Anti-proliferative effects, cell cycle G2/M phase arrest and blocking of chromosome segregation by probimane and MST-16 in human tumor cell lines

Background Anticancer bisdioxopiperazines, including ICRF-154, razoxane (Raz, ICRF-159) and ICRF-193, are a family of anticancer agents developed in the UK, especially targeting metastases of neoplasms. Two other bisdioxopiperazine derivatives, probimane (Pro) and MST-16, were synthesized at the Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China. Cytotoxic activities and mechanisms of Raz (+)-steroisomer (ICRF-187, dexrazoxane), Pro and MST-16 against tumor cells were evaluated by MTT colorimetry, flow cytometry and karyotyping. Results Pro was cytotoxic to human tumor cell lines in vitro (IC50<50 μM for 48 h). Four human tumor cell lines (SCG-7901, K562, A549 and HL60) were susceptible to Pro at low inhibitory concentrations (IC50 values < 10 μM for 48 h). Although the IC50 against HeLa cell line of vincristine (VCR, 4.56 μM), doxorubicin (Dox, 1.12 μM) and 5-fluoruouracil (5-Fu, 0.232 μM) are lower than Pro (5.12 μM), ICRF-187 (129 μM) and MST-16 (26.4 μM), VCR, Dox and 5-Fu shows a low dose-related – high cytotoxic activity. Time-response studies showed that the cytotoxic effects of Pro are increased for 3 days in human tumor cells, whereas VCR, Dox and 5-Fu showed decreased cytotoxic action after 24 h. Cell cycle G2/M phase arrest and chromosome segregation blocking by Pro and MST-16 were noted. Although there was similar effects of Pro and MST-16 on chromosome segregation blocking action and cell cycle G2/M phase arrest at 1- 4 μM, cytotoxicity of Pro against tumor cells was higher than that of MST-16 in vitro by a factor of 3- 10 folds. Our data show that Pro may be more effective against lung cancer and leukemia while ICRF-187 and MST-16 shows similar IC50 values only against leukemia. Conclusion It suggests that Pro has a wider spectrum of cytotoxic effects against human tumor cells than other bisdioxopiperazines, especially against solid tumors, and with a single cytotoxic pathway of Pro and MST-16 affecting chromosome segregation and leading also to cell G2/ M phase arrests, which finally reduces cell division rates. Pro may be more potent than MST-16 in cytotoxicity. High dose- and time- responses of Pro, when compared with VCR, 5-Fu and Dox, were seen that suggest a selectivity of Pro against tumor growth. Compounds of bisdioxopiperazines family may keep up their cytotoxic effects longer than many other anticancer drugs.


Background
Bisdioxopiperazines, including ICRF-154, razoxane (ICRF-159, Raz); ICRF-186 and ICRF-187), two stereo-isomers of Raz, and ICRF-193, developed in the UK, were some of the earliest agents found against a murine spontaneous metastatic model (Lewis lung carcinoma) in 1969 [1]. Many papers and projects have dealt with their potential use and mechanisms since that time. Three main mechanisms of bisdioxopiperazine action have been investigated, including assisting in radiotherapy, [2,3] overcoming multi-drug resistance (MDR) of daunorubicin and doxorubicin to leukemias [4,5] and inhibiting topoisomerase II [6,7]. More importantly, Raz has been licensed for cardioprotectant of anticancer anthrocyclines in more countries. Since bisdioxopiperazines represents a unique family of antimetastatic agents that are structurally conservative in their pharmacological actions, two new derivatives, probimane [1,2-bis (N 4 -morpholine-3, 5-dioxopeprazine-1-yl) propane; AT-2153, Pro] and MST-16, 1, 2-bis (4-isobutoxycarbonyloxymethyl-3, 5-dioxopiperazin-1-yl) ethane were synthesized at this institute in Shanghai, China. [8,9]. Apart from data of anti-tumor activity [10][11][12], the pharmacological mechanisms of Pro as Raz, like the detoxication of Adriamycin (ADR), induced cardiotoxicities and synergism with ADR against leukemias were reported at Henan Academy of Medicine, Henan, China [13]. As the main researchers of Pro, we reported some novel biological actions of Pro, including the inhibition of the activity of calmodulin (CaM), a cellsignal regulator, which can explain anticancer actions and the combined cytotoxic effect of Pro with ADR [13,14] inhibiting lipoperoxidation (LPO) of erythrocytes [15], down-regulating sialic acid synthesis in tumors [16] and blocking the binding of fibrinogen to leukemia cells [17]. MST-16, as a licensed drug in Japan since 1994, was permitted for direct use in leukemia chemotherapy, mainly Structural formulae of three bisdioxopiperazines Figure 1 Structural formulae of three bisdioxopiperazines

MST-16
against adult T-cell leukemia treatment [18]. Structural formulae of the three bisdioxopiperazines are represented in Figure 1.
As a new bisdioxopiperazine, the pharmacological characters and features of Pro are intriguing. Increased understanding of the advantages and disadvantages of the two compounds is a first step for promoting applications of Pro and MST-16. Therefore, in depth pharmacological evaluation was carried out. Tumors studied are from 7 different organs of origin -two gastric tumor cell line (SCG-7901, MKN-

Cytotoxic effects of Pro and MST-16 against human tumor cell lines
Data on the anticancer effects of Pro using 10 human tumor cell lines in vitro are showed in Figure 2 and

Comparison of the cytotoxic effects of bisdioxopiperazines with other drugs
The cytotoxic effects against tumor cell lines (p388, HL-60 and HeLa cells) are included in Table 1. Although IC 50 s of Dox, VCR and 5-Fu are lower than that of Pro, the greatest inhibitory rates of Pro at high concentrations are seen ( Table 2). No inhibitory difference between low and high concentrations of Dox, VCR and 5-Fu was observed (Table  3). Generally, the LD 50 of VCR and Dox in experimental 6&* +&7 0'$0% 0'$0% $ .  animals and humans are dramatically lower than Pro.
These results suggest more difficult management and wider toxicities of these drugs in their application in the clinics, suggesting Pro may avoid these drawbacks.

Comparison of anti-tumor effects of probimane and MST-16 and their time-response relationships
Cytotoxic effects (IC 50 ) of probimane and MST-16 against tumor cells were compared ( Figure 3).  The

G 2 and M phase arrests induced by Pro or MST-16
Our data shows that both probimane (Pro) and MST-16 can arrest tumor cells in G 2 and M phases of the cell cycle. Dose-and/ or concentration-dependency are observed in G 2 and M arrests (Figures 9 to 12

Chromosome segregation inhibition by Pro and MST-16
Chromosome linkages, aggregations and segregation in tumor cells were blocked by both Pro and MST-16. Figure  13 Figures 13 and 14), suggests that Pro and MST-16 act equally in this pathway.

Discussion
Increased understanding over the mechanisms of bisdioxopopiperazines can greatly improve their indications and narrow down contraindicates in clinical practice. The explanations for the anticancer actions of bisdioxopiperazine are currently focusing on anti-angiogeneses [19,20] and tumor cell DNA alterations caused by topoisomerase II. Generally speaking, most angiogenesis inhibitors often have low cytotoxicity and are ineffective against larger tumor masses, and are better combined with cytotoxic drugs clinically [21,22]. This work on the anticancer activity of Pro and MST-16 shows that they act through the blocking of chromosomal segregation and G 2 /M phase arrests, causing complete inhibition of tumor cell division. Pro, MST-16 and ICRF-187 play similar roles at equimolar concentrations. This pathway may be related to topoismerase II inhibition [23] as a possible mode of tumor growth inhibition, but is not suggested as a systematic approach through a cascade series. Two findings in this study need further discussion;

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7XPRU LQKLELWLRQV operate in the same course or cascade, and most possibly are directly linked; (ii) cyto-toxicity test (MTT) showed that Pro was more effective than MST-16. Lacking parallels in the effective dose ranges of Pro and MST-16 between cyto-toxicities and chromosome segregation -induced tumor inhibition can be explained by the fact that these effects of Pro and MST-16 do not strictly follow the same pathway given in Figure 15. Stronger cytotoxic effects of Pro against many other human tumor cell lines than original bisdioxopiperazines derivatives, especially on solid tumors, suggest some as yet undiscovered mechanism that Pro may have, and Pro may have better applications and require fewer drug combinations in the future.
This work shows that anticancer activities of Pro against lung cancer and leukemia are relatively greater than against other tumor typies. Cytotoxic and antimetastatic activities of Pro against lung tumor models in vivo have also been found [24]. Lung cancer is the most prevalent among all cancer categories, and is one of the deadliest cancers in the clinics. Targeted at lung cancers, Pro may offer better medical and economic benefits in the future.
For clinical chemotherapy, the paramount task is the balancing between treatment outcome and risks [25]. To optimize chemotherapeutic protocols containing bisdioxopiperazines, knowledge of its pharmacological parameters in terms of concentration-and time-responses are prerequisites. We found that Pro and MST-16 might act and accumulate longer in tumor cells than most of anti- Differences of anticancer effects (IC 50 ) of probimane for dif-ferent exposure intervals by a MTT method, n = 3 for 2 inde-pendent tests Figure 7 Differences of anticancer effects (IC 50 ) of probimane for different exposure intervals by a MTT method, n = 3 for 2 independent tests.
Differences of anticancer effects (IC 50 ) of MST-16 for differ-ent exposure intervals by a MTT method, n = 3 for 2 inde-pendent tests Figure 8 Differences of anticancer effects (IC 50 ) of MST-16 for different exposure intervals by a MTT method, n = 3 for 2 independent tests.
0'$0% 0'$0% ,QKLELWRU\ DFWLRQ ,& µΜ 067 GD\ 067 GD\ cancer drugs. The peak of cytotoxicity of both Pro and MST-16 is on day 3, and not usually on day 2. This result and our early work of auto-radiography that Pro [26] persists longer in tumor tissues suggest that longer intervals may be used between treatments and less nursing responsibilities may arise, while maintaining high levels of tumor growth inhibitions. The long-term cytotoxic effects of Pro and MST-16 are more obvious in high metastatic tumor cell lines, which can explain the selective effects of compounds to tumor metastases. Early reports suggest that MST-16 needs to transform into ICRF-154 to exhibit its anticancer effects [27]. This work proves that MST-16 does not degraded to ICRF-154, and has a lower G 2 /M phase arrests of human mammary tumor cell line (MDA-MB-435 cell) exposed to probimane at different con-centrations for 20 h     (Figure 15). It further suggests this mechanism is not a pivotal pathway for cytotoxic activity against tumors.

Conclusion
We suggest that Pro has a wider spectrum of cytotoxic effects against human tumor cells than other bisdioxopiperazines, especially on solid tumors. The cytotoxic pathway of Pro and MST-16 appears to be through chromosome segregation blocking and G 2 / M phase arrests. Pro may be more potent than MST-16. High dose-and time-related responses of Pro than VCR, 5-Fu and Dox are seen that suggest a selectivity by Pro against tumor growth. It suggests that the family of bisdioxopiperazines may sustain their cytotoxic effects longer than other anticancer drugs.

Methods
Pro and MST-16 were synthesized in this institute. Other chemical agents were purchased from sources stated below. The tumor cell lines were obtained from various sources and serially passaged in this lab.

Cell cycle analysis by cytometry
Tumor cells in exponential phase were exposed to Pro or MST-16. After 6 -24 h, cells were collected (300 × g, 10 min) and incubated with ice-cold PBS. Then fixed with ethanol and collected and washed with PBS by centrifugation (300 × g, 10 min). Cell deposition was added with PBS 1 ml and RNAse (5 µl) at 37°C bath for 15 min. Cells were dyed with 5 µl PI (2 mg/ml) in dark. Cells were measured for their DNA content by cytometry (Becton/Dickinson -FACS Calibur) after passing through a cell filter. MDA-MB-468) were seeded into a 6-well plate and maintained under an atmosphere of 5 % CO 2 condition. When tumor cells covered about 60-70 % of the surface, bisdioxopiperazines were added. Drug -treated cells were treated with hypotonic KCl, 0.075 M at 37°C for 30 min. Cell nuclei were fixed with fresh-prepared fixative solution [methanol/acetic acid, 3:1] for 5 min. Cell nuclei were collected by centrifugation (900 × g 15 min) and washed with fixative solution by centrifugation (1500 × g 20 min). Cell nuclei were dropped onto a cooled glass plate and placing overnight under a dehydrogenated atmosphere. The scattered chromosomes were dyed with a Giemsa solution for 15-20 min and washed with tap water. Chromosomal behaviors were viewed and photographed by microscopy with an oil-lense (LEICA, Qwin image processing analysis system, Germany).

Statistics
IC 50 of agents were calculated by software in this lab and X ± SD was calculated from data of two groups.

Blockage of chromosome segregations
Tumor cell G 2 M phase arresting Cell division inhibition