The selective cytotoxic effect of carotenoids and α-Tocopherol on human cancer cell lines in vitro

J Oral Maxillofac Surg

50:367-373,1992

The Selective Cytotoxic Effect of Carotenoids and a-Tocopherol on Human Cancer Cell

Lines In Vitro

JOEL SCHWARTZ, DMD, DMSc,* AND GERALD SHKLAR, DDS, MSt

This study compares the toxic effects of the carotenoids, P-carotene and canthaxanthin, and a-tocopherol (vitamin E) on human tumor cells and their normal counterparts in vitro. Seven different malignant cell lines were examined: oral carcinoma (two cell lines), breast (two cell lines), lung carcinoma (two cell lines), and malignant melanoma. The in vitro cell culture assays showed a consistent morphologic change in the affected tumor cells following treatment with carotenoid or vitamin E. A rounding of the tumor cells and eventual lifting off the tissue culture plate were observed. These changes were apparent after 1 to 5 hours of treatment depending on the tumor cell line. Associated with these observable cellular changes were quantitative reductions in proliferation (3H-thymidine proliferation) and succinic dehydrogenase activity (MTT assay). In addition, there was a noticeable change in protein expression, with an increased expression of a 70-kD protein following treatment with @-carotene. This protein was associated with tumor cells showing a decrease in proliferation (oral carcinoma, malignant melanoma), but not with normal keratinocytes or melanocytes. These studies substantiate a selective cytotoxic effect on human tumor cell growth by carotenoids and a-tocopherol in vitro, and may provide an explanation of the therapeutic activity of these agents and their possible use in the treatment of premalignancy or early oral carcinoma.

Previous studies in vivo have shown that the carotenoids, beta carotene @-carotene), canthaxanthin, and Ly-carotene, will inhibit tumor cell growth.‘” This laboratory has repeatedly shown that these agents will prevent and inhibit oral carcinogenesis and regress established oral invasive carcinoma using a hamster buccal pouch tumor model.‘-’ ’ Such studies indicate that these agents may act directly on tumor cells. The his- topathologic examination of oral mucosal sections, some containing oral carcinoma and others tumor-free, have never shown a toxic effect of the carotenoids or

Received from the Department of Oral Pathology, Harvard School of Dental Medicine, Boston.

* Associate Professor. t Professor and Chairman. The research project was funded in part by Cancer Prevention

Technologies Inc and the Milheim Foundation. Address correspondence and reprint requests to Dr Schwartz: De-

partment of Oral Pathology, Harvard School of Dental Medicine, 188 Longwood Ave, Boston, MA 02 I 15.

0 1992 American Association of Oral and Maxillofacial Surgeons

0278-2391/92/5004-0010$3.00/O

a-tocopherol on the oral mucosa. In contrast, the administration of 13-cis retinoic acid, a vitamin A analog, has been shown to promote the growth of oral carcinoma” and possibly damage the oral mucosa, whereas an erthyrodermic reaction was observed in non-tumor-bearing, control buccal mucosa (unpublished observation).

P-Carotene’s antitumor activity can be increased when combined with cyclophosphamide or radiation, increasing the survival of mice with breast adenocar- cinema. 12-’ 3 P-Carotene and a-tocopherol also can increase the antitumor activity of chemotherapeutic agents such as Melphalan (Sigma Chemical, St Louis, MO) and cisplatin toward human oral carcinoma cells in vitro. l4 Particularly notable was a 1 ,OOO-fold decrease in clonogenic capacity of the carcinoma cells following the combination treatment of P-carotene and Mel- phalan.14 We also have shown that both b-carotene and cY-tocopherol will reduce the proliferation and activity of succinic dehydrogenase in human oral and lung squamous cell carcinoma in vitroI while inhibiting the growth of established invasive oral carcinoma in the hamster when orally administered.” These

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368 TOXIC EFFECTS OF CAROTENOIDS AND a-TOCOPHEROL ON TUMOR CELLS

changes in cellular activity following @-carotene treatment were associated with the expression of a 70-kD protein in the treated tumor cells.15

The data presented here show that carotenoids, such as &carotene and canthaxanthin, and a-tocopherol, can exert a similar and selective debilitating effect on tumor cell growth regardless of the original origin of the tumor cell. In addition, it is shown that this reduction in tumor cell growth in vitro is not apparent in normal human cell types. It is also shown that a 70- kD protein is expressed in human oral carcinoma and malignant melanoma cells and not in normal keratinocytes or melanocytes. This protein is observed when the tumor cells are stressed by heating,i5 indicating the possibility that the 70-kD protein is also derived from the stress protein family. Taken together, these data provide a greater understanding of the therapeutic activity of carotenoids or a-tocopherol toward different human tumors.

Materials and Methods

Drugs. &Carotene was obtained from Kodak Fine Chemicals, Rochester, NY. The carotenoid was purified by column chromatography on an activated Baker (Phillipsburg, MI) 60-120 mesh silica-gel eluted with cyclohexane and characterized by ultraviolet-visible spectrophotometry. Canthaxanthin was obtained from Hoffman La Roche, Nutley, NJ, and was determined to be purified by high-pressure liquid chromatography (HPLC) as previously described.i6 Purified a-tocopherol acid succinate and reduced glutathione were obtained from Sigma Chemical Co. Quantitation of the carotenoids and a-tocopherol was by HPLC, and the experimental solutions containing these agents were produced by forming liposomelike micellar constructs using a probe sonicator.‘6 These micellar constructs were about 1 pm and were placed into the cell cultures on the day of preparation. The final concentration was 70 PM, as determined by HPLC.

Cells. Normal human keratinocytes (NHK) were obtained from either the foreskin or mammoplasties (Clonetics, Epipak, San Diego, CA). The keratinocytes from the breast skin were used in all assays, for they are a more mature cell type than the less-differentiated foreskin keratinocytes. The foreskin keratinocytes were grown according to the method of Rheinwald and Green, ” whereas the breast skin keratinocytes were grown without a feeder layer and supplemented with a modified MCDB 153 medium, containing pituitary extract, epidermal growth factor, adenine, and hydro- cortisone. The concentrations of these agents were identical to those for the preceding method.17 Human normal breast cells were kindly provided by Dr Ruth Sager, Dana Farber Cancer Institute, and grown in modified MCDB 170 medium.

Seven malignant cells were used in these studies. They included two oral carcinoma cell lines (SCC-25 derived from the hard plate, and SQ-38 derived from the buccal mucosa), two breast cell lines (MCF-7 and ZR75), two lung carcinomas (SK-MES and CALV3), and one malignant melanoma, A375. They were obtained from Dr Beverly Teicher, Dana Farber Cancer Institute, and American Tissue Type Culture collec- tion. All tumor cells were grown initially in Dulbecco’s Modified Eagle’s Medium (Sigma Chemical Co) with 10% fetal bovine serum. The oral carcinoma and breast carcinoma cell lines required insulin, whereas the lung carcinoma cell lines required sodium pyruvate and nonessential amino acids (Sigma Chemical Co).

In vitro treatment ofcells. Tumor cells or normal cells (2 X 105) were plated onto wells in a 24-well plate. The cells were treated with either P-carotene, canthaxanthin, a-tocopherol acid succinate, or reduced glutathione (70 PM) for 2 hours or 5 hours. The media in which the agents were dissolved appeared to produce no significant change in the results obtained. Therefore, the results are given as pooled results from these assays. After incubation, the supernatant containing any floating cells and agents and cells (2 X 106/3 mL of separation medium) that were removed following trypsinization + EDTA (0.25%) were placed over a separation medium. This medium, ficollpaque (Phar- amacia, Piscataway, NJ), was used to separate nonviable from viable cells using density centrifugation. This method also allowed us to separate crystalline, non- soluble precipitate of the experimental agents from the cells. It is mandatory to remove the crystalline precip- itations because they will interfere with the quantitative analysis assays based on absorbance and radioisotope incorporation. The details of the mechanism involved in this phenomenon have been previously described.18

The separation of the crystalline forms of carotenoids and ar-tocopherol will vary because of the density differences of their crystals. Therefore, the canthaxanthin- treated cells required repetition of the separation process at least twice, and a-tocopherol required repetition of the process at least three times. In contrast, P-carotene-treated cells required the separation procedure only once.

After separation, the cells were washed twice in the specific medium required for the cell type. The cells were then counted by hemocytometer and stained with trypan blue (0.25%) to determine viability. Routinely, the cells were judged to be 95% to 98% viable by this technique. The cells were replated onto wells (5 X 1 04) in a 96-well plate. These cells may contain an isolated crystal in 10 identical wells. By our quantitation there was a 90% reduction in the crystalline precipitate compared with the original observable number per millimeter squared.

SCHWARTZ AND SHKLAR 369

The replated cells were incubated for an additional 24 hours in the medium noted above without the pres- ence of the experimental agents and used for the assays detailed below. Some of the monolayer-s of the tumor cells and the normal cells in the original 24-well plates were stained with trypan blue (0.25%) (dye exclusion method) and then crystal violet to distinguish the initial effects on viability produced by the experimental agents. A comparison of the change in viability associated with the experimental incubation and the untreated control cell was determined by the formula: 1 - (Experimental - Control)/Control X 100, where control was untreated tumor cells or normal cells and experimental was experimentally treated tumor cells or normal cells. The number of viable cells was determined with the use of a calibrated micrometer of mil- limeters squared.

Tetrazolium salt assay (MTT). This assay has previously been used to quantitate the level of succinic dehydrogenase, a determinant of the viability of the mitochondria.‘6,‘s The cells were plated into 96-well plates and then analyzed for the activity of succinic dehydrogenase in identical triplicate wells for each agent (70 PM, 2 hours). MTT (3-(4,5-dimethylthiazol-

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1 2 3 4 5 6 1 2 3 4 5 6

2-yl)-2,5diphenyl-tetrazolium bromide (thiazolyl blue) (50 PL of 2 mg/mL) was added to each well and incubated at 37°C for 4 hours before processing. The activity of the enzyme was determined using a TiTertek (Plow Laboratories, McLean, VA) Multiskan, MCC/ 340 analyzer set for an absorbance spectra at 540, following the addition of dimethylsulfoxide (DM!JQ). The percentage change for the activity of succinic dehydrogenase was calculated as follows: 1 - (Experimental - Control)/Control X 100, where experimental was cells treated with agents and Control was cells that were untreated.

Thymidine incorporation (3H-thymidine). After the cells were replated onto 96-well plates, in identical triplicate wells, the cells were incubated for an additional 24 hours with 1 &i/well of 3H-thymidine (New England Nuclear, Boston, MA). After 24 hours, the cells were procured using a PHD Cell Harvester (Wa- tertown, MA) and counted in a P-scintillation counter. The number of counts per minute recorded indicated the amount of thymidine incorporated into the cells during the synthesis phase.

Protein analysis by gel electrophoresis. The oral carcinoma and malignant melanoma, as well as normal

1 2 3 4 5 6

FIGURE I. Photomicrographs of 24-well plates that contained tumor cells or normal cells. A. Normal breast cells (rows 1, 3,5) and malignant breast cell line, ZR75 (rows 2,4, 6) were plated at a density of 2 X lo5 cells per well. The cells were allowed to adhere to the wells and develop a monolayer. The normal cells were plated first, usually 24 to 48 hours before the tumor cells, to allow for the differences in doubling time and adherance to the tissue culture wells. The normal and tumor cells were treated in an identical manner with p-carotene for 5 hours (rows 3 to 6). The treated tumor cells were markedly suppressed in their ability to adhere and in their viability. B, Normal melanocytes (rows 1, 3, 5) and malignant melanoma, A375 (rows 2,4.6), were plated and treated as described above. C, Normal keratinocytes (rows 1,3,5) and oral squamous cell carcinoma, SCC-25 (rows 2, 4, 6), were plated as described above with the same results as observed above. D, Normal keratinocytes (rows 1. 3. 5) and lung carcinoma, SK-MES (rows 2,4, 6). were plated as described above with results identical to those described above.

370 TOXIC EFFECTS OF CAROTENOIDS AND c~-TOCOPHEROL ON TUMOR CELLS

human epidermal keratinocytes and melanocytes, were treated with one of the carotenoids or tocopherols for 2 hours. The cells were then incubated for 5 hours in methionine-free media (DMEM). 35S-methionine (1 &i/ X 1 O6 cells, New England Nuclear)-supplemented DMEM was added to cells for 6 hours. A cell lysate was then formed using a lysate buffer that contained 10 mM Tris, 150 mM NaCl, 7.0% Triton X-100, 100 Kallikrein units of aprotinin, 1 .O mM phenylmethyl- sulfonyl fluoride (PMSF), 1 .O% sodium deoxycholate, and 1.0% sodium dodecyl sulfate. Once added to the cells, the mixture was centrifuged at 4°C and the lysate supematant was removed from the pelleted cellular debris. A modified Lowry protein assay was performed and 5 rL of the lysate was analyzed by a denaturing polyacrylamide gel electrophoresis method according to the method of Laemmli.”

Previously, we had observed the expression of a 70- kD protein. To control for this expression and to ob- serve the protein expression following heat stress, tumor cells were heated at 45°C for 45 minutes while plated onto T25 cm* flasks (Dow-Coming, Coming, NY). Following treatment, the cells were washed and removed from the flasks by trypsinization and then ra- diolabeled with methionine lyzed as indicated above.

Statistical analysis. Significant differences between

150’

1 2 3 4 5 6 7 8

NHK SK-MES SCC-25 SQ-38 NHB ZR75 NHM A375

FIGURE 2. The viable cells per square millimeter were calculated as described in the Materials and Methods section. The percentages shown in the figure are related to the untreated value (100). Values above 100 indicate an increase in the number of viable cells per square millimeter. Values below 100 indicate a decrease in the number of viable cells per square millimeter. The figure indicates that, in general, the carotenoids &carotene and canthaxanthin produced a reduction in the number of viable cells, whereas vitamin E produced less of a decrease in tumor-cell viability. Glutathione had little effect on tumor cell viability. NHK, normal human keratinocytes; SK-ME&

lung GWcinOIIIa; SW-25, od carcinoma; S@38, oral WCiIIOtIIa; NHB,

normal human breast cells; ZR75, breast carcinoma; NHM, normal human melanccytes; and A375, malignant melanoma, corresponding to columns 1 to 8, are shown. n , BCarotene; q vitamin E; q , canthaxanthin; q glutathione.

Table 1. Relative Percentage of Change in MlT Values Following Treatment With Carotenoids, Vitamin E, and Glutathione

Agents

Normal Tumors Cells

see-25 SK-MES MCF-7 A375 NHK NHM

&Carotene 55 41 62 30 107 93 Canthaxanthin 65 24 88 48 105 104 Vitamin E 62 90 95 42 150 97 Glutathione 99 95 98 100 105 loo

The values given were obtained as indicated in the Materials and Methods section. Values below 100 indicate a reduction in succinic dehydrogenase activity as compared with the untreated controls. Values above 100 indicate a higher value of succinic dehydrogenase than with untreated controls.

groups at the P I .05 confidence limit were determined by Student’s t test.

Results

Photomicrographs of the cells plated onto the 24- well plates and then stained with trypan blue indicated a decrease in viability of the carotenoid- and tocopherol-treated tumor cell lines as compared with the untreated tumor cells (Figs 1, 2). The MTT assay confirmed this pattern of selective cellular effect. The level of succinic dehydrogenase was noticeably reduced by treatment with p-carotene and canthaxanthin, and a lower effect was seen with cY-tocopherol. There was only a small change in the level of the enzyme with (reduced) glutathione as compared with control cells. No significant decrease in succinic dehydrogenase activity was observed in the normal keratinocytes or melanocytes with any of the agents (Table 1). The level of thymidine incorporation substantiated this pattern of reduced proliferation of cell growth, with B-carotene producing the overall largest reduction in tumor cell proliferation, followed by canthaxanthin and then a-tocopherol and glutathione. Again this inhibition of proliferation was not seen with the normal cells (Fig 3). Further confir- mation of the selective effect of B-carotene on tumor cells compared with normal cells was found in the SDS- PAGE analysis for protein expression. The protein gel (Fig 4) shows the expression of a 70-kD protein in the oral carcinoma cells (lane c) and in the malignant melanoma (lane e), and the lack of expression of this protein in the normal keratinocytes or melanocytes (lanes f to i). The photomicrographs (Fig 5) show the effects of treatment with b-carotene on the oral carcinoma cell at time 0 (Fig 5A), 1 hour (Fig 5B), and 5 hours (Fig 5C). &Carotene treatment was associated with the rounding up and loss of adherance by some of the cells in some of the tissue culture plates after 1 to 2 hours, whereas after 5 hours some of the cells appeared swollen

SCHWARTZ AND SHKLAR 371

1 2 3 4 5 6

SCC-25 SK-MES MCF-7 A375 NHK NHM

FIGURE 3. ‘H-thymidine was incorporated into tumor cells SCC- 25, oral carcinoma; SK-MB, lung carcinoma; MCF-7, breast carcinoma; ~375, malignant melanoma; NHK, normal human keratinocytes; and normal human melanocytes. The number of counts per minute (cpm) of radiolabel was calculated from triplicate wells of each of the above cell lines following incubation with carotenoids, @-carotene, canthaxanthin, or vitamin E. Compared with the untreated tumor cells, p-carotene, followed by cantbaxanthin, and then vitamin E, produced a decrease in proliferation of the tumor cells. Glutathione alone produced very small changes from the untreated controls and therefore was not included in the figure. q Untreated, 0, vitamin E; q , canthaxanthin; n , p-carotene.

or shrunken and were nonviable as determined by trypan blue stain.

Discussion

The data indicated that the carotenoids and a-tocopherol could selectively alter the celhtlar state of human tumor cells from different anatomic sites. The evidence for this contention is the initially observed response following incubation with the various agents (Figs 1, 2), the quantitation of the level of succinic dehydrogenase (MTT assay) (Table I), the determination of thymidine incorporation (proliferation assay) (Fig 3), and the SDS-PAGE analysis of tumor cells and their normal counterparts (Fig 4). The reduction in tumor cell growth and expression of a 70-M> protein was not associated with an observable change in differentiation of the tumor cells.

Previous studies have indicated that carotenoids such as &carotene and a-carotene could reduce the growth of human neuroblastoma cells by inhibiting the cells at Go-G, of the cell cycle. *’ We have also observed that oral and breast carcinoma cells will accumulate at this point following P-carotene and cy-tocopherol treatment. The accumulation of these cells at this point of the cell cycle may explain the decrease in tumor cells accu- mulating at the S phase (Fig 3).

The change in adherance of the tumor cells may be related to the decreased expression of fibronectin, an extracellular adherance protein. Using flow cytometry,

we have observed and quantitated the reduction in fibronectin for oral and breast carcinoma cells (unpublished data).

The current mechanism for the antitumor activity for the carotenoid P-carotene is derived from analysis of the oxygen state of oral carcinoma cells. The data indicated a reduction in the activity of superoxide dis- mutase and glutathione-S-transferase, and lower levels of nonprotein sulthydryls. These reductions result in the loss of protection from reactive oxygen metabolites that could damage the DNA and protein synthesis of the tumor cells. The change in oxygen protection could result in the induction of oxidative stress.*’

The oxygen state of the oral tumor cell will directly alter the capacity of &carotene or cu-tocopherol to inhibit the clonogenic response of the tumor cells. We have shown that the @-carotene-treated, oxygenated tumor cell will exhibit a decreased clonogenic response as compared with the hypoxic tumor cell. To reduce clonogenic growth of the oral tumor cells in an hypoxic condition, b-carotene had to be combined with a-tocopherol. r4y2* These results support earlier findings indicating that &carotene would possibly function as a prooxidant molecule at high partial pressures of oxygen, whereas a-tocopherol would require a lower partial

PIGURE 4. Ten percent SDS-PAGE analysis of protein lysates of the tumor cells SCC-25, an oral carcinoma (rows b, c) and A375, a malignant melanoma (rows d, e), was treated with &carotene (70 PM) (rows c, e). A comparison of the marker lane (row a) with untreated tumor cells (rows b, d) indicates the expression of a 70-kD protein (row c, e). Normal keratinocytes (rows f, g) and melanocytes (rows h, i) after treatment with B-carotene (rows g, i) did not induce the expression of a 70-kD protein.

372 TOXIC EFFECTS OF CAROTENOIDS AND a-TOCOPHEROL ON TUMOR CELLS

FIGURE 5. Photomicrographs of the tumor cell line SCC-25, an oral carcinoma (A) (time 0), (B) treated with b-carotene (70 PM) for 1 hour, or (C) continued treatment for 5 hours. The arrow in Figure 5B indicate the rounding and lifting from the well of the oral carcinoma cells. Following continual treatment with p-carotene, other tumor cells are seen to be vacuolated and degenerated (C).

pressure of oxygen. 23 The prooxidant forms of $carotene and cY-tocopherol could be reactive and alter the metabolic state of the tumor cells.

It is important to recognize that oxidative stress induction has been associated with an increased expression of stress proteins, such as the 70-kD protein shown here.24 Immunofluorescent staining of human and hamster oral mucosa has shown that oral carcinomas have a d ecreased expression of stress proteins compared with non-tumor-bearing mucosa (unpublished data). Significantly, B-carotene treatment of hamster oral mucosa inhibited oral invasive carcinoma formation and enhanced expression of stress proteins.

Furthermore, immunofluorescent staining in vitro, Western immunoblotting, and immunoprecipitation of sulfated and phosphorylated protein extracts from human and hamster tumor cells have also confirmed the identity and expression of stress proteins (70 and 90 kD) induced by ,!%carotene (data not shown). Studies are now in progress detailing the capacity of stress proteins to control the expression of proto-oncogenes and mutated forms, such as ras, c-et-b B, and ~53. The completion of these studies may establish a new pathway for the triggering of tumor suppression. The sig- nificance of this tumor suppression pathway is the knowledge that readily available nutrients may trigger

the inhibitory growth of various tumors. A recent clin- ical study has indicated that the oral administration of P-carotene reverses oral leukoplakia.25

References

1. Alam BS, Alam SQ, Weir JC Jr, et al: Chemopreventative effect of &carotene and 13-cis retinoic acid in salivary gland tumors. Nutr Cancer 6:4, 1984

2. Mathews-Roth MM: Antitumor activity of p-carotene canthaxanthin and phytoene. Oncology 39:33, 1982

3. Rettura Cl, Stratford F, Levenson SM, et al: Prophylatic and therapeutic actions of supplemental @carotene in mice in- noculated with C3HBA adenocarcinoma cells: Lack of therapeutic action of supplemental ascorbic acid. JNCI 69:73, 1982

4. Seifter E, Rettura G, Padawer F, et al: Morbidity and mortality reduction by supplemental vitamin A or beta carotene in CBA mice given total gamma radiation. JNCI 73: 1167, 1984

5. Epstein JH: Effects of beta carotene on UV induced cancer formation on hairless mouse skin. Photochem Photobiol25:2 1 I, 1973

6. Shklar G, Schwartz JL, Trickier D: Prevention of experimental cancer and immunostimulation by vitamin E (immunosur- veillance). J Path01 1460, 1990

7. Schwartz JL, Suda D, Light G: Beta carotene is associated with the regression of hamster buccal pouch carcinoma and the induction of tumor necrosis factor in macrophages. Biochem Biophys Res Commun 130: 1130, 1986

8. Suda D, Schwartz JL, Shklar G: Inhibition of experimental oral carcinogenesis by topical beta carotene. Caminogenesis 7:7 11, 1986

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9. Shklar G, Schwartz JL, Trickier D, et al: Regression by vitamin E of experimental oral cancer. JNCI 78:987, 1987

10. Schwartz JL, Shklar G, Flynn F, et al: The administration of beta carotene to prevent and regress oral carcinoma in the hamster cheek pouch and the associated enhancement of the immune response, in Bendich A, Phillips M, Tengerdy RB (eds): Antioxidants Nutrients and Immune Functions. Ad- vances in Medicine and Biology. New York, NY, Plenum, 1988, pp 77-93

1 I. Schwartz JL, Sloane D, Shklar G: Prevention and inhibition of oral cancer in the hamster buccal pouch model associated with carotenoid immune enhancement. Tumor Biol 10:287, 1989

12. Seifter E, Mondecki J, Holtzman S, et al: Role of vitamin A and p-carotene in radiation protection: Relation to antioxidant properties. Pharmacol Ther 39:357, 1988

13. Seifter E, Rettura G, Padawer J, et al: Regression of C3HBA mouse tumor due to X-my therapy combined with supplemental p-carotene or vitamin A. JNCI 7 1:409, 1983

14. Schwartz JL, Tanaka J, Khandakar V, et al: &Carotene and/or vitamin E as modulators of alkylating agents in SCC-25 human squamous cells, Cancer Pharm Clin Oncol 1992 (in press)

15. Schwartz JL, Singh RP, Teicher B, et al: Induction of a 70 kD associated with selective cytotoxicity ofbeta carotene in human epidermal carcinoma. Biochem Biophys Res Commun 169: 941, 1990

16. Schwartz JL: In vitro biological methods for the determination

of carotenoid activity, in Packer L (ed): Methods of Enzy- mology. San Diego, CA, Academic, 1992 (in press)

17. Rheinwald JR, Green H: Serial cultivation of strains of human epidermal keratinocytes: The formation of keratinizing colonies from single cell colonies. Cell 6:33 1, 1975

18. CarMichael J, DeGraff WC, Gazdar AF, et al: Evaluation of a tetrazolium-based semiautomated calorimetric assay: Assess- ment of chemosensitivitv testing. Cancer Res 47:936, 1987

19. Laemrnli UK: Cleavage of &uctu~ proteins during the assembly of the head of bacteriophage T4. Nature 227:680, 1970

20. Murakoshi M, Takayasu J, Kimura 0, et al: Inhibitory effects of carotene on proliferation of the human neuroblastoma cell line GOTO. JNCI 81:1649, 1989

2 1. Benjamin IJ, Kroger B, Williams RS: Activation of the heat shock transcription factor by hypoxia in mammalian cells. Proc Natl Acad Sci USA 87:6263, 1990

22. Tanaka J, Schwartz JL, Herman TS, et al: Combinations of fl- carotene and/or vitamin E with antitumor alkylating agents (AA) in SCC-25 and SCC-25/CP cells. Proc Am Assoc Cancer kes 321373, 1991

23. Burton GW, Ingold KU: b-carotene: An unusual type of lipid antioxidant. Science 244:569, 1984

24. Young RA, Elliot TJ: Stress protein, infection, and immune sur- veillance. Cell 59:5, 1989

25. Garewal HS, Meyskens FL, Killen D, et al: Response of oral leukoplakia to beta carotene. J Clin Oncol 8: 17 11, 1990

J Oral Maxillofac Surg

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Discussion

The Selective Cytotoxic Effect of Carotenoids and a-Tocopherol on Human

Cancer Cell Lines In Vitro

No-Hee Park, DMD, PhD UCLA School of Dentistry, Los Angeles

This article by Schwartz and Shklar reports selective cytotoxic effect of carotenoids p-carotene and canthaxanthin, and a-tocopherol (vitamin E) on several human cancer cells. The authors also showed an expression of 70-kd protein from cancer cells exposed to p-carotene that was not expressed from cancer cells before exposure to Bcarotene or from normal cells treated with p-carotene. Although it is not clear, the authors believe that the expression of 70-kd protein might be associated with the selective cytotoxicity of &carotene. In view of the fact that many studies have indicated that carotenoids and retinoids might be effective in reversing a putative “filed carcinogenesis,” this report certainly provides some- what conclusive evidence that these chemicals are useful in reversing the multistep carcinogenesis.

Numerous epidemiological studies have shown that p-carotene and retinoids inhibit the development of certain carcinomas and induce remission of precancerous lesions, ie, remission of leukoplakia and reduction of micronucleated cells.‘~* Stich and his associates3 reported that administration of vitamin A for 6 months resulted in complete remission of leukoplakia in 57% and a reduction of micronucleated cells in 96% of tobacco chewers. /3-Carotene also suppressed the number of micronucleated cells and induced remission of leukoplakia.4 Furthermore, B-carotene and retinoids can temporarily inhibit the neoplastic phenotype of cancer cells in vitro, ie, induction of cell differentiation.5-7

Studies have shown a close association of squamous cell carcinoma (SCC) with the activation of cellular proto-oncogenes. For example, cellular bcl-1 (B cell lymphoma/leu- kemia-1) proto-oncogene is amplified twofold to tenfold in SCC tumors and established SCC cell lines.’ The expression of H-rus, K-rus, and myc proto-oncogenes is significantly higher in SCC cell lines compared with normal counterparts.9s’o An extremely high amount of c-e&B- 1 is expressed in SCC cell lines. The amplification and overexpression of c-erb-B-1 oncogene are closely associated with the development of SCC in human and laboratory animals, such as in established human SCC cell lines of the tongue’ ’ and 7,12- dimethylbenz(a)anthracene (DMBA)-induced hamster cheek pouch carcinoma.“*r3 More recently, an overexpression of multiple cellular proto-oncogenes, such as src, e&B-l, abl, fos. ruJ H-rus, and myc genes in human SCC cell lines was reported, indicating an involvement of multiple cellular oncogenes in carcinogenesis. I4 As indicated by the authors, the activation status of multiple cellular proto-oncogenes must be investigated in cancer cells and cells exposed to b-carotene and other chemicals to further understand the mechanism of selective cytotoxicity of these chemicals. Since the ability of cells to grow in soft agar (anchorage independency of cells) has been reported to be a reliable criterion for cell transfor- mation and a marker for in vitro malignancy,‘s a study in- vestigating affect of carotenoids P-carotene and canthaxanthin, and cu-tocopherol on in vitro anchorage independency of cancer cell lines might also provide us further information on the selective reversal activity of these chemicals. Finally, although the authors strongly suspect that 70-kd protein expressed from cancer cells exposed to /3-carotene is a heat shock protein, further study should be performed to confirm this using specific monoclonal antibody to heat shock protein.

The selective cytotoxic effect of carotenoids and α-Tocopherol on human cancer cell lines in vitro

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