@Copyright 1987 by The Humana Press Inc. All rights of any nature, whatsoever, reserved. 0163-4984/87/140(04Y329502.80
Differential Effects of Selenium on the Proliferation of Human Pulmonary Adenocarcinoma Cells and Human Embryonic Lung Diploid Cells In Vitro Ao PUNG, ZHAOMEI, AND SHU-YU YU* Cancer Institute, Chinese Academy of Medical Sciences, Beijing, China Received August 5, 1986; Accepted September 25, 1986
ABSTRACT The effect of different concentrations of Na2SeO3 on human pulmonary adenocarcinoma cells and human embryonic lung diploid cells in vitro was investigated. For human pulmonary adenocarcinoma cells, mitotic index and cell count decreased with increasing selenium concentrations. At 1 i~g/mL sodium selenite, mitotic activity and growth of human lung cancer cells were partially inhibited, and the progression of human lung cancer cell cycle was partially arrested. When human embryonic lung diploid cells were treated with 1 lag/mL sodium selenite for five continuous days, cell counts of the treated group were closely parallel to those of the control group. After treating human embryonic lung diploid cells with 1-51ag/mL sodium selenite for 1-3 d, the mitotic index (MI), labeled index (LI), and average silver grain (SG) number per 20 labeled nuclei were the same as those of the control. In mixed cultures of human embryonic lung diploid cells and human pulmonary adenocarcinoma cells, treated with 3 and 5 I~g/mL sodium selenite for 24 h, the lung diploid cells showed a normal fusiform morphology, whereas the lung cancer cells showed heavily vacuolated cytoplasms and distorted nuclei. *Author to whom all correspondence and reprint requests should be addressed. Biological Trace Element Research
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Index Entries: Selenium; human embryonic lung diploid cells; human pulmonary adenocarcinoma cells; percentage of labeled mitoses; pulse label; mitotic index (MI); labeled index (LI); average number of silver grain; vacuolated cytoplasm; progression of cell cycle.
INTRODUCTION With the increased recognition of the importance of selenium in the chemoprevention of cancer (1), research work has turned to the chemoprevention of respective cancers, such as mammary neoplasia (2,3) or human hepatoma (4). Lung cancer is of particular interest because its incidence is increasing all over the world. After surveying 27 countries, Schrauzer listed lung cancer in "Group A", which is highly inversely proportional to the dietary selenium intakes (5). Subsequently, an article comparing blood selenium levels with the incidence of lung cancer in China has been published (6). Also, in China, yeast containing Se has been used for the first time to decrease the incidence of 3-MCA-induced lung cancer in Wistar rats (7). In addition, selective inhibitory effects of selenium on the proliferation of human hepatoma cells (8) and human esophageal cancer cells have been observed (9). The present work is aimed at understanding the effects of selenium on the growth of human pulmonary adenocarcinoma cells.
/
,TERIALS AND METHODS
Cell Culture and Chemicals Human pulmonary adenocarcinoma cells LTEP-a-2 (10) and SPC-A-1 (11) were cultured in 1640-medium-supplemented, 20% heat-inactivated calf serum and penicillin and streptomycin, each at 100 U/mL. Monolayer living cells in stock culture were passaged with a mixture of 0.5% trypsin (Difco 1:250) and 0.2% EDTA. From these, counted cells were seeded into glass culture bottles for use. Human embryonic lung diploid fibroblasts were kindly provided by the Department of Cell Biology of this Cancer Institute. The complete medium used for this kind of cell was Eagle's basal medium from Japan, with 20% heat-inactivated calf serum plus 100 U of penicillin and 100 U streptomycin/mL added to the complete medium. For the experiment, the 8th -12 th passage cells were used (microphotograph 1). Sodium selenite (Beijing Chemicals factory product, C.P. 79038) was dissolved in triply distilled water at I mg/mL, the working solution at 200 ~g/mL was renewed monthly; 2 ~g/10 p.L was added into a 2-mL medium with a micropipet.
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Microphotograph 1. Embryonic lung fibroblasts of human origin, in the passage. There are two fusiform cells in the center of the field. One of them has entered prophase of division. 8 th
Mitotic Index Counted human lung cancer cells were seeded into cover-slipped glass culture bottles. Sodium selenite was added at log phase of growth. After 24 h, cover slips were fixed with methanol and stained with Giemsa. Mitotic figures were counted per 1000 cells. Human embryonic lung diploid cells were treated with 1, 3, and 5 ~g/mL sodium selenite for 3 d. Cells in control and treated groups were pulse labeled daily with 0.4 ~,Ci/mL of tritiated thymidine (specific activity, 41.3 Ci/mM) for 1 h. Mitotic indices (MIs) were counted on the autoradiography slide. Meanwhile, labeled index (LI) and the numbers of silver grains in 20 labeled nuclei were determined, and the mean values were compared with the data from the controls.
Growth Curves Counted cell suspensions were seeded into glass culture bottles, and sodium selenite was added at log phase of growth. After 24 h of exposure 1 ~g/mL, cells in three bottles of each group were trypsinized and counted; the mean value was compared with that of the control group. The Se-containing medium was then discarded from the remaining bottles, washed with prewarmed PBS, and fed with new medium. The same treatment was applied to the control group. The cells in each group were counted daily until a confluent density was reached. Human embryonic lung diploid cells were continuously treated with 1 ~g/mL sodium selenite for 5 d; cells in each group were counted daily.
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Effects on Progression of Cell Cycle Effects on S Phase Cells
Both control and treated groups of human pulmonary adenocarcinoma cells were pulse labeled with tritiated thymidine, 0.4 tzCi/mL for I h at different intervals after 1 i~g/mL sodium selenite exposure (12). Labeled index and number of silver grains in 20 labeled nuclei were counted and compared with each other. Effects on the Progression of S--G2--/vl Phases Vincristine, 0.2 l~g/mL, was screened as an optimum arrest concentration and continuous labeling and metaphase-arresting methods were performed (12). After 1 I~g/mL sodium selenite exposure, samples were taken at different intervals for autoradiography and determination of the percentage of labeled mitoses (PLM).
Morphological Observations Human embryonic lung diploid cells were planted into cover-slipped glass culture bottles. When grown to half-confluence, human pulmonary adenocarcinoma cells LTEP-a-2 were planted into the abovementioned culture bottles. The mixed culture of the two kinds of cells was treated with 3 and 5 p,g/mL sodium selenite for 24 h. Cover slips were fixed and stained for observing toxic changes in the nucleus and cytoplasm.
RESGLTS /~itotic Index Effects of various concentrations of sodium selenite on the mitotic activity of human pulmonary adenocarcinoma cells LTEP-a-2 are shown in Fig. 1. The MI in the I p,g/mL-treated group was only half of that in the
%MI 60 50 40 30 20 10
~ ~ SeConcentration 1 2 3 (l~g/mL) Fig. 1. Mitotic index in h u m a n p u l m o n a r y adenocarcinoma cell line LTEP-a-2 treated w i t h sodium selenite at 1, 2, and 3 i~g/mL for 24 h. 0
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control group. Mitotic activity decreased with increasing selenium concentrations. If treated with 1 ~g/mL for 48 h, only few mitotic figures were seen, but the cytoplasm was heavily vacuolated and the nuclei were slightly distorted (microphotograph 2). Untreated human lung cancer cells showed a normal morphology (microphotograph 3). The effects of different concentrations of sodium selenite on the mitotic activity of human pulmonary adenocarcinoma SPC-A-1 are shown in Fig. 2, similar results to these with LTEP-a-2 cells were obtained. Human lung cancer cells from different individuals showed a common sensitivity to selenium treatment. Table 1 shows the effects of 1-5 p,g/mL sodium selenite on the mitotic activity of human embryonic lung diploid cells for 3 d. It is surprising that human embryonic lung cells still actively divide though they had been treated with 5 ~g/mL sodium selenite for 72 h (microphotograph 4). Growth
Curves
The growth curve of human pulmonary adenocarcinoma LTEP-a-2 treated with 1 ~g/mL sodium selenite for 24 h is shown in Fig. 3. It can be seen from this figure that the slope of the growth curve of the treated group is lower than that of the control group within 3 d after discarding the Se-containing medium; the cells cease to grow from d 4. This means that the doubling time of the treated group was prolonged and postconfluence inhibition occurred earlier than in the control group.
Microphotograph 2. Treatment of human pulmonary adenocarcinoma cell LTEP-a-2 with 1 p,g/mL Na2SeO3 for 48 h showed multitudinous and obvious vacuoles in the cytoplasm and slightly distorted nuclei.
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Pung, /VleLand Yu
Microphotograph 3. Untreated human pulmonary adenocarcinoma cells LTEP-a-2 in control group. %MI
50 40 30 20 10 0
1
~ 2
~ 3
Se Concentration
Fig. 2. Mitotic index of human pulmonary adenocarcinoma cell line SPA-A-1 treated with sodium selenite at 1, 2, and 3 i~g/mL for 24 h. TABLE 1 Effect of Different Sodium Selenite Levels" on MI (%) of Human Embryonic Lung Diploid Fibroblasts Treated for 24, 48, and 72 h.
Time, h 24 48 72
Control group 21.5 21.5 20
Se-Treated Group, ~g/mL 1
3
22 21 21.5
20.5 20.5 23.5
5 22 22.5 20.5
'1, 3, and 5 I~g/mL. Biological TraceElementResearch
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Microphotograph 4. Treatment of human embryonic lung diploid cells with 5 p,g/mL sodium selenite for 72 h showed three metaphases in the center of the field. Figure 4 shows the growth curve of h u m a n p u l m o n a r y adenocarcinoma SPC-A-1 treated with 1 I~g/mL sodium selenite for 24 h. Similar results were obtained as with LTEP-a-2 cells. C o m m o n features are the prolonged doubling time and the earlier postconfluence inhibition in the 1 l~g/mL-sodium-selenite-treated group. Cell No.
(x 10~) 10
8
6
2
.
.............
/ f""
;
, 2
, 3
4
, 5
6
growing day after treatment Days
Fig. 3. Growth curve of human pulmonary adenocarcinoma cell line LTEP-a-2 after 24-h treatment with 1 i~g/mL sodium selenite (---) and without
(--). Biological Trace Elernent Research
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Pung, /VleL and Yu Cell No.
(x 105)
//
10
z r
z/z
0
1
2
3
, 4
5
6
growing day after treatment Days
Fig. 4. Growth curve of human pulmonary adenocarcinoma cell line SPC-A-1 after 24-h treatment with 1 I~g/mL sodium selenite (---) and without
(--).
The growth rate of h u m a n embryonic lung diploid cells treated continuously with 1 p,g/mL sodium selenite is s h o w n in Table 2. The cell n u m b e r in the treated group closely parallels that of the control group. This result is consistent with the changes of the MI.
Effects on Progression of Cell Cycle Effects on S Phase Cells The LI of the 1 i~g/mL-treated group was lower compared to that in the control group during the 12 h of treatment. This means the cell numTABLE 2 Growth Rate of Human Embryonic Lung Diploid Fibroblasts at Continuous Exposure to 1 I~g/mL Sodium Selenite Cell no. ( x 104/mL) Days of treatment 0 1 2 3 4 5 Biological Trace Element Research
Control group 10 12.5 23 41 42.5 53
Treated group 10 15 24.8 43.8 43.2 54 Vol. 14, 1987
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Effects of Selenium on Human Lung Cancer Cells In Vitro
ber entering S phase per unit of time in the Se-treated group was lower than in the controls (Fig. 5). The average value of silver grains in 20 labeled nuclei was also lower compared with that in the control group. (Fig. 6). This means the ability to synthesize DNA was partially inhibited in the treated group. For human embryonic lung diploid cells treated with 1-5 bLg/mL sodium selenite for 3 d, the LI and the mean number of silver grains in the treated group were almost identical to those in the control group. (Table 3). These results are consistent with the MI and growth curves (microphotograph 5). Effects on the Progression of S - - - G 2 ~ Phases
For human pulmonary adenocarcinoma cells LTEP-a-2 treated with 1 bLg/mL sodium selenite for 12 h, the PLM was lower than that in the control group within the treated 12 h. These data can fully explain the reason for the lower MI when they were exposed to 1 ~g/mL sodium selenite for 24 h.
Morphological Observations Microphotograph 6 shows the morphological changes observed: Mix-cultured human embryonic lung diploid cells and human pulmonary adenocarcinoma cells LTEP-a-2, treated with 5 bLg/mL sodium selenite for 24 h, showed two heavily vacuolated cytoplasms and the slightly distorted nuclei in the lung cancer cells. A human embryonic lung diploid cell with normal fusiform morphology could be seen on the right part of the picture. This picture demonstrates the great difference in sensitivity to selenium between the normal and malignant cells.
%Labeled Nuclei 80
60
40
20
Hours After Treatment 0
2
4
6
8
10
12
Fig. 5. Labeled index curve of human pulmonary adenocarcinoma cell line LTEP-a-2 treated with 1 p.g/mL sodium selenite and pulse labeling (---) and control (--). Biological Trace Element Research
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Pung, /VleL and Yu
38 S G counts/nucleus
80
60
40
9
. o
~
s ~
20
0
2
9 6
4
, 8
, 10
, Hours After Treatment 12
Fig. 6. Silver grain (S.G.) counts in nuclei in human pulmonary adenocarcinoma cell line LTEP-a-2 treated with 1 ~g/mL of sodium selenite and pulse labeling (---) and without Se (--).
DISCUSSION The selective inhibitory effects of selenium on h u m a n cancer cells in vitro have been confirmed on hepatoma cells (8) and esophageal cancer cells (9). This paper s h o w e d once again the selective inhibitory effects of selenium on h u m a n p u l m o n a r y adenocarcinoma cells in vitro. In a previous investigation (9), treatment of h u m a n embryonic lung cells with I I~g/mL sodium selenite demonstrated the absence of inhibitory effects on MI, LI, growth rate, and cycle progression. Therefore, in this experiment, the cells were treated with 1-5 ~g/mL sodium selenite for 3 d to observe differential effects. The observed significant differences of the cellular level can be explained by the results of research on the molecular level. TABLE 3 Effect of Different Sodium Selenite Levels~ on LI (%) and Mean Silver Grain Number of Human Embryonic Lung Diploid Fibroblasts Treated for 24, 48, and 72 h. Control
Se-treated group, ~g/mL
group
1
3
5
Time, h
LI
SG"
LI
SG
LI
SG
LI
SG
24 48 72
583 589 572
85.4 89.3 78.3
583 581 596
84 88.5 78.8
558.5 580 596
81 86 81
588 594 567
86.5 90.5 78.5
"1, 3, and 5 I.Lg/mL. ~ G means the mean number of silver grain in 20 randomly counted, labeled nuclei. Biological Trace Element Research
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Microphotograph 5. Treatment of human embryonic lung diploid cells with 5 ~g/mL sodium selenite for 72 h showed a heavily labeled nucleus and a metaphase in ring form. Thus, animal experiments s h o w e d that sodium selenite administered ip at 1 mg/kg bw to normal and hepatoma-bearing mice led to a significant increase in cAMP levels and a decrease in phosphodiesterase
Microphotograph 6. Mix-cultured human embryonic lung diploid cells and human pulmonary adenocarcinoma ceils LTEP-a-2 treated with 5 ~g/mL sodium selenite for 24 h showed two heavily vacuolated cytoplasms and the slightly distorted nuclei in the lung cancer cells. A human embryonic lung diploid fibroblast with normal fusiform morphology can be seen on the right part of the picture. Biological Trace Element Research
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Pung, Mei, andYu
40 PLM* 100
80
.../"
..-
t
6O f
/
.."""
40
2O ,/
I
0
2
4
i
,
10
12
Hours After Treatment
Fig. 7. The effect on progression of S~G2--M phase human pulmonary adenocarcinoma cell line LTEP-a-2 treated with 1 I~g/mL sodium selenite and 0.2 i-tg/mLvincristine as a stathnokinetic agent and continuous labeling (---) and without (--). PLM: Percentage of labeled mitoses. (PDE) activity in hepatoma cells. In contrast, in tumor host liver and normal liver, the cAMP level was reduced, and the PDE activity was slightly elevated (13). In addition, the level of cAMP-dependent protein kinase type 1 (PK0 decreased, and the level of cAMP-dependent protein kinase type 2 (PKu) increased. Generally speaking, PKI correlated with cell division and PKu with cytodifferentiation (14). The cGMP level in hepatoma cells decreased to one-half if treated in the same way (15). The glycolysis rate in mouse ascitic hepatoma is eightfold of the normal liver cells. Sodium selenite, 1-10 lag/mL, can inhibit the glycolysis rate selectively (16). The membrane fluidity in mouse ascitic hepatoma cells is higher than that of normal liver cells. Whereas mouse ascitic hepatoma cells were not killed in contact with 3 ppm sodium selenitecontaining RPMI 1640 medium in vitro for 50 rain at 37~ membrane fluidity was significantly lowered, and the life-span of the hepatomabearing mice was prolonged (17). Two human pulmonary adenocarcinoma cell lines LTEP-a-2 and SPC-A-1 established from different individuals have their heterogenecity; however they were both found to be sensitive to the selective inhibitory effects of selenium. The results suggest that selenium is not only effective in lung cancer chemoprevention, but can also be used as a powerful adjuvant chemotherapeutic agent.
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CONCLUSION In summary, Se has been shown to exert a selective effect on proliferation of human lung cancer cells as compared to human embryonic lung diploid cells. Human pulmonary ademocarcinoma cells incubated with l~g/mL of selenite for 24 hrs resulted in a reduction of mitotic index, decrease in growth rate and partial inhibiting pregression of cell cycle. Se did not exert such effects on the embryonic lung diploid cells under the same treatment. In mixed cultures of human embryonic lung diploid cells and human lung cancer cells treated with 3 and 5 ~g/mL of soodium selenite for 24 h the lung diploid cells showed a normal fusiform morphology, whereas the lung cancer cells showed heavily vacuolated cytoplasms and distorted nuclei. The results suggest that Se is not only effective in inhibiting or interrupting the promotional stage of carcinogenesis, but can also be used as a powerful adjuvant chemotherapeutic agent.
ACKNOWLEDGMENT Supported by a grant from the National Fund of Natural Sciences, PRC.
REFERENCES /. 2. 3. 4. 5. 6. 7. 8. *9. 10. 11. 12.
A. C. Griffin, Adv. Can. Res. 29, 419 (1979). D. Medina and H. W. Lane, Biol. Trace Elem. Res. 5, 297 (1983). C. Ip, Can. Res. 41, 4386 (1981). Shu-Yu Yu, Ya-Jun Chu, Xin-Lan Gong, and Chong Hou, Biol. Trace Elem. Res. 7, 21 (1985). G. N. Schrauzer, D. A. White, and C. J. Schneider, Bioinorg. Chem. 7, 36 (1977b). Ya-Jun, Qiu-Yan Liu, Chong Hou, and Shu-Yu Yu, Biol. Trace Elem. Res. 6, 133 (1984). Hong-Sheng Tian, Yun-Yan Xiong, and Wen-Qin Gao, Can. Res. Prevent. Treat. 12(3), 123 (1985). Ao Pung, Zhao Mei, and Shu-Yu Yu, Biol. Tract' Elem. Res. Vol 12, No. 1-2, 1987 (in press). Ao Pung and Pan Qiong Qing, Can. Res. Prevent. Treat. 10(1), 19 (1983). Wang Hui and Li Bai Tong, Chin. ]. Oncol. 5(2), 85 (1983). Wu Shan Fang and Wang En Zhong, Scient. Sinica 10, 913 (1982). Pan Zhen-Kun, Han Rui, and Wang Yong-Chao, Acta Biochem. Biophys. Sinica 12(1), 13 (1980).
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Pung, Mei, and Yu
13. Shu-Yu Yu and Li-Ming Wang, Biol. Trace Elem. Res. 5, 9 (1983). 14. Liu-Qiu Yan, Hang-Sheng Li, and Shu-Yu Yu, Clin. J. Oncol. 7(5), 343 (1985). 15. Chen-Huan Chao and Shu-Yu Yu, ]. Biochem. (in press). 16. Xia-Xian Min and Shu-Yu Yu, Chin. J. OncoI. 1987 (in press). 17. Lu-Xian Ping and Shu-Yu Yu, J. Biochem. (in press) 1987.
Differential Effects of Selenium on the Proliferation of Human Pulmonary Adenocarcinoma Cells and Human Embryonic Lung Diploid Cells In Vitro Ao PUNG, ZHAOMEI, AND SHU-YU YU* Cancer Institute, Chinese Academy of Medical Sciences, Beijing, China Received August 5, 1986; Accepted September 25, 1986
ABSTRACT The effect of different concentrations of Na2SeO3 on human pulmonary adenocarcinoma cells and human embryonic lung diploid cells in vitro was investigated. For human pulmonary adenocarcinoma cells, mitotic index and cell count decreased with increasing selenium concentrations. At 1 i~g/mL sodium selenite, mitotic activity and growth of human lung cancer cells were partially inhibited, and the progression of human lung cancer cell cycle was partially arrested. When human embryonic lung diploid cells were treated with 1 lag/mL sodium selenite for five continuous days, cell counts of the treated group were closely parallel to those of the control group. After treating human embryonic lung diploid cells with 1-51ag/mL sodium selenite for 1-3 d, the mitotic index (MI), labeled index (LI), and average silver grain (SG) number per 20 labeled nuclei were the same as those of the control. In mixed cultures of human embryonic lung diploid cells and human pulmonary adenocarcinoma cells, treated with 3 and 5 I~g/mL sodium selenite for 24 h, the lung diploid cells showed a normal fusiform morphology, whereas the lung cancer cells showed heavily vacuolated cytoplasms and distorted nuclei. *Author to whom all correspondence and reprint requests should be addressed. Biological Trace Element Research
29
Vol. 14, 1987
Pung, ~ei, and Yu
30
Index Entries: Selenium; human embryonic lung diploid cells; human pulmonary adenocarcinoma cells; percentage of labeled mitoses; pulse label; mitotic index (MI); labeled index (LI); average number of silver grain; vacuolated cytoplasm; progression of cell cycle.
INTRODUCTION With the increased recognition of the importance of selenium in the chemoprevention of cancer (1), research work has turned to the chemoprevention of respective cancers, such as mammary neoplasia (2,3) or human hepatoma (4). Lung cancer is of particular interest because its incidence is increasing all over the world. After surveying 27 countries, Schrauzer listed lung cancer in "Group A", which is highly inversely proportional to the dietary selenium intakes (5). Subsequently, an article comparing blood selenium levels with the incidence of lung cancer in China has been published (6). Also, in China, yeast containing Se has been used for the first time to decrease the incidence of 3-MCA-induced lung cancer in Wistar rats (7). In addition, selective inhibitory effects of selenium on the proliferation of human hepatoma cells (8) and human esophageal cancer cells have been observed (9). The present work is aimed at understanding the effects of selenium on the growth of human pulmonary adenocarcinoma cells.
/
,TERIALS AND METHODS
Cell Culture and Chemicals Human pulmonary adenocarcinoma cells LTEP-a-2 (10) and SPC-A-1 (11) were cultured in 1640-medium-supplemented, 20% heat-inactivated calf serum and penicillin and streptomycin, each at 100 U/mL. Monolayer living cells in stock culture were passaged with a mixture of 0.5% trypsin (Difco 1:250) and 0.2% EDTA. From these, counted cells were seeded into glass culture bottles for use. Human embryonic lung diploid fibroblasts were kindly provided by the Department of Cell Biology of this Cancer Institute. The complete medium used for this kind of cell was Eagle's basal medium from Japan, with 20% heat-inactivated calf serum plus 100 U of penicillin and 100 U streptomycin/mL added to the complete medium. For the experiment, the 8th -12 th passage cells were used (microphotograph 1). Sodium selenite (Beijing Chemicals factory product, C.P. 79038) was dissolved in triply distilled water at I mg/mL, the working solution at 200 ~g/mL was renewed monthly; 2 ~g/10 p.L was added into a 2-mL medium with a micropipet.
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Effects of Selenium on Human Lung Cancer Cells In Vitro
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Microphotograph 1. Embryonic lung fibroblasts of human origin, in the passage. There are two fusiform cells in the center of the field. One of them has entered prophase of division. 8 th
Mitotic Index Counted human lung cancer cells were seeded into cover-slipped glass culture bottles. Sodium selenite was added at log phase of growth. After 24 h, cover slips were fixed with methanol and stained with Giemsa. Mitotic figures were counted per 1000 cells. Human embryonic lung diploid cells were treated with 1, 3, and 5 ~g/mL sodium selenite for 3 d. Cells in control and treated groups were pulse labeled daily with 0.4 ~,Ci/mL of tritiated thymidine (specific activity, 41.3 Ci/mM) for 1 h. Mitotic indices (MIs) were counted on the autoradiography slide. Meanwhile, labeled index (LI) and the numbers of silver grains in 20 labeled nuclei were determined, and the mean values were compared with the data from the controls.
Growth Curves Counted cell suspensions were seeded into glass culture bottles, and sodium selenite was added at log phase of growth. After 24 h of exposure 1 ~g/mL, cells in three bottles of each group were trypsinized and counted; the mean value was compared with that of the control group. The Se-containing medium was then discarded from the remaining bottles, washed with prewarmed PBS, and fed with new medium. The same treatment was applied to the control group. The cells in each group were counted daily until a confluent density was reached. Human embryonic lung diploid cells were continuously treated with 1 ~g/mL sodium selenite for 5 d; cells in each group were counted daily.
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Pung,/VleL and Yu
Effects on Progression of Cell Cycle Effects on S Phase Cells
Both control and treated groups of human pulmonary adenocarcinoma cells were pulse labeled with tritiated thymidine, 0.4 tzCi/mL for I h at different intervals after 1 i~g/mL sodium selenite exposure (12). Labeled index and number of silver grains in 20 labeled nuclei were counted and compared with each other. Effects on the Progression of S--G2--/vl Phases Vincristine, 0.2 l~g/mL, was screened as an optimum arrest concentration and continuous labeling and metaphase-arresting methods were performed (12). After 1 I~g/mL sodium selenite exposure, samples were taken at different intervals for autoradiography and determination of the percentage of labeled mitoses (PLM).
Morphological Observations Human embryonic lung diploid cells were planted into cover-slipped glass culture bottles. When grown to half-confluence, human pulmonary adenocarcinoma cells LTEP-a-2 were planted into the abovementioned culture bottles. The mixed culture of the two kinds of cells was treated with 3 and 5 p,g/mL sodium selenite for 24 h. Cover slips were fixed and stained for observing toxic changes in the nucleus and cytoplasm.
RESGLTS /~itotic Index Effects of various concentrations of sodium selenite on the mitotic activity of human pulmonary adenocarcinoma cells LTEP-a-2 are shown in Fig. 1. The MI in the I p,g/mL-treated group was only half of that in the
%MI 60 50 40 30 20 10
~ ~ SeConcentration 1 2 3 (l~g/mL) Fig. 1. Mitotic index in h u m a n p u l m o n a r y adenocarcinoma cell line LTEP-a-2 treated w i t h sodium selenite at 1, 2, and 3 i~g/mL for 24 h. 0
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control group. Mitotic activity decreased with increasing selenium concentrations. If treated with 1 ~g/mL for 48 h, only few mitotic figures were seen, but the cytoplasm was heavily vacuolated and the nuclei were slightly distorted (microphotograph 2). Untreated human lung cancer cells showed a normal morphology (microphotograph 3). The effects of different concentrations of sodium selenite on the mitotic activity of human pulmonary adenocarcinoma SPC-A-1 are shown in Fig. 2, similar results to these with LTEP-a-2 cells were obtained. Human lung cancer cells from different individuals showed a common sensitivity to selenium treatment. Table 1 shows the effects of 1-5 p,g/mL sodium selenite on the mitotic activity of human embryonic lung diploid cells for 3 d. It is surprising that human embryonic lung cells still actively divide though they had been treated with 5 ~g/mL sodium selenite for 72 h (microphotograph 4). Growth
Curves
The growth curve of human pulmonary adenocarcinoma LTEP-a-2 treated with 1 ~g/mL sodium selenite for 24 h is shown in Fig. 3. It can be seen from this figure that the slope of the growth curve of the treated group is lower than that of the control group within 3 d after discarding the Se-containing medium; the cells cease to grow from d 4. This means that the doubling time of the treated group was prolonged and postconfluence inhibition occurred earlier than in the control group.
Microphotograph 2. Treatment of human pulmonary adenocarcinoma cell LTEP-a-2 with 1 p,g/mL Na2SeO3 for 48 h showed multitudinous and obvious vacuoles in the cytoplasm and slightly distorted nuclei.
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Microphotograph 3. Untreated human pulmonary adenocarcinoma cells LTEP-a-2 in control group. %MI
50 40 30 20 10 0
1
~ 2
~ 3
Se Concentration
Fig. 2. Mitotic index of human pulmonary adenocarcinoma cell line SPA-A-1 treated with sodium selenite at 1, 2, and 3 i~g/mL for 24 h. TABLE 1 Effect of Different Sodium Selenite Levels" on MI (%) of Human Embryonic Lung Diploid Fibroblasts Treated for 24, 48, and 72 h.
Time, h 24 48 72
Control group 21.5 21.5 20
Se-Treated Group, ~g/mL 1
3
22 21 21.5
20.5 20.5 23.5
5 22 22.5 20.5
'1, 3, and 5 I~g/mL. Biological TraceElementResearch
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Microphotograph 4. Treatment of human embryonic lung diploid cells with 5 p,g/mL sodium selenite for 72 h showed three metaphases in the center of the field. Figure 4 shows the growth curve of h u m a n p u l m o n a r y adenocarcinoma SPC-A-1 treated with 1 I~g/mL sodium selenite for 24 h. Similar results were obtained as with LTEP-a-2 cells. C o m m o n features are the prolonged doubling time and the earlier postconfluence inhibition in the 1 l~g/mL-sodium-selenite-treated group. Cell No.
(x 10~) 10
8
6
2
.
.............
/ f""
;
, 2
, 3
4
, 5
6
growing day after treatment Days
Fig. 3. Growth curve of human pulmonary adenocarcinoma cell line LTEP-a-2 after 24-h treatment with 1 i~g/mL sodium selenite (---) and without
(--). Biological Trace Elernent Research
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Pung, /VleL and Yu Cell No.
(x 105)
//
10
z r
z/z
0
1
2
3
, 4
5
6
growing day after treatment Days
Fig. 4. Growth curve of human pulmonary adenocarcinoma cell line SPC-A-1 after 24-h treatment with 1 I~g/mL sodium selenite (---) and without
(--).
The growth rate of h u m a n embryonic lung diploid cells treated continuously with 1 p,g/mL sodium selenite is s h o w n in Table 2. The cell n u m b e r in the treated group closely parallels that of the control group. This result is consistent with the changes of the MI.
Effects on Progression of Cell Cycle Effects on S Phase Cells The LI of the 1 i~g/mL-treated group was lower compared to that in the control group during the 12 h of treatment. This means the cell numTABLE 2 Growth Rate of Human Embryonic Lung Diploid Fibroblasts at Continuous Exposure to 1 I~g/mL Sodium Selenite Cell no. ( x 104/mL) Days of treatment 0 1 2 3 4 5 Biological Trace Element Research
Control group 10 12.5 23 41 42.5 53
Treated group 10 15 24.8 43.8 43.2 54 Vol. 14, 1987
37
Effects of Selenium on Human Lung Cancer Cells In Vitro
ber entering S phase per unit of time in the Se-treated group was lower than in the controls (Fig. 5). The average value of silver grains in 20 labeled nuclei was also lower compared with that in the control group. (Fig. 6). This means the ability to synthesize DNA was partially inhibited in the treated group. For human embryonic lung diploid cells treated with 1-5 bLg/mL sodium selenite for 3 d, the LI and the mean number of silver grains in the treated group were almost identical to those in the control group. (Table 3). These results are consistent with the MI and growth curves (microphotograph 5). Effects on the Progression of S - - - G 2 ~ Phases
For human pulmonary adenocarcinoma cells LTEP-a-2 treated with 1 bLg/mL sodium selenite for 12 h, the PLM was lower than that in the control group within the treated 12 h. These data can fully explain the reason for the lower MI when they were exposed to 1 ~g/mL sodium selenite for 24 h.
Morphological Observations Microphotograph 6 shows the morphological changes observed: Mix-cultured human embryonic lung diploid cells and human pulmonary adenocarcinoma cells LTEP-a-2, treated with 5 bLg/mL sodium selenite for 24 h, showed two heavily vacuolated cytoplasms and the slightly distorted nuclei in the lung cancer cells. A human embryonic lung diploid cell with normal fusiform morphology could be seen on the right part of the picture. This picture demonstrates the great difference in sensitivity to selenium between the normal and malignant cells.
%Labeled Nuclei 80
60
40
20
Hours After Treatment 0
2
4
6
8
10
12
Fig. 5. Labeled index curve of human pulmonary adenocarcinoma cell line LTEP-a-2 treated with 1 p.g/mL sodium selenite and pulse labeling (---) and control (--). Biological Trace Element Research
Vol. 14, 1987
Pung, /VleL and Yu
38 S G counts/nucleus
80
60
40
9
. o
~
s ~
20
0
2
9 6
4
, 8
, 10
, Hours After Treatment 12
Fig. 6. Silver grain (S.G.) counts in nuclei in human pulmonary adenocarcinoma cell line LTEP-a-2 treated with 1 ~g/mL of sodium selenite and pulse labeling (---) and without Se (--).
DISCUSSION The selective inhibitory effects of selenium on h u m a n cancer cells in vitro have been confirmed on hepatoma cells (8) and esophageal cancer cells (9). This paper s h o w e d once again the selective inhibitory effects of selenium on h u m a n p u l m o n a r y adenocarcinoma cells in vitro. In a previous investigation (9), treatment of h u m a n embryonic lung cells with I I~g/mL sodium selenite demonstrated the absence of inhibitory effects on MI, LI, growth rate, and cycle progression. Therefore, in this experiment, the cells were treated with 1-5 ~g/mL sodium selenite for 3 d to observe differential effects. The observed significant differences of the cellular level can be explained by the results of research on the molecular level. TABLE 3 Effect of Different Sodium Selenite Levels~ on LI (%) and Mean Silver Grain Number of Human Embryonic Lung Diploid Fibroblasts Treated for 24, 48, and 72 h. Control
Se-treated group, ~g/mL
group
1
3
5
Time, h
LI
SG"
LI
SG
LI
SG
LI
SG
24 48 72
583 589 572
85.4 89.3 78.3
583 581 596
84 88.5 78.8
558.5 580 596
81 86 81
588 594 567
86.5 90.5 78.5
"1, 3, and 5 I.Lg/mL. ~ G means the mean number of silver grain in 20 randomly counted, labeled nuclei. Biological Trace Element Research
VoL 14, 1987
Effects of Selenium on Human Lung Cancer Cells In Vitro
39
Microphotograph 5. Treatment of human embryonic lung diploid cells with 5 ~g/mL sodium selenite for 72 h showed a heavily labeled nucleus and a metaphase in ring form. Thus, animal experiments s h o w e d that sodium selenite administered ip at 1 mg/kg bw to normal and hepatoma-bearing mice led to a significant increase in cAMP levels and a decrease in phosphodiesterase
Microphotograph 6. Mix-cultured human embryonic lung diploid cells and human pulmonary adenocarcinoma ceils LTEP-a-2 treated with 5 ~g/mL sodium selenite for 24 h showed two heavily vacuolated cytoplasms and the slightly distorted nuclei in the lung cancer cells. A human embryonic lung diploid fibroblast with normal fusiform morphology can be seen on the right part of the picture. Biological Trace Element Research
!/ol. 14, 1987
Pung, Mei, andYu
40 PLM* 100
80
.../"
..-
t
6O f
/
.."""
40
2O ,/
I
0
2
4
i
,
10
12
Hours After Treatment
Fig. 7. The effect on progression of S~G2--M phase human pulmonary adenocarcinoma cell line LTEP-a-2 treated with 1 I~g/mL sodium selenite and 0.2 i-tg/mLvincristine as a stathnokinetic agent and continuous labeling (---) and without (--). PLM: Percentage of labeled mitoses. (PDE) activity in hepatoma cells. In contrast, in tumor host liver and normal liver, the cAMP level was reduced, and the PDE activity was slightly elevated (13). In addition, the level of cAMP-dependent protein kinase type 1 (PK0 decreased, and the level of cAMP-dependent protein kinase type 2 (PKu) increased. Generally speaking, PKI correlated with cell division and PKu with cytodifferentiation (14). The cGMP level in hepatoma cells decreased to one-half if treated in the same way (15). The glycolysis rate in mouse ascitic hepatoma is eightfold of the normal liver cells. Sodium selenite, 1-10 lag/mL, can inhibit the glycolysis rate selectively (16). The membrane fluidity in mouse ascitic hepatoma cells is higher than that of normal liver cells. Whereas mouse ascitic hepatoma cells were not killed in contact with 3 ppm sodium selenitecontaining RPMI 1640 medium in vitro for 50 rain at 37~ membrane fluidity was significantly lowered, and the life-span of the hepatomabearing mice was prolonged (17). Two human pulmonary adenocarcinoma cell lines LTEP-a-2 and SPC-A-1 established from different individuals have their heterogenecity; however they were both found to be sensitive to the selective inhibitory effects of selenium. The results suggest that selenium is not only effective in lung cancer chemoprevention, but can also be used as a powerful adjuvant chemotherapeutic agent.
Biological Trace Element Research
Vol. 14, 1987
Effects o f Selenium on H u m a n L u n g Cancer Cells In Vitro
41
CONCLUSION In summary, Se has been shown to exert a selective effect on proliferation of human lung cancer cells as compared to human embryonic lung diploid cells. Human pulmonary ademocarcinoma cells incubated with l~g/mL of selenite for 24 hrs resulted in a reduction of mitotic index, decrease in growth rate and partial inhibiting pregression of cell cycle. Se did not exert such effects on the embryonic lung diploid cells under the same treatment. In mixed cultures of human embryonic lung diploid cells and human lung cancer cells treated with 3 and 5 ~g/mL of soodium selenite for 24 h the lung diploid cells showed a normal fusiform morphology, whereas the lung cancer cells showed heavily vacuolated cytoplasms and distorted nuclei. The results suggest that Se is not only effective in inhibiting or interrupting the promotional stage of carcinogenesis, but can also be used as a powerful adjuvant chemotherapeutic agent.
ACKNOWLEDGMENT Supported by a grant from the National Fund of Natural Sciences, PRC.
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A. C. Griffin, Adv. Can. Res. 29, 419 (1979). D. Medina and H. W. Lane, Biol. Trace Elem. Res. 5, 297 (1983). C. Ip, Can. Res. 41, 4386 (1981). Shu-Yu Yu, Ya-Jun Chu, Xin-Lan Gong, and Chong Hou, Biol. Trace Elem. Res. 7, 21 (1985). G. N. Schrauzer, D. A. White, and C. J. Schneider, Bioinorg. Chem. 7, 36 (1977b). Ya-Jun, Qiu-Yan Liu, Chong Hou, and Shu-Yu Yu, Biol. Trace Elem. Res. 6, 133 (1984). Hong-Sheng Tian, Yun-Yan Xiong, and Wen-Qin Gao, Can. Res. Prevent. Treat. 12(3), 123 (1985). Ao Pung, Zhao Mei, and Shu-Yu Yu, Biol. Tract' Elem. Res. Vol 12, No. 1-2, 1987 (in press). Ao Pung and Pan Qiong Qing, Can. Res. Prevent. Treat. 10(1), 19 (1983). Wang Hui and Li Bai Tong, Chin. ]. Oncol. 5(2), 85 (1983). Wu Shan Fang and Wang En Zhong, Scient. Sinica 10, 913 (1982). Pan Zhen-Kun, Han Rui, and Wang Yong-Chao, Acta Biochem. Biophys. Sinica 12(1), 13 (1980).
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Pung, Mei, and Yu
13. Shu-Yu Yu and Li-Ming Wang, Biol. Trace Elem. Res. 5, 9 (1983). 14. Liu-Qiu Yan, Hang-Sheng Li, and Shu-Yu Yu, Clin. J. Oncol. 7(5), 343 (1985). 15. Chen-Huan Chao and Shu-Yu Yu, ]. Biochem. (in press). 16. Xia-Xian Min and Shu-Yu Yu, Chin. J. OncoI. 1987 (in press). 17. Lu-Xian Ping and Shu-Yu Yu, J. Biochem. (in press) 1987.
