Antitumor effects of two bisdioxopiperazines against two experimental lung cancer models in vivo

Background Probimane (Pro), an anti-cancer agent originating in China, was derived from razoxane (ICRF-159, Raz), a drug created in Britain, specifically targeting at cancer metastasis and as a cardioprotectant of anthrocyclines. Pro and Raz are bisdioxopiperazine compounds. In this work, we evaluated the anti-tumor and anti-metastatic effects of Pro and Raz in vivo against two lung tumor models, one of murine origin (Lewis lung carcinoma, LLC) and one of human origin (LAX-83). Results After determining the lethal dosage of Pro and Raz, we assessed and compared the inhibitory effects of Pro and Raz against primary tumor growth and metastatic occurrences of LLC at the dosage of LD5. Pro and Raz were active against primary tumor growth and significantly inhibited pulmonary metastasis of LLC at same dose-ranges (inhibitory rates > 90 %). Both Raz and Pro were effective in 1, 5, and 9 day administration schedules. Three different schedules of Raz and Pro were effective against the primary tumor growth of LLC (35–50 %). The synergistic anticancer effect of Raz with bleomycin (Ble) (from 41.3 % to 73.3 %) was more obvious than those with daunorubicin (Dau) (from 33.1 % to 56.3 %) in the LLC tumor model. Pro was also seen to have synergistic anti-cancer effects with Ble in the LLC model. Both Raz and Pro inhibited the growth of LAX 83 in a statistically significant manner. Conclusions These data suggest that both Raz and Pro may have anti-tumor potentiality and Raz and Pro have combinative effects with Ble or Dau. The potential targets of bisdioxopiperazines may include lung cancers, especially on tumor metastasis. The anti-cancer effects of Raz and Pro can be increased with the help of other anticancer drugs.


Background
Razoxane (ICRF-159) (Raz), first developed in UK, was the earliest agent against spontaneous metastasis for the murine model (Lewis lung carcinoma) in 1969 [1]. A large volume of papers and projects have been published in the utilities and mechanisms of Raz for anticancer actions, like assisting radiotherapy, [2] overcoming multi-drug resistance (MDR) of daunorubicin and doxorubicin [3], inhibiting topoisomerase II [4] and so on. More importantly, Raz, as a cardioprotectant of anthrocyclines, has been licensed in 28 countries in 4 continents. Since morpholine groups in some structures were reported to be responsible for cytotoxic or modulative actions on tumors, an anticancer agent, probimane [1,2-bis (N 4 -morpholine-3, 5-dioxopeprazine-1-yl) propane; AT-2153, Pro] was synthesized by introducing two morpholine groups into Raz in China. [5]. Raz and Pro belong to bisdiopiperazines. Like Raz, Pro also exhibits anti-tumor activity both in vivo and in vitro against experimental tumor models in a small scale investigation [6,7] and limited clinical data showed that Pro could inhibit human malignant lymphoma even for those resistant to other anticancer drugs [8]. Pro exhibits the same pharmacological effects as Raz, like detoxication of Adriamycin (ADR) induced cardiotoxicities, and synergism with ADR against tumors [9,10]. We have found some novel biological effects of Pro, like inhibiting the activity of calmodulin (CaM), a cell-signal regulator, which can explain anticancer actions and the combined cytotoxic effect of Pro and ADR [11]. Pro was also shown to inhibit lipoperoxidation (LPO) of erythrocytes [12], influence tumor sialic acid synthesis [13] and inhibit the binding of fibrinogen to leukemia cells [14].
Lung cancer is the No 1 killer among all categories of cancers in urban areas in China and many Western countries. The high mortality rate of lung cancer can easily be caused by inducing multi-drug resistance (MDR) and by high metastatic occurrence in clinics [15]. Since we assume that Pro, like Raz may possess useful therapeutic potentialities, we evaluated in vivo the chemotherapeutical parameters of Pro and Raz for lung cancer of both murine and human origins.

Lethal toxicity of Pro and Raz in mice
The lethal dosage of Pro and Raz is tabulated in Table 1. Since the toxicity of Pro and Raz seemed to lack sex specificity in mice, we were able to combine their numbers for LD 50 and LD 5 calculations. We used the approximate dosage of LD 5 of Pro (60 mg/kg ip × 7) and Raz (20 mg/kg ip × 7) as equitoxic dosages for further treatment studies.

Antitumor and antimetastatic effects of Pro and Raz on LLC
Antitumor and antimetastatic effects of Pro and Raz on LLC are tabulated in Table 2 and Table 3. Pro and Raz at equitoxic dosages (LD 5 ) showed a noticeable anticancer effect on primary tumor growth (inhibitory rates, approximately 30-45 %), and significantly inhibited the formation of tumor metastases (inhibitory rates on pulmonary metastasis > 90 %, P < 0.001). Primary tumor growth of LLC was inhibited more by Pro (48 %) than by Raz (40.3%) in a 20 day trial, whereas the inhibition of Pro (35.7%) was slightly less than that of Raz (40 %) on an 11 day trial. Pro seems to be more persistent than Raz in inhibiting primary tumor growth of LLC.

Antitumor effects of bisdioxopiperazines for different schedules and in combination with other anticancer drugs
Antitumor effects of Raz and Pro on LLC are included in Table 4, 5, 6. We evaluated 1, 5 and 9 day administration schedules in our study. We found that Raz and Pro were effective in a statistically significant manner with the 3 injection schedule of the 1, 5 and 9 day administrations on LLC. If we administered Raz to tumor-bearing mice once on day 1, 5 and 9, there was no difference between treatment and vehicle control. Antitumor effects of Raz in combination with Ble on LLC (73.3 %) were better than those in combination with Dau (56.3 %) ( Table 5 and  Table 6). Pro also showed synergistic effects in combination with Ble (Table 7).

Antitumor activity of Pro and Raz on LAX-83
The experiments showed that LAX-83 was sensitive to Raz (40-60 mgKg -1 , ip × 5) and Pro (80-100 mgKg -1 ip × 5) with inhibitory rates of 25-32 % and 55-60 % respectively (P < 0.01 vs control). CTX, as a positive anticancer drug (40 mgKg -1 ip × 5), exhibited antitumor activities against the growth of LAX-83 with an inhibitory rate of 84 %. Obvious necrosis in tumor tissues was observed by histological evaluation of CTX and Pro treatment groups, but Pro showed larger vacuoles than CTX. Drug inhibition on tumor volumes were calculated and outlined in Table 8.
We have tested the 5 most commonly used anticancer drugs -cyclophosphamide (CTX), 5-fluoruoracil (5-Fu), methotrexate (MTX), cisplatin (DDP) and vincristine (VCR) ( Table 9). In the LAX-83 model, CTX has been shown to be the most effective one. The anticancer effect of Pro was the same or better than those of MTX, DDP and as well as 5-Fu against LAX-83 tumor growth.

Discussion
Explanations of anticancer and antimetastatic mechanisms of bisdioxopiperazines are now inconclusive. The   present explanation for the anticancer mechanisms of Raz has been attributed to antiangiogenesis and topoisomerase II inhibition. [16] Since the antimetastatic activities of Raz and Pro were much stronger than those actions against primary tumor growth, this special targeting on metastasis ought to be more useful in clinical cancer treatment. Raz and Pro show typical characteristics of antiangiogenesis agents, which target small nodule of tumors. Meanwhile, recent reports on drugs targeting angiogenesis indicate that most anti-vascular drugs have low or even no effects on most cancers when they are used alone in clinics, but they show synergistic effects in combination with other anticancer drugs. [17,18] Our study shows synergistic anticancer actions of Raz and Pro with Ble or Dau basing on this theory. Previous work showed that Pro and Raz could reduce the cardiotoxicity of anthrocycline, [1,9,10] so we may reasonably deduce that they can also reduce the cytotoxicity of anthrocyclines. The data in our study suggests that the synergistic effects of Raz with anthrocyclines are present, but not as potent as those with Ble.
Since we have tested the antitumor activity of clinically available anticancer drugs (CTX, 5-Fu, MTX, DDP and VCR) against LAX-83, CTX being the best one, two bisdioxopiperazines studied on this work show overall similar anticancer effective as commonly used drugs. Although the anticancer effects of CTX and VCR are better than those of Pro, for other commonly used drugs, such as DDP, MTX and 5-Fu, the antitumor effects are no better than those of Pro. Since the antitumor effects of MTX and DDP are even less effective than those of Pro and Raz, we suggest that anticancer effects of Pro and Raz are within the effective anticancer ranges of commonly available anticancer drugs.
The other useful property of Pro is that it is the most watersoluble among the bisdioxopiperazines. Most bisdioxopiperazines are less water-soluble and given orally in clinics. Although oral administration is easy for patients, bioavailability varies from patient to patient. For some patients who have a poor absorption of bisdioxopiperazines in oral administration, Pro can be injected iv to maintain stable drug levels. Our previous work showed that Pro could strongly accumulate in tumor tissue while Pro levels in other tissues decrease rapidly [19]. Presently, a stereoisomer of Raz, (dexrazoxane, ICRF-187), a water-soluble Raz, is being reinvestigated and has aroused the interests of clinical oncologists. Phase III clinical studies are currently underway in the US. More importantly, ICRF-187 was licensed in 28 countries in 4 continents. This work shows a noticeable inhibition of Pro and Raz on lung cancers and suggests possible usage of Raz and Pro on lung cancer in clinics.

Conclusions
The

Lethal dosage determination in mice
Mice of Kun-Min strain (equal amount of male and female) were ip injected with Pro and Raz daily for 10 successive days. The deaths of mice were counted after 1 month. Lethal dosage of agents was calculated by Random Probity tests.
Antitumor and antimetastatic studies of LLC C57BL/6J mice were implanted sc with LLC (2 × 10 6 cells) from donor mice. The mice were injected intraperitoneally with drugs daily or every two days for 7 injections. On day 11 or day 20, mice were sacrificed, and locally growing tumors were separated from skin and muscles and weighed, and lungs of host mice were placed into a Bouin's solution for 24 h, and then the lung samples were submerged into a solution of 95 % alcohol for 24 h. Finally, the numbers of extruding metastatic foci in lungs were counted.

Antitumor actions of different schedules and in combinations with different drugs
C57BL/6J mice were implanted sc with LLC (2 × 10 6 cells) from donor mice. Mice were injected intraperitoneally with drugs on day 1, 5, 9. Single injection or 3 injections every 3 hours were used. Tumors were separated and weighed on day 11.

Antitumor activity study of human tumors
Nude mice were inoculated with LAX-83 under the renal capsule (SRC method). [22] Nude mice were injected intraperitoneally with drugs daily during next five days after inoculation of LAX-83. Then nude mice were sacrificed, and their kidneys were taken out for measurement of tumor sizes using a stereomicroscope a week after transplantation. Tumor volume was calculated as 1/2(ab 2 ) where a and b are their major and minor axes of the lump. Kidneys with tumors were paraffin-embedded, sliced and hematoxylin dyed. The tumor tissues were then observed from a light microscope.

Statistical analysis
Student's t-test was used to assess the differences between control and drug treatment groups of above methods.

Author's contribution
The experimental design was made by Bin Xu and Da-Yong Lu.
Experiments were performed by Da-Yong Lu (anticancer activity tests) The manuscript was written by Da-Yong Lu, and Jian Ding.
Structural formulas of razoxane and probimane