Date Published: February 23, 2013
Publisher: Impact Journals LLC
Author(s): Carolina B. Livi, Rulon L. Hardman, Barbara A. Christy, Sherry G. Dodds, Diane Jones, Charnae Williams, Randy Strong, Alex Bokov, Martin A. Javors, Yuji Ikeno, Gene Hubbard, Paul Hasty, Zelton Dave Sharp.
Chronic treatment of mice with an enterically released formulation of rapamycin (eRapa) extends median and maximum life span, partly by attenuating cancer. The mechanistic basis of this response is not known. To gain a better understanding of these in vivo effects, we used a defined preclinical model of neuroendocrine cancer, Rb1+/− mice. Previous results showed that diet restriction (DR) had minimal or no effect on the lifespan of Rb1+/− mice, suggesting that the beneficial response to DR is dependent on pRb1. Since long-term eRapa treatment may at least partially mimic chronic DR in lifespan extension, we predicted that it would have a minimal effect in Rb1+/− mice. Beginning at 9 weeks of age until death, we fed Rb1+/− mice a diet without or with eRapa at 14 mg/kg food, which results in an approximate dose of 2.24 mg/kg body weight per day, and yielded rapamycin blood levels of about 4 ng/ml. Surprisingly, we found that eRapa dramatically extended life span of both female and male Rb1+/− mice, and slowed the appearance and growth of pituitary and decreased the incidence of thyroid tumors commonly observed in these mice. In this model, eRapa appears to act differently than DR, suggesting diverse mechanisms of action on survival and anti-tumor effects. In particular the beneficial effects of rapamycin did not depend on the dose of Rb1.
Age is by far the biggest independent risk factor for a wide range of intrinsic diseases , including most types of cancer . The age-adjusted cancer mortality rate for persons over 65 years of age is 15-times greater than for younger individuals . Numerous studies demonstrate that age retarding interventions reduce cancer by decreasing incidence and/or severity (Reviewed in ). Diet restriction (DR) has a long history of retarding cancer  and most of the other age-associated diseases , consistent with life span extension in a wide range of organisms . Genetic interventions resulting in pituitary dwarfism in mice, which causes growth factor reduction (GFR) and a reduction in associated signaling, also result in maximum lifespan extension , with a concomitant reduction in cancer severity [9, 10]. Thus, factors that inhibit growth appear to extend life span and reduce cancer.
To address the question of whether eRapa mimics DR in mice deficient for a prototypical tumor suppressor gene function, we initiated chronic treatment by feeding randomly grouped males and females chow that included either eRapa at the concentration previously shown to extend life span (14 mg/kg food), [16, 17] or Eudragit (empty capsule control). Treatment was begun at approximately 9 weeks of age (>80% of animals started between 8-10 weeks (minimum at 7 weeks and maximum at 12 weeks, Table S1). Mice continued on these diets for the remainder of their lives. Based on the average amount of chow consumed per day, this delivers an approximate rapamycin dose of 2.24 mg/kg body weight/day . Blood levels of rapamycin (determined by a mass spectrometry) averaged 3.9 ng/ml for males, 3.8 ng/ml for females for Rb1+/− mice and 3.4 for males and 4.6 ng/ml for females for Rb1+/+ mice (Figure S1). Hematocrits were performed on blood from Rb1+/+ mice between 18 and 24 months of age and readings indicated normal values for mice (between 40 and 49%), indicating that long-term eRapa treatment does not adversely effect red blood cell production (data not included).
In mice, pRb1 is critical for DR-mediated lifespan extension , but not rapamycin-mediated life span extension. It is unclear why this is the case, since both of these interventions chronically inhibit mTORC1 . However, differences in the downstream in vivo effects of DR and rapamycin have been previously reported . As previously described by Harrison et al. , a distinguishing feature of eRapa is its ability to extend median and maximum life when the intervention starts at a relatively old age (600 days) in mice. By comparison, DR in most  but not all  reports shows little if any longevity benefit when started after 550 days of age (equivalent to 60 human years). DR started at 6 weeks of age reduced body growth for Rb1+/− mice but did not affect growth of Rb1−/− tumors . In contrast to DR, chronic eRapa treatment did not affect body weight of Rb1+/− mice (Livi et al., in preparation), but did reduce tumor growth. Previous studies in fruit flies show that rapamycin extends life span through a mechanism that is at least partly independent of TOR . Consistent with those results, we find that eRapa, but not DR, extended life span and reduced the growth of neuroendocrine tumors in the Rb1+/− model. It will be interesting to determine if pRb1 might be at least partially involved in those settings where responses to chronic eRapa and DR diverge.