discussed elsewhere [2]. The available data on cellular senescence in vitro and on accumulation of various human pre-malignant lesions in the cells in vivo provide evidence suggesting that senescence is an effective natural cancer-suppressing mechanism [25]. At the same time, adequate clinical application of therapy-induced ‘accelerated senescence' for prevention, progression, or recurrence of human cancers is still insufficiently understood. The mechanisms underlying the bypass of senescence response in the progression of tumors still have to be discovered. Recent studies reveal a negative side of cellular senescence, which is associated with the secreted inflammatory factors, and may alter the microenvironment in favor of cancer progression designated as syndrome of cancerophilia [26] or senescence-associated secretory phenotype (SASP) [4]. Thus, cellular senescence suppresses the initiation stage of carcinogenesis, but is the promoter for initiated cells. We believe that the similarity between two fundamental processes - aging and carcinogenesis - is a basis for understanding the two-side effects of biguanides and rapamycin (fig. 1). The reasons for the difference in response to metformin and rapamycin in different strains of mice are not well understood. Recent findings provide evidence for inhibitory effects of metformin and rapamycin on the SASP interfering with IKK-β/NF-κB [4,26] - an important step in the hypothalamic programming of systemic aging [27] and in carcinogenesis [2]. It remains to be shown whether antidiabetic biguanides and rapamycin can extend life span in humans.

This paper was supported in part by a grant 6538.212.4 from the President of the Russian Federation. The author is very thankful to Dr. Tatiana V. Pospelova and Dr. Mark A. Zabezhinski for critical reading of the manuscript and valuable comments.