hyperthermic isolated limb perfusion ( hilp ) is regarded as an established therapy for locoregional intransit metastases of malignant melanoma of the extremities .
isolation of the limb by extracorporeal circulation , that is , by using a heart - lung - machine , enables administration of high doses of cytostatic drugs without relevant systemic side effects .
simultaneous heating of the limb up to temperatures of 40.0c to 41.0c intensifies the tumortoxic action of the drug .
although hilp represents a form of regional high - dose thermo - chemotherapy , partial response or treatment failure is observed in 2550% of patients .
furthermore , up to 50% of patients with previous complete remission following hilp will undergo relapse on the treated limb , usually after a median of 912 months [ 24 ] . among
the main causes for failure of treatment is the resistance of tumor cells against the action of cytostatic drugs , which is based on the expression of certain genes such as the multidrug resistance gene ( mdr1 ) , coding for the p - glycoprotein ( p - gp ) .
the gene product functions as an energy - dependent membrane - bound transport protein for hydrophobic , cytotoxic agents such as anthracyclines , epipodophyllotoxins , vinca alcaloids and taxol , thus lowering their intracellular concentration by drug efflux . meanwhile other chemoresistance - related proteins like the multidrug resistance protein mrp1 and its isoform mrp2 ( canalicular mrp , cmrp ; canalicular multispecific organic anion transporter , cmoat ) and the lung resistance - related protein ( lrp ) have been identified [ 7 , 8 ] .
furthermore , the expression of various heat shock proteins ( hsp ) has been associated with resistance of tumor cells to thermal and chemical damage .
the function of the different families of heat shock proteins is complex and has not yet been understood in full detail .
one of the proteins highly inducible under stress conditions is the 72  kd heat shock protein ( hsp72 ) , a member of the hsp70 family . according to their function as molecular chaperones
, a cell - protective effect is ascribed to these proteins after impairment of normal cell physiology by toxic stimuli such as heat , ischemia , and inflammation , although meanwhile proapoptotic cascades have also been linked with the induction of hsp .
tumor cells respond to hsp - mediated stress in the same way and can escape the action of tumortoxic therapies .
this has prompted us to investigate the direct impact of hilp on molecular mechanisms of resistance of malignant melanoma in an experimental study .
a set of well - characterized chemoresistance - related genes ( i.e. , mdr1 , mrp1 , mrp2 , lrp ) and hsp72 were selected for targets of the effects of hilp for purposes of the study .
kb-3 - 1 cell - line ( dszm , braunschweig , frg ) : as a derivative of the human cervix carcinoma cell line hela , it is used in multiple drug resistance studies giving rise to drug - resistance mutants ( e.g. , kb - v1 , resistant to vinblastine ) .
kb-3 - 1 is negative for mdr1 mrna expression and served as a negative control in the mdr1 detection experiments .
sk - mel-3 cell - line ( american type culture collection , rockeville / md .
, us ) : cell line established from a lymph node metastasis of malignant melanoma in a 42-year - old caucasian woman . a cell suspension of sk - mel-3 was injected subcutaneously on athymic nude mice ( nmri nu / nu ) to create a solid tumor .
small particles ( approx . 5  mm ) of the tumor were implanted subcutaneously on the right hind limbs of the study animals by means of small skin incision .
animals : for experimental limb perfusions eight - week - old athymic nude rats ( rowett rnu / rnu ) weighing 200250  g and bearing the sk - mel-3 xenograft on their right hind limbs were used .
10  mg of dry substance vinblastine sulfate ( velbe , lilly , bad homburg , frg ) were dissolved in 10  ml 0.9% nacl . for experimental perfusions ,
25  g vinblastine/15  ml priming volume were injected as a fractionated bolus over 2 minutes into the arterial line .
previous dose - finding studies had shown that this concentration was well tolerated by the animals and led to complete tumor regression in single animals .
vinblastine was chosen because of its cytotoxic effect on melanoma cell lines , which has been proven in former experimental perfusion studies .
furthermore , vinblastine is one of the substrates that is eliminated by the p - glycoprotein pathway . a modified miniature equipment , first described by nagel et al .
our technique of experimental perfusion has been described in detail earlier . in brief , after induction of anesthesia , the animal was fixed in a supine position on a cork - coated operation table .
a vertical skin incision was performed in the right groin and the inguinal ligament was divided .
the external iliac and femoral vessels were exposed by retracting the peritoneal sac in craniomedial direction .
after heparinization with 1000  ie / kg body weight , all collateral vessels were temporarily clipped to reduce systemic leakage .
after central clamping of the external iliac artery and vein , both vessels were cannulated under microscopic control ( g 24 and g 20 catheters , resp . ) .
the catheters were connected with the perfusion system and the extracorporeal circulation was started with a flow rate of 0.4  ml / kg body weight . a subcutaneous temperature probe ( yellow springs instrument co. , us )
was placed near the tumor for continuous registration of tissue temperature ( stckert , frg ) .
the extracorporeal circulation was achieved using a masterflex roller pump ( reichelt , frg ) with two pump - heads running synchronously on a single axis for the arterial and venous line .
venous blood from the tumor bearing limb was directed towards a specially constructed gas dispersion oxygenator ( lettenbauer , frg ) made of high - quality acryl with foam material taken from commercially available oxygenators .
tygon tubing ( reichelt , frg ) with an internal diameter of 0.8  mm was used for circulation of the perfusate .
3-way taps in the arterial and venous line allowed injection of the drug , measurement of blood gas analysis , and registration of perfusion pressure ( if necessary ) .
hyperthermia was induced by an externally adjustable infrared light source centered onto the tumor area .
apart from the analysis of the untreated tumor ( n = 3 ) , four groups with 12 animals each were constituted .
group i comprised normothermic perfusions ( tissue temperature , 3737.5c ) without vinblastine , group ii normothermic perfusions with vinblastine ( 25  g/15 ml perfusate ) , group iii hyperthermic perfusions ( tissue temperature 40.0 - 41.0c ) without vinblastine , and group iv hyperthermic perfusions with vinblastine ( 25  g/15  ml perfusate ) .
each group ( n = 12 ) was divided into four subgroups ( n = 3 ) with variation of perfusion time .
the duration of perfusion was 30 , 60 , and 90 minutes . in the fourth subgroup
after the scheduled perfusion time , the tumor was excised and instantly frozen in liquid nitrogen .
total cellular rna was prepared from 30  mg tissue samples using the qiagen rneasy - kit .
cdna was synthesized with 1  g of total cellular rna and 50  pmol of random hexanucleotide primer ( perkin elmer ) in 20  l of a solution containing 5  mm mgcl2 , 1xpcr - buffer ii ( perkin elmer ) , 500  m each dntp , 20  u rnase inhibitor ( perkin elmer ) , and 50  u mulv reverse transcriptase ( perkin elmer ) .
the reaction was performed using a trio - thermoblock ( biometra ) with the following program : 21c for 10 minutes , 42c for 60 minutes , and denaturation at 95c for 5 minutes .
water was added to a final volume of 50  l and cdna was stored at 20c before it was used .
rt - pcr was performed with 5  l of cdna template in a 50  l reaction containing 0.15  mm mgcl2 , 10  mm tris / hcl ph 8 , 3 ; 50  mm kcl , 50  pmol 5-primer , 50  pmol 3-primer , 2.5  u ampli - taq gold ( perkin elmer ) .
after denaturation at 95c for 5 minutes , pcr was performed as follows : 45 seconds 94c , 45 seconds 60c , 90 seconds 72c for 34 cycles , with an additional elongation step at 72c for 3 minutes , pcr products were analyzed on a 2.5% agarose gel .
pcr - primer sequences were mdr1 - 51 5-gtt.caa.act.tct.gct.cct.gag-3 ; mdr1 - 31 5-acc.cat.cat.tgc.aat.agc.agg-3 ; mrp1 - 51 5-acc.gga.gga .
cca.ggt.tc-3 ; mrp2 - 31 5-ctg.gtt.gat.gaa.ggc.tct.gt-3 ; lrp1 - 51   5 tgg.agc.cat.cgg.tga.tga.gg-3 ; lrp1 - 31   5-tct.gag.cat.ggc.cgt.gga.ga-3. the expression of gapdh was used as a positive control in each rt - pcr analysis ( primer sequence : gapdh-31 5-cca.tca.cca.tct.tcc.agg.ag-3 , gapdh-51 5-cct.gct .
in addition , the gene product of mdr1 , the p - glycoprotein , was measured representatively for all chemoresistance genes by the western blot method to detect the transcriptional activity of the gene .
total protein samples were prepared from 50  mg tissue samples , adding 100  l of tris / hcl ph 7.5 , 0.1% sds , using a micro - dismembranator for 60 seconds , 2000  rpm .
500  l of 10  mm tris / hcl ph 7.5 ; 1.0% sds was added to the cell suspension and boiled for 10 minutes .
after centrifugation for 10 minutes at 13.000  rpm , supernatant was collected and the protein content was measured by biorad - protein - assay
. 25  g of protein was applied per lane to a 7.5% sds - page - gel for mdr1 protein analysis and 12% sds - page gel for hsp72 analysis .
the gel was blotted on a nitrocellulose membrane with 0.8  ma / cm gel for 1.5 hours using a semidry gel - blotting apparatus .
the membrane was blocked using a 5% solution of blocking - reagent ( amersham ) in tbs - t , for one hour at room temperature .
( amersham life science ) and  mdr ( p - glycoprotein ) ( ab-1 )  ( calbiochem ) .
after three washing steps , secondary antibodies , antimouse - antibody ( amersham ) , and antirabbit - antibody ( amersham ) , respectively , were applied .
hsp72 expression was evaluated semiquantitatively by densitometric measurement and band analysis with the imaging software tina ( raytest ) .
pixel areas of the bands in each group ( n = 3 ) were averaged and related to the mean pixel area of the untreated tumor which represented the value 1 .
all experimental perfusions could be performed as planned except the 90-minute hyperthermic perfusions ( with and without vinblastine ) which were not tolerated by the study animals ( intraoperative exitus ) .
mdr1 , mrp1 , and lrp were constantly expressed in the untreated sk - mel-3 tumor , whereas mrp2-mrna could not be detected in any of the untreated tumor samples .
this pattern of expression was neither remarkably influenced by the application of hyperthermic temperatures nor by the addition of the cytostatic drug vinblastine , nor did variation of the length of perfusion appreciably change expression of the studied chemoresistance genes . in single - tumor samples , a temporary suppression of mrp1 in groups i ,
ii , and iii was observed as well as a passing induction of mrp2 in groups ii , iii , and iv .
no later than 5 hours after the 60-minute perfusion time , the original pattern of expression was restituted as detected in the untreated tumor .
detection of the p - glycoprotein by western blot analysis corresponded to the results of mdr1 expression by rt - pcr ( data not shown ) .
variability in expression of single genes within the subgroups at different times was low : there was a congruence of expression in 51 of 60 subgroups ( 85% ) .
deviations within a group at a defined time could correspond to a true therapeutic effect , but could also be caused by the immanent variability of measurements in biological systems . a fundamental change of the intrinsic  resistance pattern  following hilp
( group i ) , only a temporary 1.5-fold induction in comparison with the base line of the untreated tumor was observed after 30 minutes .
the administration of vinblastine under normothermic conditions ( group ii ) was associated with an approximately 2.5-fold increase of hsp72 production , which dropped to a 1.5-fold rate of expression after 90 minutes .
5 hours postoperatively ( 60-minute perfusion time ) , the level of the untreated tumor was reached again .
hyperthermia alone caused a delayed increase of hsp72 expression by factor 1.5 , which was kept even for 5 hours after the perfusion had been terminated ( group iii ) . against the expectations ,
only a 1.5-fold induction was measured in combined application of hyperthermia and vinblastine ( group iv ) , though it was observed after only 30 minutes of perfusion and persisted as long as 5 hours after the perfusion had been terminated .
even with the most effective cytostatic drug , that is , dacarbazine , remissions are reported to be only as high as 1433% in the metastasized stage of disease .
one of the best - characterized mechanisms of chemoresistance is the mdr1 gene , but some studies have shown that it is rarely overexpressed in malignant melanoma [ 1518 ] . on the other hand ,
increased expression of the chemoresistance - associated transport proteins mrp and lrp was found in melanoma cell lines as well as in primary and metastatic melanoma that could explain resistance towards lipophilic , natural compounds , but not towards alkylating agents . the sk - mel-3 tumor constitutively expressed mdr1 , mrp1 , and lrp . because coexpression of several mechanisms of resistance is related to prognosis [ 1820 ] ,
the sk - mel-3 tumor can be considered as a relatively resistant variant with an unfavourable susceptibility .
this is supported by the fact that the tumor was isolated from a lymph node metastasis from a patient that had been pretreated with methyl - ccnu .
cytostatic pretreatment can also be correlated with the presence of mdr1-mrna and p - gp in the sk - mel-3 tumor because the incidence of mdr1 expression has been observed to increase following previous chemotherapy , for example , in acute myeloic leucemia , plasmocytoma , or osteosarcoma , and adversely influences adversely remission rate and prognosis of relapsed tumors .
in contrast , schadendorf et al .   did not find an enhanced induction of p - gp in vindesine- and cisplatin - pretreated metastasic melanoma .
the pattern of expression of chemoresistance genes was not changed by hyperthermic isolated limb perfusion and seems irreversibly determined . despite high concentrations of the cytostatic drug and the exposition towards hyperthermic temperatures , only temporary effects on gene expression , that is , mrp1 ( suppression ) and mrp2 ( induction ) could be observed .
apparently , even high - dose thermochemotherapy is not able to alter transcriptional activity of chemoresistance genes .  
although the results may only apply to the sk - mel-3 tumor and the number of study animals in each subgroup was rather small to draw a final conclusion , preliminary results in melanoma patients treated by hilp underline this observation .
should this be confirmed in further experimental and clinical studies , it would be desirable to define the resistance pattern preoperatively for better evaluation of the response to hilp or adjusting the drug regimen more individually to each patient , that is , a calculated choice of agents according to the individual resistance pattern .
furthermore , an additional administration of blocking agents for inhibition of resistance genes , that is , inhibition of mdr1 by verapamil- or ciclosporine - derivatives can be imagined . there are first promising clinical experiences with such modulators of chemoresistance in the systemic treatment of refractory non - hodgkin lymphoma and plasmocytoma [ 23 , 24 ] , but side effects of the therapy were limiting .
the regional action of hilp should offer an ideal indication for these substances because systemic side effects of the therapy are negligible . in the literature , increased expression of hsp ( hsp27 , hsp70 ) plays an important role in reduced sensitivity towards cytostatic drugs [ 2527 ] . a direct correlation of chemoresistance and
[ 28 , 29 ] and kioka et al .   who observed an amplification of mdr1-mrna together with an enhanced resistance of tumor cells in vitro towards vinblastine after heat exposure .
the explanation that was given for this result was the presence of heat shock - responsible consensus elements in the mdr1-promotor region . in melanoma cells
not only a chemically and heat - induced expression of hsp ( hsp72 ) has been demonstrated [ 3134 ] , but also a constitutive overexpression that was independent of stress ( hsp27 , hsp72 , hsp90 ) , as was the case in the sk - mel-3 tumor .
again , constitutive expression of hsp was associated with an advanced tumor stage and amplified resistance towards thermal and cytostatic drug damage [ 3537 ] . the cellular response to stress , that is , the increase in transscriptional activity of hsp , takes place within minutes . in vitro studies in melanoma cells showed the level of hsp expression to be dependent on time and temperature , with the maximal rate of synthesis at 42c and 9 hours exposure reaching a steady state with levels not much higher than constitutive expression following continued exposure .
these in vitro results were also obvious in the in vivo tumor model which was presented .
the sensitive stress reaction is recognized by the fact that even operative manipulation alone ( normothermic perfusion without vinblastine ) was associated with a raised hsp synthesis , probably due to limb ischemia during the clamping of the vessels for cannulation .
the administration of vinblastine in normothermic perfusions resulted in peak values of hsp72 synthesis after 30 minutes ( factor 2.5 ) , which dropped to the base line of the untreated tumor not later than 5 hours after 60-minute perfusion , maybe as a consequence of mdr1-mediated drug efflux of vinblastine .
the increase of hsp72 expression in the tumor tissue during hyperthermic temperatures was clearly evident , but with a delayed maximum for hyperthermia alone ( 60 minutes ) in comparison with the combination of hyperthermia and vinblastine ( 30 minutes ) .
the reason why the combination of both stress factors ( cytostatic drug and hyperthermia ) had a smaller effect on the level of hsp72 expression remains unclear , but a temporary paralysis of cell function including protein synthesis with gradual recovery could be discussed .
the changes in the rate of synthesis by a factor of 1.5 to 2.5 may be less impressive than in an in vitro setting , but the vascularized structure of a solid tumor undoubtedly represents a more complex system so that despite standardized experimental conditions , biologic effects are more difficult to assess .
the constitutive expression of hsp72 in sk - mel-3 tumor may add to this observation .
as has been reported earlier [ 1 , 39 , 40 ] , the proposed experimental model , that is , performance of miniaturised limb perfusion in nude rats with implanted melanoma xenografts simulating the clinical setting proved to be suitable for the investigation of the question of how well - characterized mechanisms of resistance are directly influenced by hilp .
on the one hand , the inherent , irreversible pattern of chemoresistance genes and the induction of hsp in the tumor itself during treatment offer a possible explanation for a primary ( no or partial response ) or secondary failure ( early local recurrence ) of hilp . on the other hand ,
there is clinical evidence that apart from a resistant cell phenotype there are additional factors such as tumor burden , which are important for response or resistance of the tumor towards therapy .
 hyperthermic isolated limb perfusion ( hilp ) is considered an established treatment for multiple locoregional intransit metastases in malignant melanoma of the extremities . 
 various mechanisms such as the expression of chemoresistance genes and heat shock proteins by the tumor may be responsible for varying response rates and locoregional recurrences of the treatment . 
 the aim of the experimental animal study was to investigate the direct impact of hilp on such mechanisms of resistance . 
 tissue temperature , administration of the cytostatic drug , and duration of perfusion were varied . 
 expression of the chemoresistance genes mdr1 , mrp1 , mrp2 , and lrp and of heat shock protein 72 ( hsp72 ) in the tumor tissue was analysed using rt - pcr and western blot analysis . 
 the untreated sk - mel-3 tumor expressed mdr1 , mrp1 , and lrp , but not mrp2 . 
 neither variation of temperature , administration of the cytostatic drug , nor duration of perfusion changed the expression of this  resistance pattern  . 
 in contrast to the cytostatic drug , hyperthermia causes a persistent induction of hsp72 . both observations could offer a potential explanation for failure of hilp in malignant melanoma . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusion