The Effect of Megestrol Acetate on Growth of Hepg2 Cells in Vitro and in Vivo

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5226 Vol. 10, 5226–5232, August 1, 2004 Clinical Cancer Research

The Effect of Megestrol Acetate on Growth of HepG2 Cells In vitro and In vivo

Kai Zhang1 and Pierce K. H. Chow1,2 modality of treatment that consistently prolongs survival. How- 1Department of Experimental Surgery and 2Department of General ever, only 10–15% is operable at diagnosis (2, 3), and the Surgery, Singapore General Hospital, Singapore prognosis for inoperable HCC remains dismal (4, 5). Attempts such as radiotherapy (6), chemotherapy (6–8), and immuno- therapy (9) have not been proven effective in the majority of ABSTRACT patients. Purpose: Hepatocellular carcinoma (HCC) is generally There has been sustained interest in hormonal therapy in considered as a sex hormone-dependent tumor, and hormo- HCC. Both clinical observations and laboratory investigations nal therapy has been proposed as a strategy for the treat- have suggested that HCC is a sex hormone-dependent tumor, ment of HCC. The aim of the study is to investigate the effect namely its male predominance (10), an apparent relationship to of megestrol acetate, a synthetic progesteronal agent, on steroid hormones (11, 12) and the altered status of sex hormone growth of HepG2 cells in vitro and in vivo. receptors (13, 14). New therapeutic approaches with hormonal Experimental Design: Cell growth in vitro was assessed agents have thus been attempted in the treatment of HCC. by a colormetric method, and cell growth in vivo was as- Glucocorticoid antagonists such as progesterone and RU486 sessed by tumor volumetrics. suppress ␣-fetoprotein expression in human hepatoma cell lines Results: Megestrol acetate was shown to inhibit the (15, 16). Octreotide has also been shown to be effective in growth of HepG2 cells in vitro in dose- and time-dependent inhibiting growth of HCC cell lines grown in vitro and in vivo ␮ manners with an IC 50 of 260 M (24-h incubation). The through an unknown mechanism (17). The data supporting the growth of HepG2 cell-transplanted tumors in nude mice was use of these agents in clinical practice have however remained also inhibited by i.p. injection of megestrol acetate (10 mg/ weak. kg/day). The tumor volumes of the megestrol acetate-treated Tamoxifen has similarly generated a great deal of interest group regressed to 59% of controls by week 6 and to 41% of as a possible therapeutic modality in HCC, and a number of controls by week 13. Apoptosis following G1 arrest was randomized controlled trials have been carried out. Although observed in megestrol acetate-treated cells and may be a estrogen receptor (ER)-positive HCC is known to respond to mechanism through which megestrol acetate inhibits HepG2 tamoxifen (18, 19), there is increasing evidence that many cells. Megestrol acetate was also demonstrated to have a HCCs are ER negative (20, 21). Although initial results were beneficial effect on the weight gain of tumor-bearing nude conflicting, later and larger multicenter trials have shown that mice, and the mean weight of the megestrol acetate-treated tamoxifen has no role in the treatment of HCC (4, 22–25). animals was higher than that of controls from week 4 of the Megestrol acetate is a synthetic progesteronal agent with treatment period, and the differences were statistically sig- multiple drug actions. As a potent antiestrogen agent that acts at nificant in week 5 and 6 (P < 0.05, compared with controls). the postreceptor level and thus independent of ER, megestrol No significant survival advantage was, however, demon- acetate is used in the second-line management of carcinoma of strated in the treatment group. the breast. It has been reported to cause minor reduction of Conclusions: This study showed that megestrol acetate tumor size and prolonged survival time in HCC (26). A small inhibited the growth of HepG2 cells grown in vitro and in controlled trial has shown that megestrol acetate favorably in- vivo. These data provide useful information for clinical study fluence the course of advance HCC and improve patient survival of megestrol acetate for the treatment of HCC. (27). Quite apart from its antiestrogen mechanisms, megestrol acetate impacts positively on the quality of life in patients with INTRODUCTION advanced malignancy (28). Hepatocellular carcinoma (HCC) is one of the most com- The direct effect of megestrol acetate on the growth of mon cancers in the world (1). Surgery currently remains the only HCC cells has, however, not been previously reported, although such studies are crucial to provide experimental support for the clinical studies of megestrol acetate in the treatment of HCC. We report here the results of a study aimed at investigating the Received 1/12/04; revised 4/29/04; accepted 5/7/04. effects of megestrol acetate on human HCC cells (HepG2) Grant support: Singhealth Research Committee (Singapore) Research Grant Ex025/2001. in vitro and in vivo. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to MATERIALS AND METHODS indicate this fact. Materials. Megestrol acetate was purchased from Sigma Requests for reprints: Zhang Kai, Department of Experimental Sur- gery, Singapore General Hospital, Outram Road, Singapore 160608, Chemical Co. (St. Louis, MO). Human HCC cell line, HepG2, Singapore. Phone: 65-63265550; Fax: 65-62223389; E-mail: geskai@ was obtained from American Type Culture Collection (Ameri- sgh.com.sg. can Type Culture Collection, Manassas, VA). Six-week-old

male BALB/c athymic nude mice weighing between 16 and 18 g Transplantation of HepG2 Cells as Solid Tumors in were purchased from Animal Resource Center (Canning Vale, Nude Mice. HepG2 cells (5 ϫ 106) were collected in 0.1 ml Western Australia). This animal study was approved by the of HBSS, and these cell suspensions were than injected s.c. into Institutional Animal Care and Use Committee of the Singapore the right gluteal region of each nude mouse using a 27-gauge General Hospital. needle. The ears of nude mice that received cell injections were Cell Culture. HepG2 cells were grown in MEM contain- tagged for identification. Solid tumors were observed in 18 of 19 ing1mM sodium pyruvate, 1 mM nonessential amino acids, and nude mice at day 12 after injection of HepG2 cells. 10% FCS (Life Technologies, Inc., Grand island, NY). Cells Tumor Volumetrics. The volumes of the transplanted tumors were measured weekly by the same researcher over the were maintained in humidified atmosphere of air/CO2 (19/1) and were subcultured every 2–3 days. entire experimental period. The largest and smallest diameters Cytotoxicity Assay. Approximately 1 ϫ 104 cells were were measured using a Vernier caliper, and the volumes of ϭ ϫ ϫ seeded in each well of 96-well tissue culture plates and incu- tumors were estimated according to the formula: V 1/2 a b2, as reported previously (31), where V is the tumor volume in bated in a CO incubator for 24 h. A 20 mM stock solution of 2 mm3 megestrol acetate was prepared freshly in absolute ethanol, and , and a and b are the largest and smallest tumor diameters in mm, respectively. Assuming 1 unit as the specific gravity for aliquots of the stock solution were added to wells of the plates tumor tissue, the body weights of the animals were estimated by at desired concentrations. After incubation with megestrol ace- the formula: body weight (g) ϭ total weight (g) Ϫ tumor tate for various periods of time, survival cells in plate wells volume (cm3). were determined by 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetra- Treatments of Animals. When the largest diameters of zolium bromide (Sigma) assay as reported previously (29). the tumors reached 8–10 mm, the nude mice were randomly ␮ Twenty-five l of the 5 mg/ml stock solution of 3-[4,5-dimethyl- distributed into a control group (n ϭ 9) and a test group (n ϭ 9). thiazol-2yl]-2,5-diphenyltetrazolium bromide were added to each The animals in the test group received 12 consecutive daily i.p. well, and after2hofincubation at 37°C, 100 ␮l of the extraction injections of megestrol acetate followed by injections with buffer [20% SDS w/v, in 50% N,N-dimethyl formamide v/v, 2-day intervals for 7 weeks at a dosage of 10 mg/kg given in 0.2 2.5% of 80% acetic acid and 2.5% of 1 N HCl (pH 4.7)] were ml of injection water. The animals in the control group received added. After an overnight incubation at 37°C, the absorbances injections of injection water alone following the time schedule (A) at wavelength of 570 nm were measured using a microplate for the test group. reader (Dynatech Laboratories, Chantilly, VA). When 500– Statistical Analysis. The means of tumor volumes, body 20,000 cells were seeded, there was a good correlation obtained weights, and survival time in the control and the test groups between A values (0.05–1.0) and cell numbers (r ϭ 0.99). All of were analyzed statistically for significance of the differences by the A values obtained in the study were in the linear range the Student’s t test (32), using Microsoft Excel. P Ͻ 0.05 was (0.05–1.0). The cell survival of each dose of megestrol acetate accepted as significant. was calculated as the ratio of A in wells containing drug treated cells to that in control wells with untreated cells. IC values 50 RESULTS were calculated using a linear regression from dose-dependent Inhibition of Growth of HepG2 Cells in Vitro. The curves plotted from at least five points. addition of megestrol acetate in cultures of HepG2 cells showed Cell Cycle Analysis. HepG2 cells were subcultured in dose-dependent inhibition on growth of the cells. The dose- each well (2.5 ϫ 105/well) of 6-well plates and incubated at survival curve is shown in Fig. 1A. The IC 50 for megestrol 37°C for 24 h. Aliquots of a 20 mM stock solution of megestrol acetate was calculated by a linear regression. The IC of 260 acetate were then added to the cell cultures to reach final 50 ␮M for megestrol acetate was much higher when compared with concentrations of 50–400 ␮M, and the cells were then incubated 42.8 ␮M for cisplatin (29), which is a widely used anticancer for various periods of time. Cell suspensions from both control drug for the treatment of human malignancies, including HCC. cultures and megestrol acetate-treated cultures were prepared by Incubation with 200 ␮M megetrol acetate also showed time- trypsinization and washed twice with PBS. The cells were then dependent inhibition on the growth of the human liver tumor Ϫ fixed in 85% ethanol at 20°C for 20 h and rewashed with PBS cells (Fig. 1B). to remove ethanol. Cells collected by centrifugation were Effects of Megestrol Acetate on Growth of HepG2- stained in 1 ml of propidium iodide/Triton X-100 staining Transplanted Tumors in Vivo. As shown in Fig. 2, tumor solution (40 ␮g propidium iodide/ml, 200 ␮g RNase/ml, and volumes in nude mice were not affected by treatment of meges- 0.1% Triton X-100 in PBS) at 37°C for 1 h. Cell cycle distri- trol acetate in the first 5 weeks. However, from week 6 of bution was analyzed by flow cytometry using a Becton Dickin- megestrol treatment, a significant suppression of tumor growth son FACSCalibur (San Jose, CA), and the DNA histograms was observed. The tumor volumes of the megestrol acetate- were analyzed with Winndi multicycle software. treated group regressed to 59% of controls at week 6 and to 41% Determination of Apoptosis. Detection of hypodiploid of controls at week 13 (P Ͻ 0.05 compared with controls). The

(sub-G1) apoptotic cells was performed by flow cytometry anal- megestrol acetate-induced reductions of tumors are progressive ysis as described above. DNA fragmentation was also deter- during the period of treatment. mined by ligation-mediated PCR (30) using an ApoAlert LM- Effects of Megestrol Acetate on Cell Cycle Progression. PCR Ladder Assay kit from Clontech Laboratories following Cell growth and inhibition are mediated by cell cycle progres- protocols set by the manufacturer. sion (33, 34). On the basis of the above results showing the

Fig. 2 Effects of megestrol acetate on HepG2-transplanted tumors in nude mice. HepG2 cells were transplanted as a solid tumor in nude mice, and volumes of the tumors were measured weekly as described in “Materials and Methods.” The tumor-bearing nude mice received peri- toneal injections of megestrol solution (10 mg/kg/day) and injection water for control. Points are means (ϮSD) of tumor volumes in meges- P Ͻ 0.05, compared ,ء .(trol acetate-treated and control groups (n ϭ 9 with control.

Fig. 1 Dose- and time-dependent inhibition of HepG2 cells by megestrol acetate. HepG2 cells were subcultured in each well of 96-well plates and incubated overnight for their attachment. For the dose-dependent study, aliquots of a stock solution of megestrol acetate (20 mM) were added to the wells to make final concentrations of 25, 50, 100, 200, 400, 600, and 800 ␮M, respectively, and the incubation was carried out in 37°C for 24 h (A). For the time-dependent study, 200 ␮M megestrol acetate were used, and incubation was carried out at 37°C for 6, 12, 24, 36, and 48 h, respectively (B). Cell survival was then determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method as described in “Materials and Methods.” Points are means Ϯ SD of at least triplicates.

inhibitory effects of megestrol acetate on the human liver tumor cells grown in vitro and in vivo, the effect of megestrol acetate on cell cycle progression was also examined as a sequential step to understand the possible mechanisms involved in the inhibitory effects of megestrol acetate. As shown in Fig. 3, treatment by 50–400 ␮M megestrol acetate had no significant effects on

cell cycle progression at 6 h. However, G1 arrest of the cells was Fig. 3 Flow cytometric analysis of HepG2 cells treated with megestrol demonstrated at the incubation time of 12 h in the megestrol acetate. Exponentially growing cells were incubated with 50–400 ␮M megestrol acetate for 6–48 h. Cells were fixed, stained with propidium acetate-treated group, and this megestrol acetate-induced G1 ␮ iodide, and analyzed by flow cytometry as described in “Materials and arrest was more obvious at 24 h when 50 M megestrol acetate Methods.” Cells in each phase were analyzed using Winnidi multicycle was used. When higher concentrations of megestrol acetate were software. Results were from a representative one of three independent used, megestrol acetate-treated cells revealed apparent sub-G1 experiments.

peaks at 24 h. The cell numbers in these sub-G1 peaks were increased by megestrol acetate treatment in both dose- and time-dependent manners (Fig. 3). Determination of Apoptosis in Megestrol Acetate- Treated Cells. Apoptotic cells with fragmented DNA should appear in the sub-G1 peak, and this has been considered as a marker of apoptotic cells (35). However, the sub-G1 peak does not necessarily indicate DNA oligonucleosomal fragmentation because cells with diminished amount of DNA and some cellular debris may also accumulate in the sub-G1 region (36, 37). Therefore, to confirm the presence of apoptotic cells in the sub-G1 region as showed in Fig. 3, ligation-mediated PCR was performed to detect DNA oligonucleosomal fragments, which Fig. 5 Changes of body weights of tumor-bearing nude mice under are typical features of apoptotic cells. As shown in Fig. 4, DNA treatment by megestrol acetate. Points are means (ϮSD) of body P Ͻ ,ء .(laddering was observed in the HepG2 cells treated with 300 and weights of megestrol acetate-treated and control groups (n ϭ 9 0.05, compared with control. 400 ␮M megestrol acetate for 24 h. These results agreed with the flow cytometry data and showed that megestrol acetate could induce apoptosis of HepG2 cells, and this appears to be a mechanism for the inhibitory effects of megestrol acetate on the growth of the cells. Effects of Megestrol Treatment on the Hosts of Trans- planted Tumors. As shown in Fig. 5, the mean of body weights of the megestrol acetate-treated animals were higher than that of controls from week 4 of treatment. However, significant differences in weights were only observed in weeks 5 and 6 of treatment (P Ͻ 0.05, compared with controls). The means of body weights of both control and treatment groups increased gradually until week 6 of treatment and then decreased progressively until the death of the animals The survival time of individual animal was recorded, and survival curves of the test and the control groups were shown in Fig. 6 A. No obvious changes were observed in the survival curve of the megestrol acetate-treated group when compared with the control group. There was neither any significant difference in average survival time between the two groups (Fig. 6B).

Fig. 6 Effects of megestrol acetate treatment on survival of the tumor- bearing nude mice. Survival curves show the survival animals at each week expressed as percentage of survivors (A). Average survival time was expressed as means (ϮSD) of survival weeks of nude mice in megestrol acetate treatment and control groups (B; n ϭ 9). The tumor- bearing animals received i.p. injections with megestrol acetate solution (10 mg/kg/day) or injection water for controls. Fig. 4 Induction of apoptosis in HepG2 cells by megetrol acetate. Cells were treated with 50–400 ␮M megestrol acetate for 24 h. Genomic DNA was then purified from the megestrol acetate-treated cells, and blunt-ended fragments were ligated with adapters. The adaptor-ligated DNA fragments were then used as templates to amplify blunt-ended DISCUSSION apoptotic DNA fragments using advantage cDNA polymerase mix Megestrol acetate has been used clinically for the treatment (Clontech Laboratories). Twenty ␮l of the PCR products were loaded on a 1.2% agarose/EB gel, and the electrophoresis was performed at 6v/cm of malignancies, including endometrial carcinoma, ovarian can- for 3 h. Human En-2 was used as an internal control to confirm that cer, breast cancer, prostate cancer, renal cell carcinoma, and equal amounts of DNA had been used for PCR. malignant melanoma for Ͼ30 years (28, 38, 39). Treatment with

megestrol acetate is most relevant in sex hormone-related can- which megestrol acetate inhibits the human liver tumor cells. As cers such as breast and ovarian cancers (40–42) and gives the shown in Fig. 3 in dose- and time-dependent studies, megestrol

best results. HCC has also been shown to be sex hormone acetate-treated cells were found to be arrested in G1 phase at dependent (10–14), and megestrol acetate may potentially be a incubation time points of 12 and 24 h when lower megestrol useful treatment modality for HCC. However, when compared acetate concentrations were used. When higher concentrations with ovarian and breast cancers, much fewer studies have been of megestrol acetate were used or when incubation time was

done on HCC with respect to treatment with hormonal agents. increased, cell numbers in G1 phase decreased with a progres- The results obtained from the present study will be useful for sive increase of cell numbers in sub-G1 peak, which has been consideration of clinical studies on the therapeutic effects of shown to represent apoptotic cells. The relationship between cell

megestrol acetate on HCC. We have shown that megestrol cycle arrest and apoptosis is best understood in the context of G1 acetate inhibited the growth of HepG2 cells in vitro in dose- and arrest (59, 60). Many common molecules can affect cell cycle time-dependent manners (Fig. 1, A and B). These effects were progression and apoptosis (60). The antineoplastic phospho- also observed on HepG2 cell-transplanted tumors grown in vivo lipid, ET-18-OCH3, has been shown to arrest BAC1.2 cells

(Fig. 2). Consistent with our results, megestrol acetate has been irreversibly in G1 phase by preventing their progression to S shown to inhibit growth of endometrial carcinoma and ovarian phase. This was followed by triggering of DNA fragmentation cancer cells grown as xenograft models (43–45). The impor- and morphological changes associated with apoptosis (61). It tance of sex hormone receptors in growth of sex hormone- appears in this study that megestrol acetate arrests HepG2 cells

dependent tumors and the value of hormonal manipulations for in G1 phase and consequently causes these G1-arrested cells to the treatment of these cancers are, at present, not well defined. go to apoptosis instead of entering into S phase. Clinical studies relating the expression of progesterone Clinical studies have also been designed to investigate the receptors (PgRs) to the clinical features of tumors and their effect of megestrol acetate treatment on appetite, body weight, response to hormone therapy and prognosis have yielded vary- quality of life, and survival (28, 42). Although no significant ing results (46–50). In experimental animal models, however, it survival advantage was demonstrated (62), beneficial effects on has been shown that megestrol acetate could only inhibit the appetite and body weight have been observed in megestrol acetate- growth of PgR-positive tumors but not PgR-negative tumors treated cancer patients in the majority of studies (28, 38, 42, 63, (43–45). These results suggested the roles of PgR in the regu- 64). In this study, as shown in Fig. 5, the means of the body weight lation of tumor growth, and megestrol acetate may affect the of megestrol acetate-treated animals were higher than that of con- growth of cancer cells through PgRs. In this study, however, trols from week 4 of treatment, and the differences were statisti- PgR protein could not be detected in HepG2 cells by Western cally significant in week 5 and week 6. In this aspect, the effect of blotting using an anti-PgR antibody (sc-810; Santa Cruz Bio- megestrol acetate on nude mice appear to be similar to that of technology, Inc., Santa Cruz, CA) and nor could PgR mRNA be human studies (28, 38, 42, 63, 64). Preliminary studies suggest that detected by reverse transcription-PCR in three independent ex- progestational agents down-regulate interleukin-6, an inflammatory periments (data not shown). These data suggested that PgR is cytokine widely implicated in cancer-associated anorexia and not involved in action of megestrol acetate in the HepG2 cells. weight loss. Decreased interleukin-6 may be the reason for im- Megestrol acetate is known to exhibit multiple actions in human provement of cachexia and anorexia in cancer patients, although cancer cells (51) and other mechanisms such as affinity to this was not supported in a later study (65). The mechanism androgen and glucocorticoid receptors, and action through these through which megestrol acetate improves appetite and abrogate receptors (52, 53), interactions with growth factors (54), and a weight loss remains unclear. In our study, the beneficial effects of direct cytostatic influence on tumor cells (55) have been pro- megestrol acetate on weight gain in nude mice could be partially posed to account for the antitumor action of megestrol acetate. attributed to the regression of tumors. Megestrol acetate has also been shown to alter the metabolism As discussed above, in the nude mice model, megestrol of estrogen in tissue and to suppress plasma estrogens. This acetate could inhibit growth of tumors and improve the overall antiestrogen activity may also play roles for antitumor effects of condition of the hosts. However, as shown in Fig. 6A, survival megestrol acetate (56, 57). Apoptosis may be a consequential of tumor-bearing nude mice was not affected by megestrol important step of these effects because megestrol acetate has acetate treatment, and the difference of average survival time been reported to inhibit the growth of prostatic tumors by (weeks) in megestrol acetate-treated and control groups was not induction of apoptosis of the tumor cells (58). In our study, significant (Fig. 6B). Because the life span of nude mice is only apoptosis was demonstrated in megestrol acetate-treated cells by 4–6 month, the limitation of the model is that the late stages of flow cytometry analysis. Megestrol acetate-induced apoptosis the follow-up period overlaps with the naturally declining body was also confirmed by analysis of DNA fragments using a conditions of the hosts as the animals approach the end of their ligation-mediated PCR method. However, DNA ladders were natural life spans. Tumor necrosis occurred in some of the not observed at lower concentrations of megestrol acetate (100 treated animals from week 15 of treatment (data not shown). ␮ and 200 M), whereas a significant sub-G1 peak appeared in Therefore, the effects of megestrol acetate treatment on the flow cytometry analysis at these concentrations (Figs. 3 and 4). survival of the hosts may not be reflected because of the rela- The inconsistency may be due to limitation of sensitivity of the tively short follow-up period, which is attributed to short life ligation-mediated PCR DNA ladder analysis. Particles in the span of the host animals. ␮ sub-G1 peaks could also resulted from cell debris or dead cells In this study, the IC50 of 260 M for megestrol acetate was from necrosis or other death pathways (36, 37). These results much higher when compared with 42.8 ␮M for cisplatin (29), suggest that induction of apoptosis may be a mechanism through which is a widely used anticancer drug for treatment of human

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Kai Zhang and Pierce K. H. Chow

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