Comparative Effects of Chemotherapeutic Agents on the Growth and Survival of Human Adrenal Carcinoma Cells in Culture
Authors
M. Montoya1, 2, J. W. Brown1,2, L. M. Fishman1, 2
Affiliations
1 Adrenal Research Laboratory, V. A. Medical Center, Miami, FL, USA
2 Division of Endocrinology, Diabetes and Metabolism, Department of Medicine, University of Miami Miller School of Medicine, Miami, FL, USA
Key words
· adrenal carcinoma · ara-c
2-methoxyestradiol · mitotane
· paclitaxel
Abstract
&
Adrenocortical carcinoma is an uncommon malignancy that is usually fatal within a short time after diagnosis. We have investigated the effects on the growth and survival of SW-13 human adrenal carcinoma cells in culture of some currently used and some potentially new agents in the treatment of adrenal cancer. Established drugs tested were mitotane, cisplatin, etopo- side, 5-fluorouracil, and suramin. Other agents studied included adenine arabinofuranoside, cytosine arabinofuranoside, 2-methoxyestradiol, and paclitaxel. The most potent chemothera-
peutic agents in this system were paclitaxel and 2-methoxyestradiol, with EC50 of 1.8x10-8 and 3.3 ×10-7M, respectively. Cytosine arabinofura- noside and cisplatin both had the same EC50 of 7.0×10-7M, and etoposide 1.1×10-6M. All the other agents tested required much higher doses for effect, including mitotane, the current most commonly used chemotherapy for adrenal can- cer, with an EC50 of 3.3× 10-4M. These data sug- gest that paclitaxel, 2-methoxyestradiol, and cytosine arabinofuranoside should be further evaluated for their potential in the chemother- apy of adrenal carcinoma.
received 02.10.2007 accepted 12.10.2007
Bibliography DOI 10.1055/s-2008-1073139 Horm Metab Res 2008; 40: 302-305 @ Georg Thieme Verlag KG Stuttgart . New York ISSN 0018-5043
Correspondence
Dr. J. W. Brown Adrenal Research Laboratory (151)
V. A. Medical Center
1201 N. W. 16th St. Miami 33125 Florida USA Tel .: +1/305/324 4455 (Extn 4487)
Fax: +1/305/575 3126 j.brown@miami.edu
Introduction &
Adrenocortical carcinoma is a rare malignancy, comprising approximately 0.02% of all cancers [1,2] and occurring with a frequency of about two cases per million individuals per year [3]. Recent studies have implicated novel mecha- nisms of adrenal tumorogenesis including genetic, molecular, and immune related path- ways [4-9].
Patients with functional tumors most often present with manifestations of glucocorticoid excess (i.e., Cushing’s syndrome, with central obesity, glucose intolerance, abdominal striae, protein wasting, and psychiatric disturbance), although other presentations include clinical and laboratory evidence of androgen excess (hir- sutism and virilization in women, with increased levels of testosterone and related androgens) and more rarely hyperaldosteronism (hypertension and hypokalemia) [10-12]. Estrogen and proges- terone-producing adrenocortical tumors have also been reported [13,14]. In many individuals, however, adrenal carcinomas are highly undiffer- entiated and do not produce significant quanti- ties of any biologically active steroid. Such
patients usually remain largely asymptomatic [15-17] until the effects of a rapidly growing tumor become apparent.
Progress in the chemotherapeutic approach to adrenocortical carcinoma has been slow and marked by only limited success. Mitotane (o,p’- dichlorophenyl-2,2-dichloroethane, sometimes abbreviated as o,p’-DDD), discovered over 50 years ago [18] as a breakdown product of the pes- ticide DDT, is a commonly utilized drug in the treatment of adrenal carcinoma [19,20]. High initial doses (8-12g per day orally) are required in many patients, often with debilitating side- effects. More importantly, mitotane has only modest effectiveness in prolonging the life of affected individuals [12,20,21]. Other agents currently in use include cisplatin, doxorubicin, etoposide, 5-fluorouracil (5-FU), and suramin [22-26]. As with mitotane, the clinical effective- ness of these and other chemotherapeutic drugs and drug combinations thus far tested remain marginal, as evidenced by rapid progression and high mortality rate in this disease.
In an effort to identify more effective drugs for the treatment of adrenocortical carcinoma, we have compared the effects of various established
and experimental agents on SW-13 human adrenal carcinoma cells in tissue culture [27]. The agents studied which are not cur- rently in standard use for treating adrenal carcinoma are paclit- axel (Taxol™), 2-methoxyestradiol, adenine arabinofuranoside (ara-a) and cytosine arabinofuranoside (ara-c). Paclitaxel has been shown to be extremely effective in the treatment of breast cancer and has a relatively low level of adverse side-effects. There is also clear evidence indicating that it is cytotoxic to ster- oidogenic NCI H-295 adrenocortical carcinoma cells in vitro [28]. Paclitaxel is currently one of the most successful and best known of a class of cancer drugs classified as “tubulin depolymerization inhibitors” because they block the rearrangement of cytoskeletal microtubules; the reordering of these sub-cellular scaffolding proteins is a necessary process during cell division and, accord- ingly, represents a promising target for chemotherapy. Another agent studied, 2-methoxyestradiol, is known to have tubulin and microtubule altering effects as well as other tumor growth regu- latory influences in vitro (i.e., calmodulin inhibition and inter- ference with angiogenesis [29-31]), but is not currently approved for human use (experimental human trials with this agent are now underway for treatment of both prostate and breast can- cer). Cytosine arabinofuranoside is commonly used in standard treatment protocols for a number of other forms of malignancy. Its nucleoside analogue, ara-a, although tested as a cancer drug, is most commonly used as an antiviral agent.
Materials and Methods
&
Establishment of cell cultures
Frozen SW-13 human adrenal carcinoma cells, derived from a highly undifferentiated, rapidly growing, nonsteroid producing human adrenocortical carcinoma [21], were obtained from the American Type Culture Collection (Rockville, MD) in L-15 medium, defrosted and transferred into Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum (FBS) according to protocols previously described [32,33]. Approximately 200,000 cells were plated on to each dish (result- ing in an initial attachment density of 15-20% confluence) and cultured at 37℃ in 3 cm Falcon tissue culture dishes under a humidified atmosphere of 5% CO2 and 95% air. After growth of cells reached near confluence (85-90% plate coverage), the medium was changed to DMEM containing 10% horse serum (HS) to induce a quiescent growth phase (QGP). Other cultures were grown only to 35-45% confluence to reduce potential effects of contact inhibition; fresh medium containing 10% FBS was then added to maintain these cultures in an exponential growth phase (EGP). Dulbecco’s Modified Eagle’s medium, horse serum, fetal calf serum, and other cell culture materials were obtained from Gibco (Grand Island, NY). All chemotherapy agents that were studied were obtained from Sigma Chemical Company (St. Louis, MO).
Experimental protocols
SW-13 cells were cultured for up to 6 days under QGP conditions (10% HS with experiments initiated at 85 to 90% confluence) or EGP conditions (10% FBS with experiments initiated at 35 to 45% confluence), with various pharmacologic agents present at the indicated concentrations. Cell culture density (% confluence) was determined on days 0, 2, 4, and 6 by averaging results from 3-7 culture dishes from multiple separate experiments using a light-microscopy technique previously described [34]. EC50S
100
Control
Suramin
Control
Cell Density (% Confluence)
80
Mitotane
5-FU
Mitotane
Suramin
60
Ara-A
Etoposide
40
Cisplatin
2- MeOE2
Ara-C
20
Taxol
0
0
2
4
6
Days in Culture
(effective concentration for 50% reduction in percent conflu- ence) were determined using manual logarithmic graphic inter- polation from dose-response experiments performed with drug concentrations varying between 10-8 and 10-3M. Cell viability studies were undertaken on day 6 of incubation with a 0.2% w/v trypan blue dye exclusion analysis [35]. Control cultures consist- ently showed more than 70% viability of attached cells (range 70-90%), while the viability of detached cells was found to be negligible in all cases.
In all experiments, statistical differences between experimental groups relative to appropriate controls were determined by an Analysis of Variance (ANOVA) with Dunnett’s Multiple Compari- son Post-Test analyses using the INSTAT computer program (Graph Pad Software Inc., San Diego, CA).
Results
&
Time-course of drug effects on cell growth and survival Fig. 1 shows the effects at a concentration of 10-5 M of various established and experimental agents (ara-a, ara-c, cisplatin, etoposide, 5-FU, 2-methoxyestradiol, mitotane, suramin, and paclitaxel) on the survival of SW-13 human adrenal carcinoma cells in culture over 6 days of incubation under QGP conditions. At this dose, neither mitotane, 5-FU nor suramin had any sig- nificant effect on the survival of SW-13 cells. Ara-c, cisplatin, etoposide, 2-methoxyestradiol, and paclitaxel (Taxol), however, each had a significant effect, showing at least 80% reduction in cell density (% confluence) after 4 days in culture (p<0.01) and virtually complete cell death by day 6 (p<0.001). The effect of 2-methoxyestradiol was the most rapid, followed by paclitaxel, etoposide, cisplatin, and ara-c. Adenine arabinofuranoside was found to be substantially less effective than any of these agents. To assess whether rapidly growing cells without the potential influence of contact inhibition demonstrate the same response, the effects of each of these drugs on growth and survival of SW- 13 cells were also studied under exponential growth phase (EGP) conditions ( Fig. 2). Results during EGP conditions were found
100
5-FU
80
Control
Cell Density (% Confluence)
Suramin
Mitotane
60
Ara-A
40
Ara-C
Etoposide
20
Cisplatin
Taxol
0
2- MeOF.2
0
2
4
6
Days in Culture
to be similar to those under quiescent growth phase (QGP) cell growth conditions, with mitotane, 5-FU, suramin, and ara-a hav- ing no significant effects at 10-5M. 2-Methoxyestradiol, pacli- taxel, and cisplatin had the most rapid effects (each showing significant cytotoxic influences within 2 days of addition; p<0.01), with the effects of ara-c and etoposide on cell growth and survival requiring a slightly longer time period (significant inhibition seen after 4 days of incubation; p<0.01). It should be noted that paclitaxel and cisplatin showed effects more rapidly in cells during EGP than they did under QGP conditions, but otherwise effects were similar.
Dose-response relationships of various agents on cell growth and survival
· Fig. 3 shows dose-response relationships for each of the drugs studied after 6 days of incubation under quiescent cell growth conditions; Table 1 gives the calculated EC50 [M] for each com- pound based upon these curves. The estimated EC50 of paclitaxel (1.8×10-8M) was more than an order of magnitude lower than those of the next most effective compounds: 2-methoxyestra- diol (EC50=3.3×10-7M), ara-c and cisplatin (EC50 for each =7.0 × 10-7M), and etoposide (EC50=1.1×10-6M). Mitotane had an estimated EC50 of 3.3×10-4M, while the EC50 for each ara-a, suramin, and 5-FU was also greater than 1.0x 10-4M.
Discussion and Conclusion
&
The results of these studies indicate that paclitaxel, 2-methoxy- estradiol, ara-c, cisplatin, and etoposide all demonstrate potent growth-inhibiting effects on SW-13 human adrenal carcinoma cells in culture. Of the agents in current use for treating adrenal carcinoma that we studied, cisplatin and etoposide were the most potent on a concentration basis in the SW-13 cell-line. Each of the other drugs commonly used in the treatment of adrenocortical carcinoma, including mitotane, had EC50s at least two orders of magnitude higher. Of special interest is the finding that paclitaxel, 2-methoxyestradiol, and ara-c showed greater effectiveness in the SW-13 adrenocortical carcinoma cell-line
100
Suramin
90
5-FU
Ara-A
Cell Density (% Confluence)
80
70
60
Etoposide
Mitotane
50
40
Taxol
30
Ara-C
20
2-MeOE}
Cisplatin
10
0
10 -8
10-7
10-6
10-5
10-4
10-3
Concentration (Molar)
| Agent | EC50 [M] |
|---|---|
| ara-a | >1.0×10-4 |
| ara-c | 7.0×10-7 |
| cisplatin | 7.0×10-7 |
| etoposide | 1.1×10-6 |
| 5-fluorouracil | >1.0×10-4 |
| 2-methoxystradiol | 3.3×10-7 |
| mitotane (o,p'-DDD) | 3.3×10-4 |
| paclitaxel (taxol) | 1.8×10-8 |
| suramin | >1.0×10-4 |
EC50 was determined for cells under QGP conditions after 6 days of incubation with the indicated agent
than that of most of the agents currently used in clinical practice to treat this malignancy. The effects of paclitaxel are clearly not limited to the highly aggressive, nonsteroidogenic SW-13 human adrenal carcinoma cell line studied here, since Fallo et al. [28] have previously reported comparable results in steroidogenic NCI H-295 adrenal carcinoma cultures. Similar studies in the NCI cell-line have, to our knowledge, not been reported with the other chemotherapeutic agents, but should be undertaken so that comparisons can be made between the less differentiated SW-13 cell line and the more differentiated NCI H-295 cell-line. Such studies should provide not only a better understanding of the general relevance of our findings in adrenal carcinoma but might also provide insight into differential treatment protocols for steroid-secreting and for nonsecreting adrenal carcinomas. While efficacy in vitro and low EC50s do not necessarily translate into usefulness in vivo, where complex physiological, biochemi- cal and pharmacokinetic interactions are involved, these data suggest that animal studies and more in-depth clinical trials in adrenal carcinoma of not only paclitaxel but also 2-methoxy- estradiol and ara-c may be warranted to assess their potential as supplemental chemotherapeutic agents for use with the cur- rently established treatment protocols. In vivo studies with 2- methoxyestradiol or its analogues (14-dehydro-2-methoxy-
estradiol and 2-methoxy-15-dehydroestradiol) might be particularly rewarding, since these agents are known to have antitumor effects related to a number of different mechanisms, including not only suppression of microtubule rearrangement but also angiogenesis inhibition, calmodulin signaling and meta- phase arrest [29,31].
With the exception of 2-methoxyestradiol and its analogues, all of the agents studied here are currently approved in a number of countries for treatment of malignancies other than adrenal car- cinoma. Although all of these drugs have at least some adverse side-effects in humans and/or animal models, most are gener- ally less toxic than mitotane, one of the current most commonly utilized drugs in the treatment of adrenal carcinoma.
Acknowledgments
&
These studies were supported by the U.S. Department of Veter- ans Affairs and the South Florida Veterans Affairs Foundation for Research and Education.
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