Pharmacological Inhibition of Phosphoinositide 3 and TOR Kinases Improves Survival of Drosophila melanogaster

Abstract

Recent progress in our understanding of genetic mechanisms of aging and longevity provides an opportunity to select some enzymes as targets for pharmacological correction. The phosphoinositide 3-kinase (PI3K) and TOR-kinase cascades are affected in some long-lived mutants of different animals, such as nematodes and mice. The purpose of this study was to investigate the geroprotector efficiency of the inhibitors of enzymes that are known to be affected in long-lived mutants. Experimental animals were exposed to low dozes of LY-294002 (5 μM), wortmannin (0.5 μM), and rapamycin (0.5 μM) separately during their lifetimes. We have shown that the specific PI3K inhibitors (LY-294002 and wortmannin) and the TOR-kinase inhibitor rapamycin slightly increase the median and maximal lifespan of the fruit fly, Drosophila melanogaster.

Introduction

I ncreasing the human lifespan is the most important aim of gerontology. Special attention is focused on the search for and study of chemical compounds and drugs that able to prolong the lifespan of model animals. Many potential geroprotectors have been discovered—antioxidants, hormones, adaptogens, and herbs.^1
–3 Recent progress in our understanding of genetic mechanisms of aging and longevity provides an opportunity to select some enzymes as targets for pharmacological correction. The phosphoinositide 3-kinase (PI3K) cascade and TOR signaling are affected in some long-lived mutants of different animals, such as nematodes^4

–8 and mice.⁹ Nevertheless, the genetic methods of aging correction are not applicable to humans. At the same time, cytologists know specific inhibitors of those enzymes, for instance, LY-294002 and wortmannin for PI3K^10,11 and rapamycin for TOR.¹² The purpose of this study was to investigate the geroprotector efficiency of the inhibitors of PI3K and TOR enzymes.

Materials and Methods

We carried out tests of geroprotector efficiency of LY-294002 and wortmannin (specific inhibitors of PI3K) and rapamycin (specific TOR kinase inhibitor) in Drosophila melanogaster males and females of the wild-type strain Canton-S. Flies were maintained in a thermostatically controlled room at 25 ± 0.5°C and artificial light regime 12:12 h. Lifespan was analyzed in even-aged flies, which were kept in 100-mL glass bottles, containing 20 mL of standard meal, 50 flies of the same sex per vial. Experimental animals were exposed to LY-294002 (5 μM), wortmannin (0.5 μM), and rapamycin (0.5 μM) during their lifetimes. For treatment, we greased fly medium using a paste of hydrolyzed yeast containing one of the substances. Control untreated animals were fed yeast without these substances. Flies were transferred to fresh medium every week. Lifespans were analyzed every day for both sexes separately.

We used nonparametric statistical approaches for the statistic analysis of lifespan parameters. Survival function was estimated with the Kaplan–Meyer method and presented as survival curves. To compare survival function we applied the Kolmogorov–Smirnov test. Gehan–Wilcoxon and Mantel–Cox tests were used for median lifespan comparison. In addition we estimated maximum and minimum lifespan, 90% mortality, α and R₀ parameters of the Gompertz equation (μ[x] = R₀ ^αx), and mortality rate doubling time (MRDT =ln2/α).

Results

Lifespan after LY-294002 treatment

LY-294002 treatment of Drosophila imago males (Table 1) led to an increase of the mean (8.8 %), median (14 %), and maximum (22.5 %) lifespan, as well as 90% mortality (3%). All differences between treatment and control samples were statistically significant (p < 0.001). Decreasing the α parameter and increasing the MRDT (7.4%) demonstrates the delaying of the aging rate after 5 μM LY-294002 treatment. The same data were obtained for females, where statistically significant (p < 0.001) increases of the mean (11.8%), median (14.3%), and maximum (16.3%) lifespan, and 90% mortality (11.9%) were observed. After LY-294002 treatment of females, we also found the drop of the α parameter and elevation of the MRDT (18.8%).

Table 1.

Effects of P13K and TOR-Kinase Inhibitors on Drosophila melanogaster Lifespan

Variant	\documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}\begin{align}{\bar x} \pm \delta m\end{align}\end{document} ± δm	M	Min	Max	90%	α	R₀	MRDT	N
Males
Control 1	51.1 ± 0.7	50	12	71	65	0.1038	0.00030	6.7	243
LY294002 (5 μM)^a	55.6 ± 0.8	57^*	9	87	67	0.0965	0.00030	7.2	263
Control 2	35 ± 0.6	37	5	46	43	0.16298	0.00029	4.3	217
Wortmannin (0.5 μM)^b	36.6 ± 0.6	39^***	4	64	45	0.12857	0.00068	5.4	259
Rapamycin (0.5 μM)^b	37.5 ± 0.5	39^**	7	46	46	0.19978	0.00006	3.5	253
Females
Control 1^a	54.1 ± 0.7	56	9	86	67	0.1002	0.00030	6.9	309
LY294002 (5 μM)	60.5 ± 0.8	64^*	8	100	75	0.0845	0.00030	8.2	323
Control 2	33.4 ± 0.4	34	5	48	43	0.1739	0.0003	4.0	381
Wortmannin (0.5 μM)^b	34 ± 0.6	35	5	72	43	0.07344	0.00456	9.4	291
Rapamycin (0.5 μM)^b	35.2 ± 0.5	36^*	5	64	46	0.12659	0.0009	5.5	277

LY294002 (5 μM) was compared with Control 1.

Wortmannin (0.5 μM) and Rapamycin (0.5 μM) was compared with Control 2.

p < 0.001, ** p < 0.01, ***p < 0.05 (Gehan–Wilcoxon and Mantel–Cox tests).

\documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}\begin{align*}{\bar x} \pm {\Delta m} - x\end{align*}\end{document} ± Δm − x, mean lifespan ± standard error; M, median lifespan; 90%, 90% mortality; Min and Max, minimum and maximum lifespan; α and R₀, parameters of Gompertz equation; MRDT, mortality rate doubling time (ln2/α); N, sample size.

Lifespan after Wortmannin treatment

Wortmannin treatment of males (Table 1) induced an increase of the mean (4%), median (5%), and maximum (39%) lifespan, and 90% mortality (5%). Decreasing the α parameter and increasing the MRDT (27%) demonstrate the delaying of the aging rate after 0.5 μM wortmannin treatment (Table 1). The lifespan differences in females were statistically insignificant (p < 0.05).

Lifespan after rapamycin treatment

Rapamycin treatment of males induced statistically significant (p < 0.01) increases of mean (7%) and median (5%) lifespan as well as 90% mortality (7%). In females, rapamycin treatment induced statistically significant (p < 0.01) increases of mean (5%), median (6%), and maximum (33%) lifespan, and 90% mortality (7%). After rapamycin treatment, a decrease in the α parameter was observed.

Thus, we have shown that the specific PI3K inhibitors (LY-294002 and wortmannin) and TOR-kinase inhibitor rapamycin increased Drosophila melanogaster lifespan. The progress of genetics of aging reveals many other gene products that may be important pharmacological targets in the future; e.g., NF-κB, Nrf2/SKN-1, AAK-2, JNK, FOXO, HSF-1, PTEN, MST-1, etc.

Footnotes

Acknowledgment

This work was supported by a grant from the Presidium of Russian Academy of Sciences.

References

Evason

, Huang

, Yamben

, Covey

, Kornfeld

. Anticonvulsant medications extend worm life-span. Science, 2005; 307:258–262.

Anisimov

. Premature ageing prevention: Limitations and perspectives of pharmacological interventions. Curr. Drug Targets, 2006; 7:1485–1503.

Petrascheck

, Ye

, Buck

. An antidepressant that extends lifespan in adult Caenorhabditis elegans. Nature, 2007; 450:553–556.

Friedman

, Johnson

. A mutation in the age-1 gene in Caenorhabditis elegans lengthens life and reduces hermaphrodite fertility. Genetics, 1988; 118:75–86.

Johnson

. Increased life-span of age-1 mutants in Caenorhabditis elegans and lower Gompertz rate of aging. Science, 1990; 249:908–912.

Kenyon

, Chang

, Gensch

, Rudner

, Tabtiang

. C. elegans mutant that lives twice as long as wild type. Nature, 1993; 366:461–464.

Vellai

, Takacs-Vellai

, Zhang

, Kovacs

, Orosz

, Müller

. Genetics: Influence of TOR kinase on lifespan in C. elegans. Nature, 2003; 426:620.

Ayyadevara

, Alla

, Thaden

, Shmookler Reis

. Remarkable longevity and stress resistance of nematode PI3K-null mutants. Aging Cell, 2008; 7:13–22.

Bartke

. Impact of reduced insulin-like growth factor-1/insulin signaling on aging in mammals: novel findings. Aging Cell, 2008; 7:285–290.

10.

Arcaro

, Wymann

. Wortmannin is a potent phosphatidylinositol 3-kinase inhibitor: The role of phosphatidylinositol 3,4,5-trisphosphate in neutrophil responses. Biochem J., 1993; 296:297–301.

11.

Vlahos

, Matter

, Hui

, Brown

. A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) J Biol Chem., 1994; 269:5241–5248.

12.

Wiederrecht

, Sabers

, Brunn

, Martin

, Dumont

, Abraham

. Mechanism of action of rapamycin: new insights into the regulation of G1-phase progression in eukaryotic cells. Prog. Cell Cycle Res., 1995; 1:53–71.