Alvocidib (Flavopiridol) suppresses tumor growth in SCID mice with human esophageal cancer xenografts without inducing apoptosis

ARTICLE IN PRESS Surgical Oncology (2006) 15, 107–113

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Alvocidib (Flavopiridol) suppresses tumor growth in SCID mice with human esophageal cancer xenografts without inducing apoptosis Shinsuke Satoa,, Yoshiaki Kajiyamaa, Masahiko Suganoa, Yoshimi Iwanumaa, Hiroshi Sonoueb, Toshiharu Matsumotob, Masahiko Tsurumarua a

Department of Gastroenterological Surgery, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan b First Department of Pathology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan Accepted 2 October 2006

KEYWORDS Esophageal cancer; Alvocidib; Xenograft; VEGF; Cyclin D1

Summary Alvocidib (Flavopiridol, HMR1275) is a potent inhibitor of multiple cyclin-dependent kinases and has been identified recently as an antitumor agent in several cancers. Previous studies have shown that alvocidib could potentially treat esophageal cancer in vitro. This study evaluates alvocidib for its ability to suppress tumor growth in severe combined immunodeficiency (SCID) mice bearing TE8 human esophageal squamous cell carcinoma (SCC) xenografts. Alvocidib treatment of 10 mg/kg body weight reduced tumor volume significantly. Immunohistochemistry analysis of alvocidib-treated tumor sections showed significant reductions in cyclin D1, VEGF, and Rb levels. Alvocidib treatment did not cause a marked increase in apoptotic tumor cells by terminal deoxynucleotidyl transferasemediated dUTP nick end labeling (TUNEL) analysis, yet hematoxylin and eosin staining revealed tumor necrosis. In vivo investigation of alvocidib treatment confirmed antitumor activity in TE8 esophageal xenografts. These findings suggest that alvocidib could be a useful anti-cancer agent for esophageal cancer. & 2006 Elsevier Ltd. All rights reserved.

Introduction Esophageal cancer has one of the poorest prognoses of any human cancer despite recent advances in its diagnosis and Corresponding author. Tel.: +81 3 3813 3111;

fax: +81 3 5802 1951. E-mail address: [email protected] (S. Sato). 0960-7404/$ - see front matter & 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.suronc.2006.10.001

treatment, which have only led to small but significant improvements in survival [1,2]. Given the poor outcome of surgery or radiotherapy alone, physicians often use combined therapy, including chemotherapy and/or radiotherapy and/or surgery in a variety of schedules. However, combined therapies using fluorouracil and cisplatin have been statistically insignificant and have failed to demonstrate any survival benefit [3,4]. Therefore, an alternative form of systemic treatment, using more effective anticancer agents, should be explored.

ARTICLE IN PRESS 108 Basic cancer treatment is changing in response to advances in molecular targeting. Genetic changes in the Rb pathway, which are common in tumor cells, lead to hyperactivation of cyclin-dependent kinase (CDK) and progression through the cell cycle. Consequently, many scientists have sought to develop CDK inhibitors as therapeutic agents in cancer. Alvocidib (Flavopiridol, HMR1275) is a semisynthetic flavonoid and is the first CDK inhibitor to be tested in clinical trials. The drug inhibits multiple CDKs, leading to a reduction in cyclin D1 mRNA transition and cell cycle arrest in the G1 or G2/M transition phase [5]. Recent studies have shown that alvocidib inhibits P-TEFb (CDK9/cyclin T1), which controls the elongation phase of transcription by RNA polymerase II [6,7]. In addition to inhibiting cell cycle progression, alvocidib exhibits a variety of other activities including the induction of apoptosis and antiangiogenesis [8]. The mechanism by which alvocidib induces apoptosis remains to be elucidated. It is unclear whether the inhibition of CDK activity by alvocidib is required for the induction of apoptosis [9]. In several phase I and II trials, alvocidib has had no cytotoxic effect in several cancers [10,11]. Many of these trials, however, have not clearly demonstrated the antitumor effect of alvocidib in esophageal cancer specifically. Alvocidib has previously demonstrated cell cycle arrest and apoptosis in esophageal adenocarcinoma and epidermoid carcinoma in vitro [12]. The antiproliferative and apoptotic effects of alvocidib on esophageal squamous cell carcinoma (SCC) cell lines in vitro have been reported [13]. Recently, alvocidib was also found to reduce the prevalence of murine esophageal adenocarcinoma and SCC in p27 knockout mice, which were exposed to the carcinogen, N-methyl-N-benzylnitrosamine [14]. As a result of these earlier findings, this study investigates the effect of alvocidib in esophageal SCC in vivo and considers alvocidib in a treatment approach for patients with esophageal SCC.

Materials and methods Cell lines and cell culture The human esophageal TE8 SCC cell line was obtained from the Cell Resource Center for the Biomedical Research Institute of Development, Aging and Cancer at Tohoku University (Sendai, Japan) [15]. Cells were maintained in RPMI 1640 medium (R8758; Sigma Chemical Co., St. Louis, MO) supplemented with 10% heat-inactivated fetal bovine serum and penicillin at 37 1C in an atmosphere of 5% CO2.

Materials Alvocidib was obtained from Sanofi-aventis (Paris, France). A 10 mg/ml stock solution was prepared in 0.05 M acetic acid and stored at 4 1C. This solution was further diluted in the same medium to appropriate concentrations.

S. Sato et al. viable cells/ml. Female 8-week-old severe combined immunodeficiency (SCID) mice, weighing between 16 and 20 g, received an injection of 2.5  105 cells (200 ml) in the right flank and started treatment 15 days after tumor implantation. Mice were randomly divided into a control group of 5 mice and an alvocidib group of 10 mice. Mice bearing TE8 cells approximately 100 mm3 in volume received an intravenous (IV) injection of the vehicle control (saline) or alvocidib once a day for five days. Subcutaneous tumors were measured with a caliper, and animal weights were recorded every five days. Tumor volumes were regularly calculated using the following formula: 0.5  length (mm)  width (mm)  width (mm). Animals from the control and alvocidib groups were killed 5 days after the last alvocidib injection. Tumor tissue was retrieved for analysis and the tumor was weighed immediately. All procedures involving animals were performed in accordance with international laws and policies.

Histology and immunohistochemistry After the animals were sacrificed, their tumors were excised, fixed in formalin, and embedded in paraffin for hematoxylin, eosin, and immunohistochemical staining. The avidin–biotin–peroxidase complex method detected expression of cyclin D1 (P2D11F11, Novocastra Laboratories, Burlingame, CA), Rb (1F8, Novocastra Laboratories), bcl-2 (bcl-2/100/D5, Novocastra Laboratories), Mcl-1 (554103, BD Pharmingen, San Diego, CA), E2F1 (554213, BD Pharmingen), and VEGF (555036, BD Pharmingen).

Evaluation of immunohistochemical results by scoring The nuclear immunoreactivity of cyclin D1, Rb, and E2F1 and cytoplasmic immunoreactivity of VEGF, Mcl-1, and bcl-2 were evaluated. For each tumor, the percentage of immunopositive cells was calculated for at least 500 cells and expressed as the mean percentage in 4 different wellstained fields for each section at 200  magnification. This percentage was used to classify expression levels of cyclin D1, VEGF, Rb, bcl-2, Mcl-1, and E2F1 (‘‘’’ ¼ o10% positive tumor cells with strong staining; ‘‘+’’ ¼ 10–20% positive tumor cells with strong staining; and ‘‘++’’ ) 20% positive tumor cells with strong staining).

Evidence of drug-induced apoptosis To determine if tumor cells underwent drug-induced apoptosis, the Apoptosis in situ Detection Kit (295-53501, Wako Pure Chemical Industries, Ltd., Osaka, Japan) was utilized on paraffin sections. These sections were analyzed with light microscopy.

Statistical analysis Animal studies TE8 cells were cultured to subconfluency, trypsinized, and resuspended in RPMI at a concentration of 1.25  106

The data were presented as the means7S.D. and analyzed by the statistical software, SPSS 11.0 for Windows program, using repeated measured analysis of variance (ANOVA).

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Differences were considered significant when the p-value was o0.05.

Results Therapeutic effect of alvocidib against TE8 esophageal SCC xenografts Alvocidib efficiently controlled tumor growth in a dosedependent manner (Fig. 1). A dose of 5 mg/kg did not control tumor growth effectively, yet a dose of 10 mg/kg inhibited tumor growth. The SCID mice that received 10 mg/ kg alvocidib showed a reduction in tumor growth by 50% compared with the control group. Drug toxicity, having been assessed by weight loss, was minimal during treatment since no significant difference in weight reduction was observed between the control group and alvocidib group (Fig. 2). After treatment, when the mice were sacrificed, there was a substantial difference in weight loss between the control group and the group that received 10 mg/kg alvocidib (Fig. 3).

Figure 2 Effect of alvocidib on tumor weight in mice implanted with TE8 cells. (n ¼ 5 in each group; S.E.—Standard Error).

Effect of alvocidib on the expression of cyclin D1, VEGF, Rb, Mcl-1, E2F1 and bcl-2 To understand the processes by which alvocidib inhibits tumor growth in vivo, tumors that received the vehicle control or alvocidib were stained for markers of cell proliferation and apoptosis (Table 1). Staining for the proliferation markers cyclin D1, VEGF, and Rb in alvocidibtreated tumors revealed a noticeable decrease in the amount of proliferating cells in a dose-dependent manner (Fig. 4). In contrast, expression levels of Mcl-1 and E2F1 were the same in both the control and alvocidib-treated tumors. Additionally, both the control and alvocidib-treated TE8 tumors did not express bcl-2. Therefore, Mcl-1, E2F1, and bcl-2 were not evaluated further. Figure 3 Effect of alvocidib on tumor weight in mice sacrificed 5 days after the last alvocidib injection. (n ¼ 5 in each group; *po0:05; N.S.—Not Significant).

Table 1 Effect of alvocidib on expression of cyclin D1, VEGF, Rb, bcl-2, Mcl-1, and E2F1. Markers

Cyclin D1 VEGF Rb Bcl-2 Mcl-1 E2F-1

Figure 1 Efficacy of alvocidib in TE8 esophageal SCC xenograft models in vivo. (n ¼ 5 in each group; **po0:01, *po0:05; N.S.—Not Significant).

Untreated group

++ ++ ++  ++ 

Treated group 5 mg/kg

10 mg/kg

 +   ++ 

    ++ 

 o10% positive tumor cells with strong immunohistochemical staining. + 10–20% positive tumor cells with strong immunohistochemical staining. ++ 420% positive tumor cells with strong immunohistochemical staining.

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Figure 4 Immunohistological evaluation of TE8 tumors in the control and alvocidib groups. Paraffin sections, probed and stained with the appropriate antibodies and analyzed under light microscopy, are shown at 100  .

Analysis of esophageal SCC xenograft for apoptosis and necrosis The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was utilized to determine if apoptotic cells were found in tumors after in vivo treatment with alvocidib. Hematoxylin and eosin staining was also examined for evidence of necrosis. TUNEL staining did not detect an increased number of apoptotic cells in alvocidibtreated tumors as compared with the control tumors (Fig. 5). TUNEL-positive cells were rarely found in both the control tumor and alvocidib-treated tumor sections. In contrast, hematoxylin and eosin stained sections of alvocidib-treated tumors demonstrated significant necrosis. The amount of necrosis was contingent on the amount of alvocidib administered (Fig. 6). The area of necrosis in the 10 mg/kg alvocidib-treated group was approximately 5 times larger than the area of necrosis in the control group.

Discussion This study demonstrates that alvocidib (a) has a therapeutic effect against esophageal cancer xenografts, (b) triggers decreased expression of cyclin D1, VEGF, and Rb protein, and (c) does not bring about an increase in the amount of apoptotic cells, but induces necrosis in the xenografts. Previous studies have reported the inhibitory effect of alvocidib in several cancer xenografts [16–19]. However, the precise acting mechanisms of alvocidib have not been fully elucidated. It is well accepted that alvocidib induces apoptosis in many cancers both in vitro and in vivo. A previous study has reported that alvocidib induces cell cycle arrest and apoptosis in TE8, TE9, and KE4 esophageal SCC cell lines in vitro [13]. In this study, the most interesting

finding was that apoptosis failed to be a prominent feature in alvocidib-treated TE8 tumors in SCID mice. In support of this finding, other reports have shown that alvocidib failed to induce apoptosis in AS283 lymphoma in vivo and A549 human lung carcinoma cells in vitro [19,20]. A recent study showed that alvocidib treatment did not succeed in reducing the Mcl-1 level in H1299 and NIH3T3 fibroblast cell lines [21]. Additionally, these cell lines were resistant to apoptosis after alvocidib induction. The authors suggested that apoptosis was dependent on enhancement of the E2F1 level and the repression of Mcl-1. In our study, the data show that alvocidib did not enhance E2F1 or repress the Mcl-1 levels. The role of bcl-2 in the apoptotic action of alvocidib is not well known. Alvocidib has been shown to reduce bcl-2 levels in several cell lines [22,23]. Alonso et al. has also showed that expression of the bcl-2 protein decreased and expression of the Bax protein increased in response to alvocidib treatment in glioma cell lines [24]. These findings intimate that this change in the proteins’ expression levels could be one of the mechanisms for inducing apoptosis. Previous work suggests that alvocidib induces apoptosis in TE8, TE9, and KE4 esophageal SCC cell lines; however, alvocidib decreased bcl-2 protein expression in KE4 cells only [13]. Yet, in other reports, the cytotoxicity of alvocidib did not correlate with changes in bcl-2 protein expression alterations [12,25,26]. Rb is an important tumor suppressor and a cell cycle regulator. Obvious diminution of Rb protein levels was detected after alvocidib treatment of TE8 esophageal SCC in vivo and in a previous in vitro study [13]. Yet the activity of alvocidib in relation to Rb expression remains controversial. No significant difference in the cytotoxic activity of alvocidib has been found between cell lines that express pRb and cell lines that do not [12,27]. Conversely, a previous

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Figure 5 TUNEL evaluation of tumor sections from control and alvocidib-treated TE8 xenograft tumor models. Sections are shown at 200  .

Figure 6 Hematoxylin and eosin staining of tumor sections from control and alvocidib-treated TE8 xenograft tumor models. Sections are shown at 40  .

ARTICLE IN PRESS 112 study proposed that Rb plays a regulatory role in the process of alvocidib-related apoptosis [12]. Apoptotic activity, after exposure to alvocidib, seemed more pronounced in SKGT-2 and SKGT-4 esophageal adenocarcinoma cell lines that lacked detectable Rb expression levels. It remains unclear why alvocidib fails to induce apoptosis in the TE8 xenograft, and yet induces apoptosis in TE8 cells in vitro. Important factors, such as hypoxia and angiogenesis, must be considered in an in vivo experiment. The mechanism of resistance acquisition to apoptosis in solid cancers has not been completely understood. Yet, studies have already shown that, in hypoxia and hypoglycemia, leukemia cells readily undergo apoptosis, whereas solid cancer cells resist apoptosis [28]. It has also been stated that hypoxia is involved in the selection of apoptosisresistant cells [29]. Thus, the environment for tumor development may itself cause resistance to apoptosis. When tumor formation causes hypoxia, the accumulation of HIF-1a activates transcription of the VEGF family, resulting in angiogenesis [30]. It has been reported that the expression of VEGF in esophageal cancer correlates with the tumor stage, number of positive lymph nodes, patient survival, and recently an association with VEGF-C [31,32]. Therefore, the VEGF family might become an important target in the treatment strategy for esophageal cancer. To date, alvocidib has been shown to suppress the hypoxia-induced expression of VEGF in vitro [33,34]. The in vivo data from the alvocidib-treated groups confirm alvocidib’s ability to suppress VEGF expression. Since the suppression of VEGF production in solid tumors results in the inhibition of angiogenesis, necrosis should readily occur, even if apoptosis is suppressed in hypoxia. Therefore, this drug may be clinically useful for treating esophageal cancer, even when apoptosis is not induced. Another important action of alvocidib is its ability in lowering the expression levels of cyclin D1, an oncogene that is overexpressed in many human cancers. It has been suggested that the reduction of cyclin D1 expression might be an essential mechanism by which alvocidib induces cytotoxicity [35]. This study supports this hypothesis given that cyclin D1 levels also declined after alvocidib treatment. The significance of cyclin D1 in human esophageal cancer has been studied extensively, and its expression has been shown to correlate with poor prognosis in esophageal cancer patients [36–38]. Not only in esophageal SCC, but also in esophageal adenocarcinoma, was increased expression of cyclin D1 seen in 64% of tumors [39]. In addition, reports have demonstrated that cyclin D1 overexpression starts early in dysplasia and could play important roles in esophageal carcinogenesis and progression [40,41]. Therefore, cyclin D1 may be a new therapeutic target for esophageal cancers, and alvocidib may have therapeutic potential. Our presented data illustrates that alvocidib exhibits potent activity in TE8 esophageal SCC xenografts and reduces the expression of cyclin D1, VEGF and Rb. Alvocidib may, therefore, be a useful targeting agent in the treatment of esophageal cancer.

Acknowledgements We would like to thank Naomi Sato and Tamami Sakanishi for their excellent technical assistance. This work was sup-

S. Sato et al. ported in part by Grants-in-Aid from the Japanese Ministry of Education, Culture, Sports, Science and Technology.

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