BACKGROUND
razoxane  , first developed in uk, was the earliest agent against spontaneous metastasis for the murine model  in  <dig>  <cit> .
a large volume of papers and projects have been published in the utilities and mechanisms of raz for anticancer actions, like assisting radiotherapy,  <cit>  overcoming multi-drug resistance  of daunorubicin and doxorubicin  <cit> , inhibiting topoisomerase ii  <cit>  and so on.
more importantly, raz, as a cardioprotectant of anthrocyclines, has been licensed in  <dig> countries in  <dig> continents.
since morpholine groups in some structures were reported to be responsible for cytotoxic or modulative actions on tumors, an anticancer agent, probimane  was synthesized by introducing two morpholine groups into raz in china.
<cit> .
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  <cit>  and limited clinical data showed that pro could inhibit human malignant lymphoma even for those resistant to other anticancer drugs  <cit> .
pro exhibits the same pharmacological effects as raz, like detoxication of adriamycin  induced cardiotoxicities, and synergism with adr against tumors  <cit> .
we have found some novel biological effects of pro, like inhibiting the activity of calmodulin , a cell-signal regulator, which can explain anticancer actions and the combined cytotoxic effect of pro and adr  <cit> .
pro was also shown to inhibit lipoperoxidation  of erythrocytes  <cit> , influence tumor sialic acid synthesis  <cit>  and inhibit the binding of fibrinogen to leukemia cells  <cit> .
lung cancer is the no  <dig> 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  and by high metastatic occurrence in clinics  <cit> .
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.
RESULTS
lethal toxicity of pro and raz in mice
the lethal dosage of pro and raz is tabulated in table  <dig>  since the toxicity of pro and raz seemed to lack sex specificity in mice, we were able to combine their numbers for ld <dig> and ld <dig> calculations.
we used the approximate dosage of ld <dig> of pro  and raz  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  <dig> and table  <dig>  pro and raz at equitoxic dosages  showed a noticeable anticancer effect on primary tumor growth , and significantly inhibited the formation of tumor metastases .
primary tumor growth of llc was inhibited more by pro  than by raz  in a  <dig> day trial, whereas the inhibition of pro  was slightly less than that of raz  on an  <dig> 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  <dig>   <dig>   <dig>  we evaluated  <dig>   <dig> and  <dig> day administration schedules in our study.
we found that raz and pro were effective in a statistically significant manner with the  <dig> injection schedule of the  <dig>   <dig> and  <dig> day administrations on llc.
if we administered raz to tumor-bearing mice once on day  <dig>   <dig> and  <dig>  there was no difference between treatment and vehicle control.
antitumor effects of raz in combination with ble on llc  were better than those in combination with dau  .
pro also showed synergistic effects in combination with ble .
antitumor activity of pro and raz on lax-83
the experiments showed that lax- <dig> was sensitive to raz  and pro  with inhibitory rates of 25– <dig> % and 55– <dig> % respectively .
ctx, as a positive anticancer drug , exhibited antitumor activities against the growth of lax- <dig> with an inhibitory rate of  <dig> %.
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  <dig>  we have tested the  <dig> most commonly used anticancer drugs – cyclophosphamide , 5-fluoruoracil , methotrexate , cisplatin  and vincristine  .
in the lax- <dig> 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- <dig> 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.
<cit>  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.
<cit>  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, <cit>  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  against lax- <dig>  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 water-soluble 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  <cit> .
presently, a stereo-isomer of raz, , 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- <dig> was licensed in  <dig> countries in  <dig> 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 advantages of bisdioxopiperazines in clinical treatment of lung cancers are as follows:  pro and raz can inhibit the growth of lung cancers, with and without the help of other anticancer drugs, like dau and ble;  like raz, pro strongly inhibits spontaneous pulmonary metastasis of llc;  since pro can inhibit cam  <cit> , a calcium activated protein that's associated with mdr and metastatic phenotypes, synergistic anticancer effects of pro and raz can be expected in combination with other anti-cancer drugs, like dau or ble.
now, new concepts of the relationship between tumor metastasis and mdr in cancers have been stated, <cit>  whereas bisdioxopiperazines can inhibit both tumor metastasis and mdr.
as a counterpart of raz, pro might be of interest and have chemotherapeutic potential in clinics.

 razoxane, first developed in uk, was the earliest agent against spontaneous metastasis for the murine model in. a large volume of papers and projects have been published in the utilities and mechanisms of raz for anticancer actions, like assisting radiotherapy, overcoming multi-drug resistance of daunorubicin and doxorubicin, inhibiting topoisomerase ii and so on. more importantly, raz, as a cardioprotectant of anthrocyclines, has been licensed in countries in continents. since morpholine groups in some structures were reported to be responsible for cytotoxic or modulative actions on tumors, an anticancer agent, probimane was synthesized by introducing two morpholine groups into raz in china.. 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 and limited clinical data showed that pro could inhibit human malignant lymphoma even for those resistant to other anticancer drugs. pro exhibits the same pharmacological effects as raz, like detoxication of adriamycin induced cardiotoxicities, and synergism with adr against tumors. we have found some novel biological effects of pro, like inhibiting the activity of calmodulin, a cell-signal regulator, which can explain anticancer actions and the combined cytotoxic effect of pro and adr. pro was also shown to inhibit lipoperoxidation of erythrocytes, influence tumor sialic acid synthesis and inhibit the binding of fibrinogen to leukemia cells. lung cancer is the no 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 and by high metastatic occurrence in clinics. 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 since the toxicity of pro and raz seemed to lack sex specificity in mice, we were able to combine their numbers for ld and ld calculations. we used the approximate dosage of ld of pro and raz 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 and table pro and raz at equitoxic dosages showed a noticeable anticancer effect on primary tumor growth, and significantly inhibited the formation of tumor metastases. primary tumor growth of llc was inhibited more by pro than by raz in a day trial, whereas the inhibition of pro was slightly less than that of raz on an 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 we evaluated and day administration schedules in our study. we found that raz and pro were effective in a statistically significant manner with the injection schedule of the and day administrations on llc. if we administered raz to tumor-bearing mice once on day and there was no difference between treatment and vehicle control. antitumor effects of raz in combination with ble on llc were better than those in combination with dau. pro also showed synergistic effects in combination with ble. antitumor activity of pro and raz on lax-83 the experiments showed that lax- was sensitive to raz and pro with inhibitory rates of 25– % and 55– % respectively. ctx, as a positive anticancer drug, exhibited antitumor activities against the growth of lax- with an inhibitory rate of %. 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 we have tested the most commonly used anticancer drugs – cyclophosphamide, 5-fluoruoracil, methotrexate, cisplatin and vincristine. in the lax- 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- 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. 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. 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, 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 against lax- 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 water-soluble 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. presently, a stereo-isomer of raz,, 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- was licensed in countries in 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.  the advantages of bisdioxopiperazines in clinical treatment of lung cancers are as follows: pro and raz can inhibit the growth of lung cancers, with and without the help of other anticancer drugs, like dau and ble; like raz, pro strongly inhibits spontaneous pulmonary metastasis of llc; since pro can inhibit cam, a calcium activated protein that's associated with mdr and metastatic phenotypes, synergistic anticancer effects of pro and raz can be expected in combination with other anti-cancer drugs, like dau or ble. now, new concepts of the relationship between tumor metastasis and mdr in cancers have been stated, whereas bisdioxopiperazines can inhibit both tumor metastasis and mdr. as a counterpart of raz, pro might be of interest and have chemotherapeutic potential in clinics. 
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 ld pro and raz were active against primary tumor growth and significantly inhibited pulmonary metastasis of llc at same dose-ranges. three different schedules of raz and pro were effective against the primary tumor growth of llc. the anti-cancer effects of raz and pro can be increased with the help of other anticancer drugs. both raz and pro inhibited the growth of lax in a statistically significant manner. the potential targets of bisdioxopiperazines may include lung cancers, especially on tumor metastasis.
 probimane, an anti-cancer agent originating in china, was derived from razoxane, 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 and one of human origin.  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 ld pro and raz were active against primary tumor growth and significantly inhibited pulmonary metastasis of llc at same dose-ranges. both raz and pro were effective in and day administration schedules. three different schedules of raz and pro were effective against the primary tumor growth of llc. the synergistic anticancer effect of raz with bleomycin was more obvious than those with daunorubicin 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 in a statistically significant manner.  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. 