Stefano Di Biase1, Lizzia Raffaghello2, Giovanna Bianchi2, Sebastian Brandhorst1-3, Min Wei 1,Valter D. Longo1-3. 1Andrus Gerontology Center, Department ...
Fasting increases rapamycin cancer therapeutic index in the presence or absence of chemotherapy Stefano Di Biase1, Lizzia Raffaghello2, Giovanna Bianchi2, Sebastian Brandhorst1-3, Min Wei 1,Valter D. Longo1-3 1Andrus Gerontology Center, Department of Biological Sciences, University of Southern California, Los Angeles, USA. 2Laboratory of Oncology, G. Gaslini
Institute, Genova, Italy 3L-nutra inc. ,Culver city, USA
ABSTRACT
Short term starvation (STS) augments the efficacy of rapamycin and chemotherapy in reducing tumor progression SH-SY5Y
Ad lib
AP A
A +
R
AP
R
D +
ST S ST
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***
80
150 100 50
A R
ST
S One w ay ANOVA p value £ 0.0001
DXR 8 m g/kg
1500 1000 500
AL
STS
t test p value = 0.0020
R +
XR D +
A
L A 1000 500
Tumor Volume (mm3)
1500
1500 1000 500
Percent survival
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0
4
8
12
16
20
24
28
32
Days after injection One w ay ANOVA p value £ 0.0001
The experiment has been repeated 2 times
The schedule for the stress resistance experiment is reported in the figure on the top. The mice were injected ip with rapamycin for a period of 14 days prior the administration of a lethal dose of the chemo-drugs doxorubicin (DXR). Mice undergoing STS were also fasted for 48h prior DXR injection. In the days following doxorubicin injection the survival of C57BL/6 and Balb/c mice was recorded.
AL + DXR
STS + DXR
t test p value = 0.042
AL + Rapa
STS + Rapa
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2.0
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200 100 0
KB
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2.5
***
1.5 1.0 0.5
AL
STS
Rapa
Rapa + STS
Results from measurement done in 3/5 different experiments
0.0
AL
STS
Rapa
Rapa + STS
Results from measurement done in 2/3 different experiments
1000
500
0
0
0
0
In vivo fasting: Animals were fasted for a total of 48 to 60 hours to allow a 20% bodyweight loss by complete deprivation of food but with free access to water. In addition, in order to avoid dehydration, the mice were fed a low caloric hydrogel. Mice were individually housed in a clean new cage to reduce cannibalism, coprophagy, and residual chow. Body weight was measured immediately before, during and after fasting.
STS + Rapa (n=10)
One Way anova p value £ 0.0001
1500
2000
Tumor Volume (mm3)
Tumor Volume (mm3)
Tumor Volume (mm3)
*
2000
1000
L
+ L A
Green shaded areas represent STS cycles
2000
32
AL + Rapa (n=10) STS (n=10)
300
X + + R R ap Rap a + a D XR
44
**
28
AL (n=10)
DXR 16 m g/kg
1000
Days after Tumor Inoculation
3000
24
Days after injection
400
D
40
100 90 80 70 60 50 40 30 20 10 0
2000
L
36
20
Blood Glucose
A
32
16
AP
AP
R + S S
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***
***
0 28
12
3
Tumor Volume (mm )
2000
24
8
A
A
XR D
+ ST
3000
Ad Lib Ad Lib + Rapa Ad Lib + DXR Ad Lib + Rapa + DXR STS + Rapa + DXR STS STS + Rapa STS + DXR
16 20 1st day Rapa injection
4
Survival Balb/c
mg/dl
2500
0
0
Tumor size day 45 Ad lib STS
Tumor growth (4T1) in 16 months old mice DXR 8 m g/kg
In vitro Short Term Starvation (STS): Cellular fasting was done by glucose and serum restriction to achieve blood glucose levels typical of fasted and normally fed mice. The fasting medium contained 0.5 g/L of glucose and 1% of serum (FBS) while the normal medium (or Ad lib) contained 2 g/L glucose and 10% serum (FBS). For human cell lines, normal glucose was considered to be 1.0 g/liter and fasting conditions were achieved by reducing serum (FCS) from 10% (normal) to 1% (low). Cells were washed twice with PBS before changing to fasting medium.
*
b -HB (mM)
AL
+
D
ST
S
XR
+
+
AP
AP
R
D +
S ST
+ XR
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D
A
XR
S
R
AP
ST
A
A
R +
+ AL
AL
AP
XR D
AL
0
DXR 8 m g/kg
ST
Four cancer cancer cell lines were tested for the response to rapamycin (RAPA) and chemotherapy drugs doxorubicin (DXR) and TVD (topotecan + vincristine + DXR) under ad lib feeding (AL) or STS regimen. The cell line tested were NXS2 (murine neuroblastoma), ACN and SHSY5Y (Human neuroblastoma) and 4T1 (murine breast cancer). The cell viability was determined by Trypan blue staining for the neuroblastoma cell lines and MTT assay for the breast cancer cell line. The effect of the treatment was also tested in MEF cells (mouse embryonic fibroblasts) and the damage is reported as LDH release
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200
S S ST
+ XR
D + AL
+ S ST
*
METHODS
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One w ay ANOVA p value £ 0.0001
STS
** ***
*
DXR 24 m g/kg
ST S S TS ST S + D X S TS + + R R ap Rap a + a D XR
250
ST
A R
R
AL
+
+ AL
AP
XR D
+ R D XR AP A + R AP A
D XR S ST
+
D
XR
ST
+
S
+
R
ST
AP A + R AP A
XR D
+ AL
AL
MEF
AL (n=19) AL + Rapa (n=19) STS (n=10) STS + Rapa (n=10)
The experment has been repeated 3 times A
0 AL
0
100 90 80 70 60 50 40 30 20 10 0
One w ay ANOVA p value £ 0.0001
AP
MTT
20
S
20
AL
40
AL
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60 40
• The increased blood glucose levels observed during rapamycin administration may be the reason of the increased sensitivity to doxorubicin.
TV
S
ST
STS
100
60
Check survival
Survival C57BL/6
Percent survival
120
80
Ad lib
LDH % to Ad lib
+
+
+
+ +
S
AL
4T1
Ad lib
STS
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INTRODCUTION
Tumor Volume (mm3)
TV D
S ST
A R AP A
+ AL
TV +
D
AL
A R
AP
A D
+ S ST
+ AL
ST
one w ay ANOVA p value £ 0.0001
One w ay ANOVA p value £ 0.0001
Short-term starvation (STS) have been shown to be an effective practice in the sensitization of a variety of cancer cell types to chemotherapy (Differential Stress Sensitization), while protecting normal cells and tissues (Differential Stress Resistance). Multiple changes occur during STS at the molecular and physiological levels including (but not exclusively) the down-regulation of IGF-I, and signal transduction proteins such as mTOR and PKA. In addition, our dietary intervention causes reduction in a variety of amino acids, insulin and glucose and the increase of ketone bodies, forcing cells to switch and rely on respiration for energy production and survival. However, virtually all cancer cells have mutations in oncogenes, which make them unresponsive to the changes outside, but also inside, the cell and unable to switch to an alternative pathway. All these molecules, actors and proteins modulate normal-cell growth in part by regulating serine/threonine protein kinases, which lead to the idea of administrating STS with or without kinase inhibitors. Whereas the specific kinase and other growth inhibitors can reduce cancer progression, particularly if the inhibitor acts downstream of the oncogenic mutation, the multiple changes induced by STS can promote an additional growth inhibitory effect. Thus, the combination of STS with specific kinase inhibitor is expected to play an additive or synergistic pro-death role in tumor cell growth, which could be further potentiated by chemotherapy. In addition, kinases and other signal transduction inhibitors can delay cancer growth but also cause major side effects to normal cells. STS is expected to reverse a number of these side effects.
Daily injection (14 days) with Rapamycin 1.5 mg/Kg/mouse
Time
NXS2
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***
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***
one w ay ANOVA p value £ 0.0001
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• The toxic effect observed during the administration of rapamycin in combination with doxorubicin can be reversed by STS.
STS (48h)
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TV +
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AL
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A AP R
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Single injection of doxorubicin
40 20
Ad lib
Inject mice daily with Rapamycin (ip)
• Short-term starvation (STS) increases the efficacy of rapamycin and doxorubicin in decreasing tumor progression.
60
20
AL
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TV
40
80
D
60
AL
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R AP
% viability
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80
120
STS can reverse the sensitization of normal tissue to chemotherapy induced by rapamycin treatment
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120 100
STS
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TV
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100
% viability
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STS
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120
CONCLUSIONS
ACN
Ad lib
% viability
Short-term starvation (STS) has been shown to be effective in both protection of normal cell and tissues and the sensitization of a variety of tumors during chemotherapy treatment. This condition of protection/sensitization of different cell types under the same condition has been named differential stress resistance (DSR). Although the effects of fasting have been mainly attributed to reduced glucose and circulating IGF1 levels, the involvement of signaling genes regulated by glucose and growth factors in these effects of fasting remain poorly understood. In this study we have administered the mTOR inhibitor rapamycin in combination with STS and with the chemotherapy drug doxorubicin to shed light on the role of mTOR in the STS response and on its role in mediating DSR. In a mouse allograft model for breast cancer (4T1) we show that STS and rapamycin have an additive effect in reducing tumor progression, which confirms the possibility that STS-mediated effects on cancer cells are not due to the up-regulation of mTOR. Surprisingly, we observed that the administration of rapamycin during chemotherapy sensitizes the mice to the drug leading to an increased mortality. However, this sensitizing effect could be reversed by STS.
RESULTS
AL + DXR + Rapa
STS + DXR + Rapa
Tumor progression was observed in middle aged (16-month-old) female Balb/c mice treated with Rapamycin, STS or the combination of the two treatments. In addition, the effect of chemotherapy was evaluated. Rapamycin was daily administrated intraperitoneally for the whole duration of the experiment.
Rapamycin treatment induce an insulin-resistance-like state causing an increase of blood glucose (left). This increased glucose level may partially mediate the sensitization to chemotherapy seen in animals treated with Rapa. The decreased blood glucose in Rapa + STS mice supports this hypothesis (mice treated with Rapa + STS are more resistant than mice treated with rapamycin alone). As expected, the ketone bodies (KB) levels were significantly increased during STS, with or without rapamycin administration (b).
Tumor progression: For our murine breast cancer model, female Balb/c mice were injected subcutaneously s.c. in the right flank with 20.000 4Tl cancer cells and ad lib fed with standard diet until the tumor was palpable. At this point the tumor volume was determined and the mice were arranged in the following experimental groups; ad lib (ad libitum feeding), STS, DXR, STS + DXR. In order to observe the response to every treatment in presence or not of rapamycin, each group was present as duplicate but only one underwent rapamycin treatment. The mice were subject to a daily injection of rapamycin and to a multi-cycle treatment (for a total of three) where each cycle was composed as follows: In short, mice belonging to the STS and STS + DXR groups underwent 48 to 72h of complete food deprivation with free access to water and then re-fed with standard chow diet for l0 days between cycles. The animals from the STS + DXR group were also injected iv with Doxorubicin at the end of the first, second and third cycle respectively. The animals from DXR group were ad lib fed and injected with Doxorubicin as well. Stress resistance: 12 weeks old female BALB/c and C57BL/6 mice were divided in the following experimental groups; ad lib (ad libitum feeding), STS, DXR, STS + DXR. In order to observe the response to every treatment in presence or not of rapamycin, each group was present as duplicate but only one underwent rapamycin treatment. The administration of rapamycin was performed for a period of l4 days at the end of which a high dose of doxorubicin was administrated iv. The animals belonging to the STS + DXR groups were fed a very low calorie and no protein for 48h prior the injection of doxorubicin. Following doxorubicin injection the animal were monitored every day and the survival was recorded.
