BRIEF COMMUNICATION

Inorganic Arsenite-Induced Malignant Transformation of Human Prostate Epithelial Cells

William E. Achanzar, Eduardo M. Brambila, Bhalchandra A. Diwan, Mukta M. Webber, Michael P. Waalkes

Affiliations of authors: W. E. Achanzar, E. M. Brambila, M. P. Waalkes, Inorganic Carcinogenesis Section, Laboratory of Comparative Carcinogenesis, National Cancer Institute at National Institute of Environmental Health Sciences, Research Triangle Park, NC; B. A. Diwan, Intramural Research Support Program, SAIC-Frederick, NCI-Frederick, Frederick, MD; M. M. Webber, Departments of Medicine and Zoology, Michigan State University, East Lansing, MI.

Correspondence to: Michael P. Waalkes, Ph.D., Inorganic Carcinogenesis Section, Laboratory of Comparative Carcinogenesis, National Cancer Institute at National Institute of Environmental Health Sciences, MD F0–09, 111 Alexander Dr., Research Triangle Park, NC 27709 (e-mail: waalkes{at}niehs.nih.gov).

ABSTRACT

Although several epidemiologic studies show an association between arsenic exposure and prostate cancer, it is still unknown whether human prostate epithelial cells are directly susceptible to arsenic-induced transformation. This study was designed to determine whether the nontumorigenic human prostate epithelial cell line RWPE-1 could be malignantly transformed in vitro by arsenite. RWPE-1 cells were continuously exposed to 5 µM arsenite and monitored for signs of transformation, assessed as changes in matrix metalloproteinase-9 levels. After 29 weeks of exposure, the arsenite-exposed RWPE-1 cells (referred to as CAsE-PE) showed a marked increase in matrix metalloproteinase-9 secretion, a common finding in prostate malignancies. Malignant transformation was confirmed when CAsE-PE cells produced aggressive undifferentiated malignant epithelial tumors in nude mice. The tumors stained positive for human prostate-specific antigen, confirming their origin. These results are the first report of arsenite-induced malignant transformation of a human epithelial cell line and provide an important in vitro model for studying the mechanisms underlying arsenic-induced carcinogenesis in humans.


Prostate cancer is a leading cause of cancer-related death in the United States (1), yet the underlying etiology of this disease is poorly understood. Evidence, however, indicates that both endocrine and environmental factors play a role in the development of prostate tumors (1,2). An important environmental contaminant associated with prostate cancer is inorganic arsenic. Indeed, several epidemiologic studies have suggested a possible association between exposure to inorganic arsenic and prostate cancer (3,4), including a recent study of populations residing in the United States (5). Inorganic arsenic is a toxic metalloid long known to be carcinogenic to humans, especially to tissues such as lung, skin, bladder, and liver (6,7). It occurs in the environment both naturally and as a result of human endeavors (6,7). Although, for unknown reasons, development of animal models to study inorganic arsenic-induced carcinogenesis has been problematic, we have successfully induced malignant transformation of a rat liver epithelial cell line by long-term exposure to arsenite in vitro (8). This in vitro model has proven valuable for studying the molecular mechanisms underlying arsenite-induced malignant transformation (911). To our knowledge, there are no published reports of human epithelial cells being malignantly transformed by arsenic. Because it is unknown whether arsenic can directly malignantly transform human prostate cells, we set out to develop an in vitro model of arsenic-induced prostatic carcinogenesis by subjecting the nontumorigenic human prostate epithelial cell line RWPE-1 to long-term arsenite exposure in vitro. The development of such a model would validate human prostate epithelial cells as a viable and direct target for arsenic-induced carcinogenesis and would allow for in-depth molecular examination of the changes underlying arsenic-induced carcinogenesis in a model of clear relevance to human exposure.

Development and culture of the RWPE-1 cell line has been described previously (12,13). RWPE-1 cells were derived from normal human prostate epithelium, were immortalized with human papillomavirus 18, and are nontumorigenic (12,13). For up to 29 weeks, cells were cultured continuously in keratinocyte serum-free medium supplemented with 50 µg/mL bovine pituitary extract, 5 ng/mL epidermal growth factor, antibiotics and 5 µM as sodium arsenite. Parallel cultures maintained in arsenite-free medium served as passage-matched controls. Cells were passaged weekly, with new cultures seeded with 1 x 106 cells in 75-cm2 flasks. Previous studies (14,15) revealed that increased secretion of matrix metalloproteinase-9 (MMP-9) is associated with Ras-induced or cadmium-induced malignant transformation of RWPE-1 cells. In addition, increased MMP-9 secretion occurs in prostate tumors and primary cultures derived from prostatic cancers and is associated with aggressive malignancies (16,17). Thus, we periodically assayed the long-term arsenite-exposed RWPE-1 cells (referred to as CAsE-PE cells) for MMP-9 activity by zymography as described (15).

Compared with MMP-9 activity from passage-matched RWPE-1 cells, no discernible increases in MMP-9 activity from CAsE-PE cells were detected until after 29 weeks of continuous arsenite exposure. At this time, CAsE-PE cells had a 2.2-fold (95% confidence interval [CI] = 2.0 to 2.5) increase in MMP-9 activity compared with passage-matched control RWPE-1 cells (Fig. 1Go). Because the observed increase in MMP-9 activity suggested that arsenic-induced malignant transformation had occurred, the CAsE-PE cells and passage-matched RWPE-1 cells were inoculated into the renal capsules of male nude mice (NCr-nu) to test their ability to form tumors. Male NCr-nu mice were obtained at 8 weeks of age from the NCI-Frederick Animal Production Area breeding colony and were housed in an American Association for Accreditation of Laboratory Animal Care (AAALAC) accredited facility under conditions that met or exceeded recommendations outlined in the Guide for Care and Use of Laboratory Animals (National Institutes of Health Publication No. 86–23, 2985). Within 10 weeks, five of five mice inoculated with the CAsE-PE cells developed tumors, whereas none of five mice inoculated with the control RWPE-1 cells developed tumors, a statistically significant difference (P = .008, two-sided Fisher’s exact test). After staining sections of the tumors with hematoxylin and eosin, histologic examination of the tumors revealed them to be undifferentiated epithelial tumors (Fig. 2, AGo), which is consistent with the majority of epithelial prostate tumors and with previous reports of tumors formed by malignantly transformed prostate epithelial cells (12,15,18). The CAsE-PE-derived tumors were highly aggressive and invaded the surrounding capsular muscle and adjacent normal renal parenchyme (Fig. 2, AGo). The invasive nature of the tumors is consistent with the increased MMP-9 secretion observed in CAsE-PE cells in vitro because MMP-9 appears to facilitate prostate tumor invasion and metastasis (16). By immunohistochemical analysis, tumor sections stained strongly positive with an antibody specific for human prostate-specific antigen (Fig. 2, BGo), confirming that the tumors were of human prostatic origin.



View larger version (29K):
[in this window]
[in a new window]
 
Fig. 1. Analysis of matrix metalloproteinase-9 (MMP–9) activity in conditioned medium from passage-matched, human immortalized, nontumorigenic prostate RWPE-1 cells and RWPE-1 cells exposed long-term to 5 µM sodium arsenite (CAsE-PE). A) Culture supernatants were collected and analyzed for MMP-9 activity by zymography as described (15). Zymogram gel shows increased MMP-9 activity in CAsE-PE conditioned medium, which was collected from cells that had been exposed to sodium arsenite for 29 weeks. Each lane represents medium from an independent culture. B) Changes in MMP-9 activity were quantified by photographing the gel and densitometric analysis of the bands using 1D version 2.0 software (Kodak, Rochester, NY). Data are expressed as fold increase relative to MMP-9 levels from RWPE-1 culture supernatants and are represented as mean (n = 3) with upper 95% confidence intervals. *, Statistically significant difference (P < .001, unpaired Student’s t test).

 


View larger version (89K):
[in this window]
[in a new window]
 
Fig. 2. Histologic examination of tumors formed by human prostate epithelial cells exposed long-term to sodium arsenite (CAsE-PE cells) after inoculation into the renal capsules of male NCr-nu nude mice. Ten weeks after inoculation, the tumors were removed, fixed in formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (A) or stained for human prostate-specific antigen (PSA) by immunohistochemistry (B). A) A representative tumor section. The staining pattern revealed the tumors to be undifferentiated epithelial tumors that frequently invaded into the surrounding capsular muscle tissue. Muscle fibers (arrowhead) can be seen in the upper left portion of the photomicrograph. Bar = 50 µm. B) A representative tumor section. The section was incubated with a mouse anti-human PSA antibody (Novocastra Laboratories, Newcastle upon Tyne, U.K.) at a 1 : 600 dilution and the antigen-antibody complexes were detected histochemically, with diaminobenzadine as the chromogen following the protocol recommended by Novocastra. The section was counterstained with hematoxylin. Positive staining for human PSA, indicated by the reddish-brown color, confirmed that the tumors were derived from human prostate epithelial cells. Bar = 100 µm.

 
To our knowledge, there are no published reports of arsenic-induced malignant transformation of human epithelial cells. In fact, the only available report described induction of anchorage-independent growth, an event sometimes associated with transformation, in human osteosarcoma cells exposed long-term to arsenic (19); however, no evidence of tumorigenicity was provided. There is little evidence that bone is a target for arsenic-induced carcinogenesis in humans (20), and defining the events relevant to arsenic-induced carcinogenesis in a tumor-derived, albeit nontumorigenic, cell line may be problematic. Thus, our study not only represents the first, to our knowledge, description of arsenic-induced malignant transformation of a human epithelial cell line, but also of arsenic-induced transformation in a cell line analogous to a potential in vivo target cell population relevant to arsenic-induced carcinogenesis in humans. The finding that human prostate epithelial cells are directly susceptible to the transforming effects of inorganic arsenite is biologically significant, given that there is epidemiologic evidence suggesting that the human prostate may be a possible target of arsenic-induced carcinogenesis (35). More importantly, development of CAsE-PE cells provides a model in which the molecular and genetic events associated with arsenic-induced carcinogenesis in humans can now be examined.

Understanding the mechanisms involved in arsenic-induced carcinogenesis has been hampered by the lack of suitable animal models. Inorganic arsenic is recognized as a human carcinogen even though, in the absence of treatment with other agents, arsenic-induced tumors have been difficult to produce in rodents (7,2123). Consequently, mechanistic studies rely heavily on experiments performed using in vitro systems, the validity of which is always a critical issue. Our laboratory has previously demonstrated arsenite-induced malignant transformation of rat liver epithelial cells (8), and has identified several important molecular events associated with arsenite-induced transformation (911). More importantly, several of these changes correspond with alterations found in the livers of humans exposed long-term to inorganic arsenic (24). Nevertheless, there are differences between humans and rodents with respect to susceptibility to arsenic-induced tumors, highlighting the need for human cell-based in vitro models of arsenic-induced carcinogenesis. The development of a model such as the one described herein will allow further analysis of the molecular events associated with inorganic arsenic-induced carcinogenesis in a human system. This will permit additional validation of results obtained in rodent cells and potentially provide clues to understanding why humans are apparently more sensitive than rodents to the carcinogenic effects of inorganic arsenic.

NOTES

This project was funded in part by contract NO-1-CO-12400 from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services.

The authors are grateful to Dr. Jerrold M. Ward for assistance with the pathological analysis. The authors also thank Drs. Jie Liu and Dr. Hua Chen for their critical evaluation of the manuscript.

REFERENCES

1 Jemal A, Thomas A, Murray T, Thun M. Cancer statistics, 2002. CA Cancer J Clin 2002;52:23–47.[Abstract/Free Full Text]

2 Key TJ. Hormones and cancer in humans. Mutat Res 1995;333:59–67.[Medline]

3 Wu MM, Kuo TL, Hwang YH, Chen CJ. Dose-response relation between arsenic concentration in well water and mortality from cancers and vascular diseases. Am J Epidemiol 1989;130:1123–32.[Abstract]

4 Chen CJ, Wang CJ. Ecological correlation between arsenic level in well water and age-adjusted mortality from malignant neoplasms. Cancer Res 1990;50:5470–4.[Abstract]

5 Lewis DR, Southwick JW, Ouellet-Hellstrom R, Rench J, Calderon RL. Drinking water arsenic in Utah: a cohort mortality study. Environ Health Perspect 1999;107:359–65.[Medline]

6 IARC. Some Metals and Metallic Compounds. In: IARC Monograph on the evaluation of the carcinogenic risk of chemicals to humans. Vol. 23. Lyon (France): IARC; 1980. p. 39–142.

7 IARC. Some metals and metallic compounds. In: IARC monograph on the evaluation of the carcinogenic risk to humans–overall evaluations of carcinogenicity: an update of IARC Monographs 1 to 42. Suppl. 7. Lyon (France): IARC; 1987. p. 100–3.

8 Zhao CQ, Young MR, Diwan BA, Coogan TP, Waalkes MP. Association of arsenic-induced malignant transformation with DNA hypomethylation and aberrant gene expression. Proc Natl Acad Sci U S A 1997;94:10907–12.[Abstract/Free Full Text]

9 Chen H, Liu J, Merrick BA, Waalkes MP. Genetic events associated with arsenic-induced malignant transformation: applications of cDNA microarray technology. Mol Carcinog 2001;30:79–87.[Medline]

10 Chen H, Liu J, Zhao CQ, Diwan BA, Merrick BA, Waalkes MP. Association of c-myc overexpression and hyperproliferation with arsenite-induced malignant transformation. Toxicol Appl Pharmacol 2001;175:260–8.[Medline]

11 Liu J, Chen H, Miller DS, Saavedra JE, Keefer LK, Johnson DR, et al. Overexpression of glutathione S-transferase {pi} and multidrug resistance transport proteins is associated with acquired tolerance to inorganic arsenic. Mol Pharmacol 2001;60:302–9.[Abstract/Free Full Text]

12 Bello D, Webber MM, Kleinman HK, Wartinger DD, Rhim JS. Androgen responsive adult human prostatic epithelial cell lines immortalized by human papillomavirus 18. Carcinogenesis 1997;18:1215–23.[Abstract]

13 Webber MM, Bello D, Kleinman HK, Hoffman MP. Acinar differentiation by non-malignant immortalized human prostate epithelial cells and its loss by malignant cells. Carcinogenesis 1997;18:1225–31.[Abstract]

14 Webber MM, Waghray A, Bello D, Rhim JS. Proteases and invasion in human prostate epithelial cell lines: implications in prostate cancer prevention and intervention. Radiat Oncol Invest 1996:3:358–62.

15 Achanzar WE, Diwan BA, Liu J, Quader S, Webber MM, Waalkes MP. Cadmium-induced malignant transformation of human prostate epithelial cells. Cancer Res 2001;61:455–8.[Abstract/Free Full Text]

16 Hamdy FC, Fadlon EJ, Cottam D, Lawry J, Thurrell W, Silcocks PB, et al. Matrix metalloproteinase 9 expression in primary human prostatic adenocarcinoma and benign prostatic hyperplasia. Br J Cancer 1994;69:177–82.[Medline]

17 Festuccia C, Bologna M, Vicentini C, Tacconelli A, Miano R, Violini S, et al. Increased matrix metalloproteinase-9 secretion in short-term tissue cultures of prostatic tumor cells. Int J Cancer 1996;69:386–93.[Medline]

18 Prasad SC, Thraves PJ, Dritschilo A, Rhim JS, Kuettel MR. Cytoskeletal changes during radiation-induced neoplastic transformation of human prostate epithelial cells. Scanning Microsc 1996;10:1093–102.[Medline]

19 Rossman TG, Visalli MA, Uddin AN, Hu Y. Human cell models of arsenic carcinogenicity and toxicity: transformation and genetic susceptibility. In: Chappell WR, Abernathy CO, Calderon RL, editors. Arsenic exposure and health effects IV. Amsterdam (The Netherlands): Elsevier; 2001. p. 285–95.

20 National Research Council. Arsenic in the drinking water. Washington (DC): National Academy Press; 1999.

21 Kitchin KT. Recent advances in arsenic carcinogenesis: modes of action, animal model systems, and methylated arsenic metabolites. Toxicol Appl Pharmacol 2001;172:249–61.[Medline]

22 Basu A, Mahata J, Gupta S, Giri AK. Genetic toxicology of a paradoxical human carcinogen, arsenic: a review. Mutat Res 2001;488:171–94.[Medline]

23 Rossman TG, Uddin AN, Burns FJ, Bosland MC. Arsenite is a cocarcinogen with solar ultraviolet radiation for mouse skin: an animal model for arsenic carcinogenesis. Toxicol Appl Pharmacol 2001;176:64–71[Medline]

24 Lu T, Liu J, LeCluyse EL, Zhou YS, Cheng ML, Waalkes MP. Application of cDNA microarray to the study of arsenic-induced liver diseases in the population of Guizhou, China. Toxicol Sci 2001;59:185–92.[Abstract/Free Full Text]

Manuscript received May 23, 2002; revised September 19, 2002; accepted October 4, 2002.


This article has been cited by other articles in HighWire Press-hosted journals:


             
Copyright © 2002 Oxford University Press (unless otherwise stated)
Oxford University Press Privacy Policy and Legal Statement