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Departments of
*
Food Science and Nutrition and
Exercise Science, University of Rhode Island, Kingston, RI 02881 and the
**
Department of Pathology, Rhode Island Hospital and Brown University, Providence, RI 02903
2To whom correspondence should be addressed. E-mail: pprakash__mail{at}yahoo.com
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • ß-carotene • NCI-H69 • lung cancer cells • electron microscopy • image analysis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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,2
). The beneficial effects of ß-carotene on cancer have been found in a number of studies including epidemiological studies (1
,2
), clinical studies (3
,4
), animal model studies (5
–7
), and cell culture studies (8
–10
). The overall results of these studies indicate that ß-carotene provides a protective effect on the incidence of lung cancer. However, either no protective effect (11
,12
) or a negative effect (13
,14
) of supplemental ß-carotene was observed in a few clinical intervention trials conducted in the recent past to determine its effect on the incidence of lung cancer.
Carotenoids have been shown to induce morphological changes in cancer cells in a number of studies. ß-carotene induced differentiation in MCF-10A mammary epithelial cells in culture by the formation of cytoplasmic vacuoles (15
) and induced cellular differentiation and caused inhibition of growth in mouse B-16 melanoma cells (16
). Crocin, its derivative dimethylcrocetin, and ß-carotene induced erythroid differentiation and efficiently inhibited cell growth of K562 tumor cells (17
). Inhibition of growth and induction of differentiation of promyelocytic leukemic HL-60 cells by carotenoids from Crocus sativus L. was reported (18
). In the human cervical cancer cell line CICCN-2, 10 µmol/L of ß-carotene caused chromatin condensation, a characteristic of apoptosis (19
).
In a previous investigation, we reported that 20 µmol/L of ß-carotene significantly reduced the growth of NCI-H69 small cell lung cancer cells on d 11 and 15 of treatment by >50% (20
). In the same study, we also reported that ß-carotene is primarily localized to the nucleus after its uptake by these cells and is metabolized to retinoic acid, retinol and retinal in the cells. Another previous investigation by our group (21
) demonstrated that ß-carotene at a dose of 20 µmol/L arrested NCI-H69 cells in the G0/G1 stage of the cell cycle and also decreased N-myc and c-Jun mRNA. Because these gene expression activities take place in the nucleus, it is imperative that the effect of ß-carotene on the nuclear morphology be determined in addition to its effect on the general morphology of the cells. This would further define the role of the NCI-H69 nucleus, because it is the accumulation site of ß-carotene in the cells (20
) and also the site for ß-carotene action in inhibiting cell growth (21
). This study was designed to address these issues and to explore the morphological changes, if any, in NCI-H69 small cell lung cancer cells that had undergone ß-carotene-induced growth reduction. Specifically, ß-carotene in crystalline form was delivered to NCI-H69 cells in culture and the cells showing a reduction in proliferation because of treatment were critically viewed under a transmission electron microscope. In addition, a morphometric analysis of the cells and organelles was done using computer image analysis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The human small cell lung cancer line NCI-H69 was obtained from the American Type Culture Collection (Rockville, MD). RPMI 1640 growth medium and other growth regulators were purchased from Gibco (Gaithersburg, MD). Crystalline ß-carotene was kindly provided by Hoffman-LaRoche (Nutley, NJ). Tetrahydrofuran (THF)3 containing butylated hydroxytoluene (BHT) was purchased from Aldrich (Allentown, PA). The fixatives and the stains required for the electron microscopy work were purchased from EM Science (Gibbstown, NJ).
Cell culture.
NCI-H69 cells were grown in RPMI 1640 medium, supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin and 1% glutamine in a humidified atmosphere of 5% CO2 in air at 37°C. Cells were grown and maintained at a concentration of 1 x 108/L in the medium for the experiments. The viability of the cells was determined using trypan blue (0.4%) exclusion method before and during the carotenoid treatment of the cells in all experimental groups.
Treatment of cells with ß-carotene.
ß-carotene was used at a concentration of 20 µmol/L in the medium. This concentration was selected based on our previous dose-response experiments conducted using 2–20 µmol/L of ß-carotene (21
). THF containing BHT was used as a delivery vehicle of crystalline ß-carotene to the cells. Based on the molecular weight of ß-carotene, the appropriate amount of ß-carotene was dissolved in THF to make a stock solution of 20 mmol/L. Stock ß-carotene solution (1 µL) was added to each 1 mL medium to achieve a final concentration of 20 µmol/L ß-carotene in the medium as described (10
). An equivalent amount of THF was added to the medium for control cells. In this way, the concentration of THF in the control and experimental medium was 0.1%. Cells were diluted from stock cultures to 
1 x 108 cells/L in 10 mL of treatment or control medium. Three replications were used in each group. The cells were grown in either control or ß-carotene-treated medium for 16 d and the medium was changed at d 5 and 11 of cell growth. Red light was used while working with ß-carotene solutions to prevent photodamage to the carotenoid. Solutions of ß-carotene were made in the cell culture hood that was routinely purged with ultra violet light to maintain sterility. A new bottle of tetrahydrofuran was used for each experiment, purged and filled with nitrogen after each usage.
Preparation of cells for morphological analysis.
Cells grown at 1 x 108/L and treated with 20 µmol/L ß-carotene for 16 d, which showed a reduction in proliferation compared with THF-supplemented control cells were used for this study. Cells were harvested by washing with PBS four times and fixed by chemical fixation for transmission electron microscopy by modifying the described procedure (22
). Cells were prefixed in 2% glutaraldehyde and postfixed in 1% osmium tetroxide at 4°C for 2 h each and rinsed three times after each fixation with 0.2 mol/L sodium cacodylate buffer at 4°C for 20 min. Cells were then dehydrated in ethanol and infiltrated with LX112 and ethanol in various proportions. Samples were embedded with LX112 and polymerized at 45°C for 24 h followed by 60°C for 24 h. Ultrathin sections were collected on 200 mesh copper grids and stained with 10% uranyl acetate for 15 min followed by 0.2% lead citrate for 10 min (22
,23
). A Philips 301 transmission electron microscope (Amsterdam, The Netherlands) was used for the morphological analysis of cells.
Image analysis.
Electron micrographs taken at various magnifications were captured on a computer using NIH Image 1.55 software (Bethesda, MD) for the morphometric measurements of control and ß-carotene-treated cells. The images were transferred to the computer using a DAGE series 72 CCD camera and Data Translation image grabbing board. A calibration slide was used to ensure that the pixel values in a selected area were correct. Sharpness and smoothness of each captured image was either increased or decreased to enhance the quality for measurement. For the measurement of nuclei, extra care was taken to choose nuclei within a predefined area of the cytoplasm. Nuclei were selected for characteristics that would enhance ease of measurement: nonoverlapping, nonfragmented cells with clearly identifiable and complete nuclear outlines (24
). Clearly incomplete or partial nuclei were not measured. The size (area), perimeter, major diameter and minor diameter of the cell, nucleus and nucleolus were measured in 100 control and 100 treated cells. The area ratio of nucleus to cytoplasm was determined using these morphometric parameters. In addition, the nuclear roundness factor (NRF) was determined by the following formula (24
): (4
) (area)/perimeter2. This shape factor estimates the degree of variance from a circle. A value of 1.00 is a perfect circle, and 0.00 is a line. All measurements were expressed in µm.
Statistical analysis.
Treated and untreated cells were compared using Student’s t test and differences with P < 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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On d 16 of ß-carotene treatment, the treated cells were 3.6 x106 compared with a control population of 8.2 x 106 cells, indicating a growth inhibition of 56% (data not shown). Viability determination using 0.4% trypan blue solution revealed >90% of the cells in all groups to be viable throughout the experiment. Control cells contained a round nucleus and few vacuoles in the cytoplasm (Fig. 1
A). The treated cells exhibited an irregular morphology of the nuclei and typically had more vacuoles in the cytoplasm than control cells (Fig. 1
B). This qualitative analysis of cells indicated an altered morphology of the lung cancer cells treated with ß-carotene. Carotenoid-treated cells contained more vacuoles in the cytoplasm and more irregularly shaped nuclei than control cells.
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The NRF was significantly smaller (P < 0.05) in the ß-carotene-treated cells compared with the control cells (Table 1
), indicating a less round nuclear morphology of the treated cells. The major diameter of the cells and the minor diameter of the nuclei were significantly smaller (P < 0.05) in ß-carotene-treated cells. The nuclei perimeter was greater (P < 0.05) in the treated cells. The morphometric parameters did not differ in the nucleoli of control and treated cells. The percentage of cells containing nucleoli did not differ between control (61%) and treated (56%) cells. The area ratio of nucleus to cytoplasm was significantly smaller (P < 0.05) in ß-carotene-treated cells compared with control cells (Table 1)
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). ß-carotene (7 µmol/L) dissolved in THF induced differentiation in MCF-10A mammary epithelial cells in culture by the formation of cytoplasmic vacuoles (15
). Inhibition of growth and induction of differentiation of promyelocytic leukemic HL-60 cells by carotenoids from Crocus sativus L. has been reported (18
). Crocin, its derivative dimethylcrocetin, and ß-carotene induced erythroid differentiation and efficiently inhibited cell growth of K562 tumor cells (17
). In HL-60 leukemic cells, 10 µmol/L ß-carotene or lutein caused differentiation as determined by the nitroblue tetrazolium assay (26
). In B-16 mouse melanoma cells, 37.2 µmol/L ß-carotene caused cell enlargement with cytoplasmic processes (16
). ß-carotene and canthaxanthin at 10 µmol/L caused F9 mouse teratocarcinoma cells to become a flat polygonal shape (27
). ß-carotene at a high 70 µmol/L dose caused rounding and lifting of the SCC-25 oral cancer cells along with vacuolization (28
). Carotenoids also have been observed to induce morphological apoptosis in cancer cells. Crocin extracted from saffron, at 10 mmol/L, caused cell shrinkage, widened cytoplasmic vacuole-like areas and reduced cytoplasm and pyknotic nuclei in HeLa human cervical cancer cells (29
). In another human cervical cancer cell line, CICCN-2, 10 µmol/L ß-carotene caused chromatin condensation, a characteristic of apoptosis (19
).
The quantitative image analysis methods used in this study indicated a significant reduction (P < 0.05) in the nuclear roundness of ß-carotene-treated cells compared with control cells. The shape of cell nuclei changed from round to irregular in a majority of cells after ß-carotene treatment. Nuclear shape is controlled by the nuclear matrix, the RNA protein skeleton of the nucleus, and its interactions with cytoskeletan systems such as intermediate filaments and actin microfilaments. The nuclear matrix plays an important role in cell function and gene expression because active genes are bound to the nuclear matrix, whereas inactive genes are not (30
). The association of nuclear shape and cellular differentiation as a result of retinoic acid treatment has been suggested (31
,32
). Additionally, an irregularity of nuclei was observed in the basal segment of maturing and differentiating epithelial cells of the colorectal crypt, possibly due to an increase in the functional and metabolic requirements of these cells, which were active in cell division and differentiation (33
).
In this study, the altered nuclear morphology of the treated cells seems to be a part of the differentiation process; however, a more careful investigation with sufficiently supporting evidence is required to prove this association. A higher incidence of cytoplasmic vacuolization in ß-carotene-treated cells reported here supports other studies reporting differentiated phenotypes in carotenoid-treated cancer cells. To our knowledge, this study is the first of its kind to determine the effect of ß-carotene on lung cancer cells, both qualitatively and quantitatively. However, the effects of ß-carotene administration on morphological features other than the nucleus were not of the magnitude of the effects in nucleus, although these cytoplasmic changes were statistically significant. We have previously reported that ß-carotene is metabolized to retinoic acid, retinol and retinal after its uptake by NCI-H69 cells (20
). The assessment of the effect of these metabolites on the morphology of these cells was not undertaken in this investigation. Therefore, whether the effects on the cell morphology due to ß-carotene treatment should be attributed to carotenoid or retinoids is unknown. The observed decrease in the ratio of nucleus to cytoplasm in the ß-carotene-treated cells indicates a less malignant phenotype as reported previously (34
). The overall morphology of ß-carotene-treated cells seems to indicate a less aggressive tumor cell growth.
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FOOTNOTES |
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3 Abbreviations used: NRF, nuclear roundness factor; THF, tetrahydrofuran.
Manuscript received 25 May 2001. Revision accepted 10 October 2001.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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