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A potent chemotherapeutic strategy in prostate cancer: S-(methoxytrityl)-L-cysteine, a novel Eg5 inhibitor.

Docetaxel-based combination chemotherapy remains the predominant treatment for castration-resistant prostate cancer. However, taxane-related drug resistance and neurotoxicity have prompted us to develop substitute treatment strategies. Eg5 (kinesin spindle protein), which is crucial for bipolar spindle formation and duplicated chromosome separation during the early phase of mitosis, has emerged as an attractive target for cancer chemotherapy. The aim of this study was to investigate the anticancer efficacy of S -(methoxytrityl)- L -cysteine [S(MeO)TLC], a novel Eg5 inhibitor in prostate cancer. Eg5 expression was examined in human prostate cancer cell lines and tissue microarrays constructed from clinical specimens. Anti-proliferative activity of S(MeO)TLC in prostate cancer cells was assessed by a cell viability assay. The anticancer effect and inhibitory mechanism of S(MeO)TLC in prostate cancer cells was further explored by Hoechst staining, flow cytometry and immunofluorescence. In addition, the antitumor effect of S(MeO)TLC on subcutaneous xenograft models was assessed. Eg5 expression was identified in PC3, DU145 and LNCaP cells. More than half of prostate cancer clinical specimens displayed Eg5 expression. S(MeO)TLC exhibited more powerful anticancer activity in prostate cancer cells compared with the other four Eg5 inhibitors tested. S(MeO)TLC induced cell death after arresting dividing cells at mitosis with distinct monopolar spindle formation. S(MeO)TLC exhibited its significant inhibitory activity ( P < 0.05) on subcutaneous xenograft models also through induction of mitotic arrest. We conclude that Eg5 is a good target for prostate cancer chemotherapy, and S(MeO)TLC is a potent promising anticancer agent in prostate cancer.


Introduction
Prostate cancer is one of the most commonly diagnosed malignancies and the second highest cause of cancer death in American men, with an estimated 27 360 patients dying of castration-resistant prostate cancer (CRPC) in 2009 1 . Furthermore, in recent years, there has been a more rapid increase in the incidence and mortality rates in Asian countries 2 . Androgen deprivation therapy (ADT), including surgical orchiectomy and luteinizing hormone-releasing hormone agonists are the primary treatments for patients with advanced prostate cancer. These patients respond initially to ADT within 1-2 years, but eventually they progress to CRPC. Eventually CRPC develops followed by ADT after 1 to 2 years' response time 3 . Combination chemotherapy based on docetaxel is the standard treatment for patients with CRPC 4,5 .
However, no standard treatment options are established once patients with CRPC progress. Moreover, docetaxel as an anti-microtubule drug has its limitations, including drug resistance and side effects caused by drug-binding site mutations on β-tubulin 6 , overexpression of P-glycoprotein 7 and β-tubulin 8 , and neurotoxicity 9 .
Therefore, there is an urgent need to develop alternative treatment strategies.
Eg5, also known as kinesin spindle protein 10 , is a member of the kinesin-5 motor proteins, and plays a crucial role in bipolar spindle formation and duplicated chromosome separation during the early phase of mitosis 11,12 . Inhibition of Eg5 can arrest dividing cells in mitosis followed by cell death, without affecting the function of interphase microtubules 13 , thus offering an encouraging alternative to microtubule-targeted cancer chemotherapy. The first Eg5-specific inhibitor was identified in 1999 on the basis of phenotype-screening, and named monastrol because it can induce monoastral spindle formation 12 . Inactivation of Eg5 cannot cause peripheral neuropathy because of Eg5 localization to mitotic spindle microtubules but not to interphase microtubules 14 . Eg5 inhibitors can overcome multidrug resistance to taxanes caused by overexpression of P-glycoprotein because Eg5 cannot be transported out of cells by P-glycoprotein 15,16 17 , a derivative of S-trityl-L-cysteine (STLC) 18 , can specifically inhibit Eg5, as does STLC 19 , and induce monopolar mitotic spindle formation, but it is more than 10 times as potent as STLC 17 . Meanwhile, the functionalized indoles, such as KPYC10665 (2-(trifluoromethyl)-9H-carbazole), KPYC10666 (2-(tert-butyl)-9H-carbazole) and KPYC10728 (7-(trifluoromethyl)-9H-pyrido [3,4-b]indole), which are synthesized by one-pot N-arylation-oxidative biaryl coupling of anilines and phenyl triflates 20 , are also potent Eg5 inhibitors 21 .
In our previous study, S(MeO)TLC displayed its potent anticancer efficacy in bladder cancer in vitro and in vivo 22 . In the present study, we examined Eg5 expression in prostate cancer cells. We also analyzed the relationship between Eg5 expression in clinical samples and clinicopathological characteristics. We assessed the potent anticancer efficacy of S(MeO)TLC both in vitro and in vivo.

Cell culture and drug compounds
The prostate cancer cell lines PC3, DU145 and LNCaP were obtained from the American Type Culture Collection (ATCC; Rockville, MD, USA). PC3 Luc cell line was also constructed and used. PC3 cells were lipofected with pGL3-control plasmid vector (firefly luciferase gene; Promega, Madison, WI, USA) and pSV2Neo plasmid (a vector for neomycin resistance; ATCC), as described previously 23 . The clone that stably expressed strongest luciferase activity was designated PC3 Luc . The cells were routinely propagated in RPMI 1640 supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin, and incubated at 37°C with a humidified 5% CO 2 atmosphere.
Five Eg5 inhibitors were used in our study. KPYC10665, KPYC10666 and KPYC10728 were synthesized in our institute, and STLC and S(MeO)TLC were purchased from Bachem (Bubendorf, Switzerland). All compounds were dissolved in DMSO and stored at -20°C.

Eg5 expression in prostate cancer tissue microarrays (TMAs) and cells
After obtaining approval from the Institutional Review Board at Kyoto University Hospital and informed consent, a total of 80 archival specimens of clinically localized prostate cancer patients who underwent radical prostatectomy at our institute were used in the study. TMAs were constructed from these specimens as previously described 24 . Standard indirect immunoperoxidase assays using primary anti-Eg5 rabbit antibody (1:300; Cytoskeleton, Denver, CO, USA) were performed to detect Eg5 antigen. Eg5 immunostaining was considered positive if the cytoplasm of ≥ 5% cancer cells showed weak or greater intensity. Ki67 labeling index was determined as previously described 24  Eg5 expression in prostate cancer cell lines was determined by western blot analysis using standard methods as described previously 27 . Briefly, 25 µg protein extracts were added to each lane and separated by SDS-PAGE (30 mA for 120 min), then blotted onto a polyvinylidene difluoride membrane (16 V for 120 min). After incubated with 5% non-fat dry milk for an hour at room temperature, membranes were incubated with rabbit primary antibody (1:1 000; Cytoskeleton) for Eg5 detection and anti-β-actin mouse antibody (1:20 000; Abcam, Cambridge, UK), which was employed as an internal control. After incubation with corresponding horseradish peroxidase-conjugated secondary antibodies (anti-rabbit, 1:25 000; anti-mouse, 1:10 000), ECL solution (GE healthcare, Buckinghamshire, UK) was applied to react with A Self-archived copy in Kyoto University Research Information Repository https://repository.kulib.kyoto-u.ac.jp the membranes and reactive bands were imaged by a PhosphorImager (GE Healthcare, Buckinghamshire, UK).
After that, cells were administered varying concentrations of Eg5 inhibitors, or DMSO alone as a control, for additional 72 h incubation. The concentrations of 50% cell growth inhibition (IC 50 ) were assessed according to the dose-response curve.
Trypan blue staining was also used to assess the percentage of viable cells, as described previously 27 .

Flow cytometry assay and Hoechst staining
To assess the cell cycle distribution, DNA content of PC3 cells treated with S(MeO)TLC or vehicle was stained by 7-amino-actinomycin D (BD Biosciences, SanDiego, CA, USA) and assayed by flow cytometry, as described previously 28   at the end of observation were compared using one-way ANOVA. The least squares method was used to evaluate the difference between each two groups when ANOVA was significant (P < 0.05).

Expression of Eg5 in prostate cancer
We evaluated Eg5 expression in prostate cancer cell lines using western blot analysis.
Eg5 expression was identified in all three cell lines, LNCaP, PC3 and DU145 ( Figure   1A). We investigated Eg5 expression in prostate cancer clinical samples by immunohistochemistry using TMAs. Eg5 immunostaining was mainly observed in the cytoplasm ( Figure 1B). As shown in Table 1, more than half (55.6%, 44/80) of prostate cancer clinical specimens exhibited Eg5 expression. Statistical analysis revealed that Eg5 expression was significantly associated with Ki67 LI (P = 0.004), although there were no significant relationships between Eg5 expression and the other clinicopathological parameters (P > 0.05).

Anti-proliferative activity of Eg5 inhibitors against prostate cancer cells
In our study, the anti-proliferative activity of five Eg5 inhibitors on prostate cancer cells was assessed using MTT assay. As shown in Figure 2A and Table 2 MTT assay and trypan blue staining verified that Eg5 inhibitors can substantially inhibit prostate cancer cell proliferation and promote cell death. To explore the was further visualized by immunofluorescence assay (Figure 3E), and such cells also increased significantly after 16 h treatment ( Figure 3F, P < 0.01). Collectively, these data confirmed that the anti-proliferative activity of S(MeO)TLC in prostate cancer cells is induced by mitotic arrest followed by apoptosis.

Efficacy of S(MeO)TLC in the prostate cancer xenograft model
Our previous study showed that S(MeO)TLC (400 μg kg -1 to 20 mg kg -1 ) exhibited significant anticancer efficacy (P < 0.05) in the human bladder cancer (KU7) subcutaneous xenograft models in a dose-dependent manner 22 . Also, there was no A Self-archived copy in Kyoto University Research Information Repository https://repository.kulib.kyoto-u.ac.jp significant difference (P > 0.05) in tumor growth between the 20 and 30 mg kg -1 groups. Among the five Eg5 inhibitors tested, S(MeO)TLC was the most effective one for the inhibition of KU7 xenograft tumor growth, and the IC 50 of S(MeO)TLC on KU7 and PC3 cells was almost the same. Therefore, in our present study, we only evaluated the anticancer efficacy of S(MeO)TLC in PC3 luc subcutaneous xenograft models using doses of 10 and 20 mg kg -1 and an equal amount of vehicle as a control.
As shown in Figure 4A, on day 24 after the first treatment, S(MeO)TLC considerably inhibited subcutaneous tumor growth in a dose-dependent manner (P < 0.05). At the end of the observation, we also compared mean final tumor weights between treatment and control groups. The data confirmed that difference in the mean final tumor weights between the two groups was statistically significant ( Figure 4B, P < 0.05). No obvious body weight changes and other toxic events such as motor disorder and mortality were seen in any group (data not shown). The microscopic appearance of the tumors is shown in Figure 4C. Characteristic monopolar spindle mitotic cells were observed in H&E-stained tumors from the S(MeO)TLC-treated mice, but not in the control group ( Figure 4C).

Discussion
Cancer cells enter into the cell cycle and undergo uncontrolled mitosis permanently, which results in unlimited tumor growth. Thus, drugs that interfere with mitosis have emerged as the most successful chemotherapeutic agents in clinical use today 29,30 .
Among them, anti-microtubule agents, including taxanes and vinca alkaloids that A Self-archived copy in Kyoto University Research Information Repository https://repository.kulib.kyoto-u.ac.jp disturb microtubule dynamics of mitosis, represent the most effective anticancer drugs 29,30 . Given the success of taxanes in cancer therapy by perturbing mitosis, Eg5, which is more highly expressed in proliferating tissues during mitosis 31 , is considered as an attractive target for cancer chemotherapy.
Accumulating evidence also indicates that Eg5 inhibition is especially relevant in the treatment of CRPC. In addition to sharing the target molecules with taxanes, Eg5 inhibitors can also exhibit powerful anticancer activity in taxol-resistant cancer cells, and they can overcome taxane resistance 16 . In addition, they can overcome taxane-related peripheral neurotoxicity 13,32 . It has been also reported that, in androgen-independent progression of prostate cancer cells, the androgen receptor distinctly upregulates M-phase cell-cycle genes to promote androgen-independent cell proliferation 33 . These observations provide us with a strong rationale to utilize Eg5 inhibitors in the treatment of CRPC.
In the present study, we found a correlation between Eg5 immunohistological expression and clinicopathological characteristics, using TMAs of prostate cancer clinical specimens. Although Eg5 expression was not significantly (P > 0.05) associated with age, initial PSA, pathological T stage, positive surgical margin status, nor Gleason score, we did observe a significant correlation (P = 0.004) between Eg5 expression and Ki67 LI (the proliferation marker), which is consistent with preferential expression of Eg5 in proliferating tissues 31 . Furthermore, more than half the prostate cancer specimens showed Eg5 expression. Meanwhile, western blotting analysis showed that Eg5 expression was also present in all three prostate cancer cell To the best of our knowledge, there have been two systematic studies involving Eg5 inhibition in prostate cancer 34,35 . These have revealed that docetaxel-resistant and non-resistant prostate cancer cell lines showed almost the same sensitivity to antisense oligonucleotide or STLC treatment 34 . In contrast to the antisense oligonucleotide and STLC used in those two studies, in our previous study, our target