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Inhibition of the growth of Ehrlich ascites tumors by treatment with the respiratory inhibitor rotenone
Europ. J. Cancer Vol. 9, pp. 529-531. Pergamon Press 1973. Printed in Great Britain
Letter to the Editor Inhibition of the Growth of Ehrlich Ascites Tumors by Treatment with the Respiratory Inhibitor Rotenone* M. J. FIGUERAS and MARIO GOSALVEZ Citogene'tica and Bioqulmica Experimental, Cllnica Puerta de Hierro, UniversidadAutonoma, Madrid, Spain
Ix WAS suggested, in a previous paper [1], that the anti-cancer agent VM-26 could exert its antimitotic action through the inhibition of cellular respiration with NADH-linked substrates. In order to investigate whether the inhibition of NADH-linked respiration could represent a new approach to experimental anti-cancer chemotherapy, we have studied the effects of the known mitochondrial inhibitor rotenone [2] on the growth, survival, and mitotic fraction of Ehrlich ascites tumors. Thirty-six mice (Swiss strain) weighing 25 g each were injected intraperitoneally with a massive inoculation of l0 x l06 washed Ehrlich ascites cells. Beginning with the 24th hr after inoculation, 15 mice were injected daily with 3 mg of rotenone (K & K laboratories) per kg of animal weight dissolved in 0.1 ml of sunflower oil. Fifteen controls were injected with 0-1 ml of the solvent alone. The treatment was continued until the animals died, and the weight and survival times of the animals in both groups were recorded. The 6 remaining mice were allowed to develop a tumor during 6 days and 19 hr, after which 3 mice were injected with a single dose of 80 #g of rotenone dissolved in 0.1 ml of 25% ethanol. The other 3 mice were injected with the solvent alone. Samples of ascites liquid were collected from these 6 mice
t.5.
ntrols
80~ \ ~ 1 \
\Rotenone \ tre°ted
E
~:
3s
| g 30 ted
2
/4
6
8 10 12 V~ 15 Doys
°1
2
~
6
8 10 12 16 |6 Ocys
Fig. 1. (Left) : Average weight of two groups of fifteen mice during 16 days after inoculation with 10x 10 e Ehrlich ascites cells. Controls: group injected intraperitoncally with the solvent daily. Rotenone treated: groups injected intraperitoneally with 80 pg of rotenone per animal daily. The standard deviation (not shown) of each value of average weight ranged between 0"5 and I g, depending on the number of surviving animals. Total packed cell volume reached 3"5 ml in the controls on the 12th day while it was undetectable at necropsy in the treated group. (Right) : Percentage of surviving animals in the same experiment as that shown by the figure on the left.
at 1, 2, 4 and 6 hr after the injection ofrotenone, and cell smears were stained with May Grunwald-Giemsa for evaluation of the mitotic fraction and percentage of mitotic phases. Figure 1 (left) shows the average weight of both groups of 15 mice during 16 days following the transplant of the tumor. It can be observed that the control group gained weight progressively until the 12th day due to the growth of the tumor in each animal. In comparison, the average weight of the 15 mice treated with rotenone was maintained at steady
Accepted 7 May 1973. *This work has been supported by grant 12-079-72 from the Instituto Nacional de Previsi6n.
529
530
M . J. Figueras and Mario Gosalvez Controls
shows 100% survival up to the 10th day, which compares favorably with the 13% survival in the control group at the same time. After the 10th day, a high mortality rate in the rotenone treated group can be seen due to drug toxicity, and on the 16th day the last animal died. The toxicity of rotenone seems to be due to severe hepatic injury (fat degeneration). The mean life span of the control group was 6 days and of the treated group, 12.7 days, Thus, there was a 110% increase of mean life span in the treated group. Figure 2 shows the average mitotic fraction and mitotic phases per thousand cells in the two groups of 3 animals treated with a single injection the 7th day after transplant (3000 cells in each sample were counted for each animal). It can be observed that in the control group the mitotic fraction and the percentage of mitotic phases remain almost steady during the 6 hr of observation. The rotenone treated group shows a marked increase in the percentage of cells in metaphase at the 6th hr after the injection, and this increase in the main cause of the increase in the mitotic fraction at this moment. A discrete increase in prophases and a discrete decrease in telophases and anaphases are also observed in the samples of the rotenone-treated group. Figures 1 and 2 represent the typical results of experiments realized under optimal conditions. Doses of rotenone less than 3 mg per kg of weight, subcutaneous injections of the drug, and shorter treatments show essentially similar results. Table 1 shows a dose-response relationship for the effects of rotenone against the growth ofEhrlich ascites tumors. From our results it can be concluded that rotenone inhibits the growth of Ehrlich ascites tumors and increases temporarily the survival
20!
"~ (3
11.
hr
o
3
N
.
2~ ° m -...:-
20
¢'
....
..---
hr
Fig. 2. Evolution during 6 hours of the average mitotic fraction and mitotic phases per thousand Ehrlich ascites cells in two groups of 3 mice. Controls : group treated with a single injection of the solvent at zero time. Rotenone treated: group treated with a single injection of 80 Itg of rotenone per animal at zero time. Closed circles: prophases; open circles: metaphases; open squares: telophases; closed triangles: anaphases; closed squares: mitoticfraction.
values due to the absence of tumor growth in this series. Ascitic fluid and cells were undetectable in these animals at necropsy. Rotenone does not cause a weight loss in normal mice at the dose used (3 mg/kg), which is ~ of the LDso. Figure 1 (right) shows the percentage of surviving animals. It can be observed that the control group shows a high mortality rate from the 6th to the 7th day, and on the 15th day the last animal died. The rotenone-treated group Table 1.
Inoculum
I0 x 106 cells i.p. 10 x 106 cells i.p. 10 x I06 cells i.p. 3 x l0 s cells i.p.
Dose-response relationship of the effect of rotenone on Ehrlich ascites tumors
Host
Swiss Swiss Swiss Swiss
Treatment Start day Injection 2nd 2nd 2nd 2nd
daily, 8 days daily, 8 days daily, 8 days daily, 16 days
Dose (mg/kg) % Anti-tumor activity By tumor By packed weight cell volume 2 3 4 3.5
66 74.8 83.9 100
67 83.3 68 100
Mean increase life span
No. of exp.
(%) 83 110 100 100
2 3 2 2
Each experiment included 30 treated animals and 30 controls. Fifteen animals of each group were used for evaluation by tumor weight and survival time and the other fifteen for evaluation by total packed celt volume. Tumor weight was calculated by difference between inoculated controls and non inoculated rotenone treated animals (10 normal animals for each dose level). Rotenone did not cause weight loss at the used doses. The figures of antitumor activity and increase of mean life span are the average of 2 or 3 experiments as indicated.
Inhibition of the Growth of Ehrlich Ascites Tumors time of the animals. The inhibition of growth seems to be due to the arrest of mitosis in metaphase. Rotenone has been shown to be a very potent agent which blocks electron transport in the mitochondrial respiratory chain at site I of oxidative phosphorylation [2]. Therefore, its main action is the inhibition of respiration with NADH-linked substrates, as we reported for the anti-cancer agent V M - 2 6 [1]. One of the effects of low doses of V M - 2 6 is a transient arrest of mitosis in metaphase [3]. Meisner and Sorensen [4] showed in vitro that rotenone blocks mitosis in metaphase. Due to the lack of release of the mitotic inhibition with the partial release of respiration adding succinate, these authors estimated that the action of rotenone on mitosis did not appear to be related
to its action on mitochondrial respiration. However, Tobey et al. [5], more recently, showed that several mitochondrial inhibitors such as rotenone, antimycin, oligomycin, dinitrophenol, prevent the completion of mitosis and specified that a respiration-linked process is required during mitosis for progress through metaphase. The antimitotic action ofrotenone could, consequently, be explained by the inhibition of the production of the energy necessary for the metaphase-anaphase transition [5]. The high toxicity of rotenone and its low therapeutic index make it a poor candidate for further studies as a chemotherapeutic agent. However, the action of rotenone reported here indicates that an anti-cancer chemotherapy based on the inhibitors of NADH-linked respiration may be worth investigating.
REFERENCES 1. 2. 3. 4. 5.
531
M. GOSALVEZ,J. PEREz-GAREIA and M. LOPEZ, Inhibition of NADH-linked respiration with the anti-cancer agent 4'-demethylepipodophyllotoxin thenylidene glucoside (VM-26). Europ. or. Cancer. 8, 471 (1972). B. CHANCEand G. HOLLUNGER, Inhibition of electron and energy transfer in mitochondria. I. Effects of amytal, thiopental, rotenone, progesterone and methylene glycol. J. biol. Chem. 278, 418 (1963). H. ST~,HU.Lm,4'-Demethyl-epipodophyllotoxin thenylidene glucoside (VM-26), a podophyllum compound with a new mechanism of action. Europ. J. Cancer. 6, 303 (1970). H . M . M~.ISNERand L. SO~NSEN, Metaphase arrest of Chinese hamster cells with rotenone. Exp. Cell Res. 42, 291 (1966). R . A . TOBEY, D. F. PEa'EmEN and E. C. ANDERSON,Energy requirements for mitosis in Chinese hamster cells. In Biochemistry of Cell Division (Edited by R. BASEROA),pp. 39--56. Ch. Thomas (1969).
Letter to the Editor Inhibition of the Growth of Ehrlich Ascites Tumors by Treatment with the Respiratory Inhibitor Rotenone* M. J. FIGUERAS and MARIO GOSALVEZ Citogene'tica and Bioqulmica Experimental, Cllnica Puerta de Hierro, UniversidadAutonoma, Madrid, Spain
Ix WAS suggested, in a previous paper [1], that the anti-cancer agent VM-26 could exert its antimitotic action through the inhibition of cellular respiration with NADH-linked substrates. In order to investigate whether the inhibition of NADH-linked respiration could represent a new approach to experimental anti-cancer chemotherapy, we have studied the effects of the known mitochondrial inhibitor rotenone [2] on the growth, survival, and mitotic fraction of Ehrlich ascites tumors. Thirty-six mice (Swiss strain) weighing 25 g each were injected intraperitoneally with a massive inoculation of l0 x l06 washed Ehrlich ascites cells. Beginning with the 24th hr after inoculation, 15 mice were injected daily with 3 mg of rotenone (K & K laboratories) per kg of animal weight dissolved in 0.1 ml of sunflower oil. Fifteen controls were injected with 0-1 ml of the solvent alone. The treatment was continued until the animals died, and the weight and survival times of the animals in both groups were recorded. The 6 remaining mice were allowed to develop a tumor during 6 days and 19 hr, after which 3 mice were injected with a single dose of 80 #g of rotenone dissolved in 0.1 ml of 25% ethanol. The other 3 mice were injected with the solvent alone. Samples of ascites liquid were collected from these 6 mice
t.5.
ntrols
80~ \ ~ 1 \
\Rotenone \ tre°ted
E
~:
3s
| g 30 ted
2
/4
6
8 10 12 V~ 15 Doys
°1
2
~
6
8 10 12 16 |6 Ocys
Fig. 1. (Left) : Average weight of two groups of fifteen mice during 16 days after inoculation with 10x 10 e Ehrlich ascites cells. Controls: group injected intraperitoncally with the solvent daily. Rotenone treated: groups injected intraperitoneally with 80 pg of rotenone per animal daily. The standard deviation (not shown) of each value of average weight ranged between 0"5 and I g, depending on the number of surviving animals. Total packed cell volume reached 3"5 ml in the controls on the 12th day while it was undetectable at necropsy in the treated group. (Right) : Percentage of surviving animals in the same experiment as that shown by the figure on the left.
at 1, 2, 4 and 6 hr after the injection ofrotenone, and cell smears were stained with May Grunwald-Giemsa for evaluation of the mitotic fraction and percentage of mitotic phases. Figure 1 (left) shows the average weight of both groups of 15 mice during 16 days following the transplant of the tumor. It can be observed that the control group gained weight progressively until the 12th day due to the growth of the tumor in each animal. In comparison, the average weight of the 15 mice treated with rotenone was maintained at steady
Accepted 7 May 1973. *This work has been supported by grant 12-079-72 from the Instituto Nacional de Previsi6n.
529
530
M . J. Figueras and Mario Gosalvez Controls
shows 100% survival up to the 10th day, which compares favorably with the 13% survival in the control group at the same time. After the 10th day, a high mortality rate in the rotenone treated group can be seen due to drug toxicity, and on the 16th day the last animal died. The toxicity of rotenone seems to be due to severe hepatic injury (fat degeneration). The mean life span of the control group was 6 days and of the treated group, 12.7 days, Thus, there was a 110% increase of mean life span in the treated group. Figure 2 shows the average mitotic fraction and mitotic phases per thousand cells in the two groups of 3 animals treated with a single injection the 7th day after transplant (3000 cells in each sample were counted for each animal). It can be observed that in the control group the mitotic fraction and the percentage of mitotic phases remain almost steady during the 6 hr of observation. The rotenone treated group shows a marked increase in the percentage of cells in metaphase at the 6th hr after the injection, and this increase in the main cause of the increase in the mitotic fraction at this moment. A discrete increase in prophases and a discrete decrease in telophases and anaphases are also observed in the samples of the rotenone-treated group. Figures 1 and 2 represent the typical results of experiments realized under optimal conditions. Doses of rotenone less than 3 mg per kg of weight, subcutaneous injections of the drug, and shorter treatments show essentially similar results. Table 1 shows a dose-response relationship for the effects of rotenone against the growth ofEhrlich ascites tumors. From our results it can be concluded that rotenone inhibits the growth of Ehrlich ascites tumors and increases temporarily the survival
20!
"~ (3
11.
hr
o
3
N
.
2~ ° m -...:-
20
¢'
....
..---
hr
Fig. 2. Evolution during 6 hours of the average mitotic fraction and mitotic phases per thousand Ehrlich ascites cells in two groups of 3 mice. Controls : group treated with a single injection of the solvent at zero time. Rotenone treated: group treated with a single injection of 80 Itg of rotenone per animal at zero time. Closed circles: prophases; open circles: metaphases; open squares: telophases; closed triangles: anaphases; closed squares: mitoticfraction.
values due to the absence of tumor growth in this series. Ascitic fluid and cells were undetectable in these animals at necropsy. Rotenone does not cause a weight loss in normal mice at the dose used (3 mg/kg), which is ~ of the LDso. Figure 1 (right) shows the percentage of surviving animals. It can be observed that the control group shows a high mortality rate from the 6th to the 7th day, and on the 15th day the last animal died. The rotenone-treated group Table 1.
Inoculum
I0 x 106 cells i.p. 10 x 106 cells i.p. 10 x I06 cells i.p. 3 x l0 s cells i.p.
Dose-response relationship of the effect of rotenone on Ehrlich ascites tumors
Host
Swiss Swiss Swiss Swiss
Treatment Start day Injection 2nd 2nd 2nd 2nd
daily, 8 days daily, 8 days daily, 8 days daily, 16 days
Dose (mg/kg) % Anti-tumor activity By tumor By packed weight cell volume 2 3 4 3.5
66 74.8 83.9 100
67 83.3 68 100
Mean increase life span
No. of exp.
(%) 83 110 100 100
2 3 2 2
Each experiment included 30 treated animals and 30 controls. Fifteen animals of each group were used for evaluation by tumor weight and survival time and the other fifteen for evaluation by total packed celt volume. Tumor weight was calculated by difference between inoculated controls and non inoculated rotenone treated animals (10 normal animals for each dose level). Rotenone did not cause weight loss at the used doses. The figures of antitumor activity and increase of mean life span are the average of 2 or 3 experiments as indicated.
Inhibition of the Growth of Ehrlich Ascites Tumors time of the animals. The inhibition of growth seems to be due to the arrest of mitosis in metaphase. Rotenone has been shown to be a very potent agent which blocks electron transport in the mitochondrial respiratory chain at site I of oxidative phosphorylation [2]. Therefore, its main action is the inhibition of respiration with NADH-linked substrates, as we reported for the anti-cancer agent V M - 2 6 [1]. One of the effects of low doses of V M - 2 6 is a transient arrest of mitosis in metaphase [3]. Meisner and Sorensen [4] showed in vitro that rotenone blocks mitosis in metaphase. Due to the lack of release of the mitotic inhibition with the partial release of respiration adding succinate, these authors estimated that the action of rotenone on mitosis did not appear to be related
to its action on mitochondrial respiration. However, Tobey et al. [5], more recently, showed that several mitochondrial inhibitors such as rotenone, antimycin, oligomycin, dinitrophenol, prevent the completion of mitosis and specified that a respiration-linked process is required during mitosis for progress through metaphase. The antimitotic action ofrotenone could, consequently, be explained by the inhibition of the production of the energy necessary for the metaphase-anaphase transition [5]. The high toxicity of rotenone and its low therapeutic index make it a poor candidate for further studies as a chemotherapeutic agent. However, the action of rotenone reported here indicates that an anti-cancer chemotherapy based on the inhibitors of NADH-linked respiration may be worth investigating.
REFERENCES 1. 2. 3. 4. 5.
531
M. GOSALVEZ,J. PEREz-GAREIA and M. LOPEZ, Inhibition of NADH-linked respiration with the anti-cancer agent 4'-demethylepipodophyllotoxin thenylidene glucoside (VM-26). Europ. or. Cancer. 8, 471 (1972). B. CHANCEand G. HOLLUNGER, Inhibition of electron and energy transfer in mitochondria. I. Effects of amytal, thiopental, rotenone, progesterone and methylene glycol. J. biol. Chem. 278, 418 (1963). H. ST~,HU.Lm,4'-Demethyl-epipodophyllotoxin thenylidene glucoside (VM-26), a podophyllum compound with a new mechanism of action. Europ. J. Cancer. 6, 303 (1970). H . M . M~.ISNERand L. SO~NSEN, Metaphase arrest of Chinese hamster cells with rotenone. Exp. Cell Res. 42, 291 (1966). R . A . TOBEY, D. F. PEa'EmEN and E. C. ANDERSON,Energy requirements for mitosis in Chinese hamster cells. In Biochemistry of Cell Division (Edited by R. BASEROA),pp. 39--56. Ch. Thomas (1969).