A novel tubulin inhibitor STK 899704 induces tumor regression in DMBA / TPA induced skin carcinogenesis model ( Short title : STK 899704 induces tumor regression in DMBA / TPA induced carcinogenesis model )

Skin cancer is the most common type of cancer. The incidence rate of skin cancer has continuously increased over the past decades. In an effort to discover novel anticancer agents, we identified a novel tubulin inhibitor STK899704 which is structurally distinct from other microtubule-binding agents such as colchicine, vinca alkaloids, and taxanes. STK899704 inhibited microtubule polymerization leading to mitotic arrest, and suppressed the proliferation of various cancer cell lines as well as multidrug-resistance cancer cell lines. In this study, our investigation is further extended into animal model to evaluate the effect of STK899704 on skin carcinogenesis in vivo. Surprisingly, almost 80% of the tumors treated with STK899704 were regressed with a one fifth reduction in tumor volume. Furthermore, the efficacy of STK899704 was nearly two times higher than that of 5-fluorouracil, a widely used skin cancer therapeutic. Overall, our results suggest that STK899704 is a promising anticancer chemotherapeutic that may replace existing therapies, particularly for skin cancer. BACKGROUND Skin cancer is the most common type of cancer worldwide. The incidence rate of skin cancer continues to rise with over 3.5 million cases diagnosed annually, which is higher than the combined incidence rates of breast, prostate, lung and colon cancer (1). The general prevalence of skin cancer is higher in Caucasians and increases with age, in part due to accumulative ultraviolet (UV) exposure. In South Korea, the number of patients with skin A cc ep te d A rt ic le This article is protected by copyright. All rights reserved. cancer has grown at a rapid rate with a 44.1 % increase in the five-year period from 2009 to 2013, mainly as a result of increased UV exposure and an aging population (2, 3). Thus, the prevention and protection against skin cancer is a global issue, and the demand for an advanced therapy with convenient accessibility and low side effects is high. In an effort to discover novel anticancer agents, we previously identified the small molecule STK899704 which has an acylhydrazone moiety known as a useful scaffold for drug development, especially for anticancer therapy due to its antimitotic activity (4, 5). STK899704 inhibited microtubule polymerization leading to mitotic arrest and cell death. It is proposed to bind microtubulin at the interface between the αβ-tubulin heterodimer, overlapping with the colchicine-binding site. STK899704 suppressed the proliferation of various cancer cell lines as well as multidrugresistance (MDR) cancer cell lines, unlike many anticancer drugs such as Taxol and doxorubicin. QUESTIONS ADDRESSED The aim of this investigation was to evaluate the effect of STK899704 treatment on tumor regression in comparison with 5-fluorouracil (5-FU) or colchicine treatment in an in vivo animal model. The changes in the total number and volume of tumors were monitored for statistical analyses, and histological analyses were performed to verify the cellular effects of STK899704 on skin tumors. In addition, the adverse effects of STK899704 treatment on normal skin were also evaluated using real-time detection methods. EXPERIMENTAL DESIGN The male FVB/N mice were purchased from Jackson Laboratory, and all animal studies were conducted in accordance with the guidelines approved by the Institutional Animal Care and Use Committee of KRIBB (permit number: KRIBB-AEC-16081). The dorsal skin area of the A cc ep te d A rt ic le This article is protected by copyright. All rights reserved. 6–7 week old mice was shaved 2 days before the start of the two-stage carcinogenesis as previously described (6, 7). Tumorigenesis was induced by both a single topical treatment with 100 μg of DMBA (7,12-dimethylbenz[α]anthracene) in 0.2 ml of acetone over a period of 1 week and a treatment with 5 μg of TPA (12-O-tetradecanoylphorbol-13-acetate) in 0.2 ml of acetone twice a week for 12 weeks. In the DMBA/TPA-induced tumorigenesis model, the average number of tumors per mouse was approximately 25 at week 12. After tumor formation, the mice were divided into five groups; and each group consisted of more than 20 mice. Each compound (STK899704, 5-FU, or colchicine) at a concentration of 500 nM in 0.2 ml of acetone was co-treated with TPA three times a week, for a total 20 treatments. The control groups received only TPA or acetone (vehicle). In order to measure the number and volume of skin tumors, mice were weighed and photographed 24 hours after each treatment. The formula: 4π/3(l/2)(w/2)(h/2), where l is the length, w is the width, and h is the height was used to calculate tumor volume (8). Upon the completion of all treatments, the mice were euthanized with CO2, and tumors and skin samples were collected for further histological examination. Collected tumor and skin samples were fixed at 4°C in 4% paraformaldehyde/PBS or 10% formalin and then sectioned for histological analysis. The sections (10 μm thickness) were stained with primary antibodies overnight at 4°C for immunofluorescent staining as previously reported (9). The antibodies and dilutions used were: anti-histone H3 (1:300), antiphosphorylate histone H3 at Ser10 (1:300), and Alexa Fluor® 488 goat anti-mouse or rabbit IgG (1:400). The slides were mounted with Vector Shield (Vector Laboratories Inc.) and observed using a 710 Meta (Zeiss) laser scanning confocal microscope.