A phase II study of the vitamin D analogue Seocalcitol in patients with inoperable hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a common malignant tumour, which has a poor prognosis. Surgical resection can be curative but most patients are inoperable and most chemotherapy agents have minimal activity in this disease. Seocalcitol, a vitamin D analogue, induces differentiation and inhibits growth in cancer cell lines and in vivo. The vitamin D receptor is expressed in hepatocytes and more abundantly in HCC cells. In total, 56 patients with inoperable advanced HCC were included in an uncontrolled study of oral Seocalcitol treatment for up to 1 year (with possible extension for responders). The dose was titrated according to serum calcium levels. The treatment effect was evaluated by regular CT scans. Out of 33 patients evaluable for tumour response, two had complete response (CR), 12 stable disease and 19 progressive disease. The CRs appeared after 6 and 24 months of treatment, and lasted for 29 and at least 36 months (patient still in remission when data censored). Seocalcitol was well tolerated; the most frequent toxicity was hypercalcaemia and related symptoms. Most patients tolerated a daily dose of 10 micro g of Seocalcitol. This is the first study showing activity, by reduction in tumour dimensions, of a differentiating agent in patients with an advanced bulky, solid tumour. Seocalcitol may have an effect in the treatment of HCC, especially in early disease when a prolonged treatment can be instituted. The survival benefit with or without tumour response should be determined in controlled studies.

Irinotecan combined with bolus 5-fluorouracil and folinic acid Nordic schedule as first-line therapy in advanced colorectal cancer.

BACKGROUND: This multicentre phase II study evaluated the efficacy and safety of irinotecan combined with the Nordic schedule of 5-fluorouracil (5-FU) and folinic acid (FA) as first-line therapy in patients with advanced colorectal cancer. PATIENTS AND METHODS: Seventy-four patients with measurable disease and a WHO performance status of 2 or less were treated with irinotecan 210 mg/m(2) as a 30-90 min intravenous infusion on day 1, followed by 5-FU 500 mg/m(2) and FA 60 mg/m(2) bolus on days 1 and 2, every 2 weeks, until disease progression or unacceptable toxicity. The primary end point was the objective response rate. RESULTS: Twenty-nine out of 68 evaluable patients achieved a complete (n = 7) or partial (n = 22) response, leading to an overall response rate of 43% [95% confidence interval (CI) 31% to 55%]. The median duration of response was 10 months. The estimated median time to progression and survival were 6.4 months (95% CI 5.4-9.0) and 15.6 months (95% CI 13.3-19.0), respectively, in the intention-to-treat population. A total of 860 cycles were administered to 74 patients. Neutropenia was the main adverse event with grade 3-4 toxicity in 66% of patients and 17.5% of cycles. Grade 3-4 non-haematological toxicities were infrequent and included diarrhoea in 16% of patients and 2% of cycles and nausea/vomiting in 10% of patients and 1% of cycles. CONCLUSIONS: Irinotecan combined with the bolus Nordic schedule of 5-FU/FA is active in advanced colorectal cancer with an easily managed safety profile which ensures good schedule compliance. The low incidence of grade 3-4 non-haematological toxicity justifies the further evaluation of this combination in the context of randomised clinical trials.

Functional monitoring of anthracycline cardiotoxicity: a prospective, blinded, long-term observational study of outcome in 120 patients.

BACKGROUND: With increasing doses the highly tumoricidal anthracycline drugs cause heart damage. Based on empirical drug limitations about 10-15% of patients will develop congestive heart failure (CHF) with a mortality of -50% within 2 years on digitalo-diuretic therapy alone. To avoid CHF there is a consensus recommendation that cardiac function should be monitored in close connection with anthracycline administration. As no prospective studies in a larger series have been performed, these recommendations are based on retrospective data on small numbers of patients. PATIENTS AND METHODS: In a prospective, blinded observational study 120 patients with advanced breast cancer were followed before, during, and a median 3 years after treatment with epirubicin. They had 604 serial radionuclide measurements of left ventricular ejection fraction (LVEF) that were stored without calculations except in patients who developed a well-defined CHF. RESULTS: Anthracycline cardiotoxicity was closely correlated with the cumulative dose, with a great variability in individual susceptibility and a dramatic increase with advancing age. With a delayed onset of 3 months or more, epirubicin induced a threatening, slowly progressive deterioration of cardiac function continuing years after treatment. An actuarial estimation of 59% of the patients experienced a 25% relative reduction in LVEF 3 years after 850-1000 mg/m2 of epirubicin and 20% had deteriorated into a CHF. The patients did not spontaneously regain cardiac function whereas continued therapy with a circadian angiotensin-converting enzyme inhibitor for more than 3 months caused a remarkably potent and long-lasting recovery. CONCLUSIONS: Due to the displaced cardiotoxic manifestation, functional monitoring in close connection with anthracycline administration appears to be a poorly effective method while later monitoring is essential. Current monitoring recommendations should therefore be revised.

Trimetrexate as biochemical modulator of 5-fluorouracil/leucovorin in advanced colorectal cancer: final results of a randomised European study.

BACKGROUND: Trimetrexate (TMTX) is a biochemical modulator of 5-fluorouracil (5-FU) and leucovorin (LV). Phase II trials have shown promising activity of 5-FU/LV/TMTX in patients with advanced colorectal cancer (ACC). This trial evaluated the effect of TMTX in combination with 5-FU/LV as first-line treatment in ACC. PATIENTS AND METHODS: Patients with ACC were randomised to receive either intravenous LV 200 mg/m2/5-FU 600 mg/m2 or TMTX 110 mg/m2 followed 24 h later by LV 200 mg/m2/5-FU 500 mg/m2 plus oral LV rescue. Both schedules were given weekly for 6 weeks every 8 weeks. Patients were evaluated for progression-free survival (PFS), overall survival (OS), tumour response, quality of life (QoL) and toxicity. RESULTS: A total of 365 patients were randomised. A statistically significant prolongation of median PFS was seen in patients treated with TMTX/5-FU/LV compared with 5-FU/LV (5.4 months versus 4.1 months, respectively; P = 0.03), and a trend towards a significant benefit for OS (13.4 months versus 10.5 months, respectively; P = 0.08). Tumour response, QoL and toxicity were comparable between the two arms. Diarrhoea was the most frequently occurring grade 3 or 4 toxicity (22% and 30%, respectively). CONCLUSIONS: The addition of TMTX to a weekly regimen of 5-FU/LV results in a small but significant improvement in PFS without adding toxicity or worsening QoL in patients with ACC.

Expression of P-glycoprotein and multidrug resistance associated protein in Ehrlich ascites tumor cells after fractionated irradiation.

PURPOSE: To characterize irradiated murine tumor cells with respect to drug resistance, drug kinetics, and ATPase activity, and to evaluate the possible role of P-glycoprotein (PGP) and murine multidrug resistance associated protein (Mrp1) in the drug-resistant phenotype of these cells. METHODS AND MATERIALS: Sensitive Ehrlich ascites tumor cells (EHR2) were in vitro exposed to fractionated irradiation (60 Gy). Western blot analysis was performed for determination of PGP and Mrp1, reverse transcriptase-polymerase chain reaction (RT-PCR) for determination of mdr1a + b mRNA, and semiquantitative RT-PCR for Mrp1 mRNA. The clonogenic assay was applied to investigate sensitivity, whereas the steady-state drug accumulation of daunorubicin (DNR), 3H-vincristine (VCR), and 3H-etoposide (VP16) was measured by spectrofluorometry and scintillation counting, respectively. For determining of ATPase activity, the release of inorganic phosphate from ATP was quantified using a colorimetric method. RESULTS: Compared with EHR2, the irradiated cell line EHR2/irr showed increased expression of PGP (threefold), Mrp1 (eightfold), and Mrp1 mRNA (sixfold), and a slight reduction of mdr1b mRNA, whereas mdr1a was present in EHR2 but could not be detected in EHR2/irr. EHR2/irr developed sixfold resistance to VP16, twofold resistance to vincristine, but remained sensitive to DNR. Addition of the PGP inhibitor, verapamil (VER) or depletion of glutathione by buthionine sulfoximine (BSO) partly reversed the resistance in EHR2/irr. In EHR2/irr, the steady-state accumulation of 3H-VCR and 3H-VP16 was significantly decreased as compared with EHR2, whereas the accumulation of DNR was unchanged. The ATPase activity of plasma membrane vesicles prepared from EHR2/irr cells was similar to that of wild-type EHR2 cells. The ATPase activity was neither stimulated by vinblastine nor VER. CONCLUSION: Irradiation induced a multidrug-resistant phenotype in sensitive tumor cells. This phenotype was characterized by increased expression of Mrp1 mRNA, Mrp1, and PGP but decreased expression of mdr1a + b mRNA. The influence of irradiation on PGP and Mrp1 expression seemed to be different.

A phase II study of oral eniluracil/fluorouracil in patients with anthracycline-refractory or anthracycline- and taxane-refractory advanced breast cancer.

BACKGROUND: Eniluracil is an effective inactivator of dihydropyrimidine dehydrogenase, the initial and rate limiting enzyme in the catabolism of fluorouracil. The current study was done to determine the objective tumour response of a 28-day oral regimen of eniluracil-fluorouracil in patients with advanced breast cancer. PATIENTS AND METHODS: This was a multicentre, phase II study in patients with anthracycline-refractory (AR) or anthracycline- and taxane-refractory (ATR) advanced breast cancer. Oral eniluracil (10 mg/m2) and fluorouracil (1 mg/m2) were taken twice daily for 28 days of each 35-day treatment course. RESULTS: In this study, 106 patients received treatment: 62 patients in the AR stratum and 44 patients in the ATR stratum. The objective tumour response rate in the intent-to-treat population was 18% (95% confidence intervals (CI): 11%-27%), including three complete responses. The response rate was similar in both strata: 19% in the AR and 16% in the ATR stratum. The overall median duration of response was 23.6 weeks. The treatment was well tolerated with a low incidence of grade 3 or 4 toxicities that were considered attributable to study medication. CONCLUSION: Treatment with oral eniluracil-fluorouracil was well tolerated by patients with advanced breast cancer. The efficacy data were comparable with those of other published studies in this group of refractory patients.

Prognostic factors and long-term survival in 585 patients with metastatic breast cancer treated with epirubicin-based chemotherapy.

BACKGROUND: Analysis of prognostic factors in patients with metastatic breast cancer treated with epirubicin-based chemotherapy. PATIENTS AND METHODS: Data from 469 patients treated with epirubicin-based chemotherapy for metastatic breast cancer were used. Prognostic factors were identified (Cox multivariate analysis). A prognostic index was compiled and risk groups were established accordingly. The applicability of the index was investigated in a series of 116 patients. RESULTS: The prognostic factors identified were: liver, pleural, soft tissue, lung and bone metastases, performance status > 2, advancing age, abnormal elevation of serum lactate dehydrogenase and negative/unknown oestrogen receptor status. Four risk groups were established: good, intermediate I, intermediate II and poor. The median and five-year survivals in percentage were: good: 34 months (26%); intermediate I: 19 months (6%), intermediate II: 12 months (0%); poor: 7 months (1%). The corresponding values in the applicability group were: 32 months (23%); 28 months (22%); 18 months (5%); and 6 months (0%). CONCLUSIONS: It is more the number and impact on the organs involved, that predict the patients' survival. The construction of a prognostic index could be helpful in assessing the outlook for patients, especially the quite dramatic difference in long-term survival between the good and poor risk patients.

Characterisation of multidrug-resistant Ehrlich ascites tumour cells selected in vivo for resistance to etoposide.

An Ehrlich ascites tumour cell line (EHR2) was selected for resistance to etoposide (VP16) by in vivo exposure to this agent. The resulting cell line (EHR2/VP16) was 114.3-, 5.7-, and 4.0-fold resistant to VP16, daunorubicin, and vincristine, respectively. The amount of salt-extractable immunoreactive topoisomerase IIalpha and beta in EHR2/VP16 was reduced by 30-40% relative to that in EHR2. The multidrug resistance-associated protein (MRP) mRNA was increased 20-fold in EHR2/VP16 as compared with EHR2, whereas the expression of P-glycoprotein was unchanged. In EHR2/VP16, the steady-state accumulation of [(3)H]VP16 and daunorubicin was reduced by 64% and 17%, respectively, as compared with EHR2. Deprivation of energy by addition of sodium azide increased the accumulation of both drugs to the level of sensitive cells. When glycolysis was restored by the addition of glucose to EHR2/VP16 cells loaded with drug in the presence of sodium azide, extrusion of [(3)H]VP16 and daunorubicin was induced. Addition of verapamil (25 microM) decreased the efflux of daunorubicin to the level of sensitive cells, but had only a moderate effect on the efflux of [(3)H]VP16. The resistant cells showed moderate sensitisation to VP16 on treatment with verapamil, whereas cyclosporin A had no effect. Compared with that of sensitive cells, the ATPase activity of plasma membrane vesicles prepared from EHR2/VP16 cells was very low. Vanadate inhibited the ATPase activity of EHR2/VP16 microsomes with a K(i) value of 30 microM. ATPase activity was slightly stimulated by daunorubicin, whereas vinblastine, verapamil, and cyclosporin A had no effect. In conclusion, development of resistance to VP16 in EHR2 is accompanied by a significant reduction in topoisomerase II (alpha and beta) and by increased expression of MRP mRNA (20-fold). MRP displays several points of resemblance to P-glycoprotein in its mode of action: 1) like P-glycoprotein, MRP causes resistance to a range of hydrophobic drugs; 2) MRP decreases drug accumulation in the cells and this decrease is abolished by omission of energy; and 3) MRP increases efflux of drug from cells. However, compared with that of P-glycoprotein-positive cells, the ATPase activity of MRP-positive cells is found to be low and not able to be stimulated by verapamil.

Characterisation of non-P-glycoprotein multidrug-resistant Ehrlich ascites tumour cells selected for resistance to mitoxantrone.

An Ehrlich ascites tumour cell line (EHR2) was selected in vivo for resistance to mitoxantrone (MITOX). The resistant cell line (EHR2/MITOX) was 6123-, 33-, and 30-fold-resistant to mitoxantrone, daunorubicin, and etoposide, respectively, but retained sensitivity to vincristine. The resistant cells showed moderate sensitisation to mitoxantrone on treatment with verapamil or cyclosporin A. Compared with EHR2, the multidrug resistance-associated protein mRNA was increased 13-fold in EHR2/MITOX. Western blot analysis showed an unchanged, weak expression of P-glycoprotein. Topoisomerase IIalpha was reduced to one-third in EHR2/MITOX relative to EHR2 cells, whereas topoisomerase IIbeta was present in EHR2 but could not be detected in EHR2/MITOX. In the resistant subline, net accumulation of MITOX (120 min) and daunorubicin (60 min) was reduced by 43% and 27%, respectively, as compared with EHR2. The efflux of daunorubicin from preloaded EHR2/MITOX cells was significantly increased. EHR2/MITOX microsomes had a significant basal unstimulated ATPase activity. The apparent K(i) value for vanadate inhibition of the ATPase activity in EHR2/MITOX microsomes was not significantly different from the K(i) value for P-glycoprotein-positive cells. However, whereas verapamil (50 microM) inhibited the ATPase activity of EHR2/MITOX microsomes, it stimulated the ATPase activity of microsomes derived from P-glycoprotein-positive cells. In conclusion, the resistance in EHR2/MITOX was multifactorial and appeared to be associated with: 1) a quantitative reduction in topoisomerase IIalpha and beta protein; 2) reduced drug accumulation, probably as a result of increased expression of a novel transport protein with ATPase activity; and 3) increased expression of MRP mRNA.

Epirubicin or epirubicin and cisplatin as first-line therapy in advanced breast cancer. A phase III study.

PURPOSE: To compare the efficacy and toxicity of epirubicin to that of the combination of epirubicin and cisplatin in patients with advanced breast cancer. PATIENTS AND METHODS: A total of 155 patients were randomized to receive either epirubicin (70 mg/m2) days 1 and 8 every 4 weeks or epirubicin (60 mg/m2) days 1 and 8 plus cisplatin (100 mg/m2) day 1 every 4 weeks. Epirubicin was continued until disease progression or to a cumulative dose of 1000 mg/m2. Cisplatin was discontinued after six cycles. In 45 premenopausal women an oophorectomy was performed. None of the evaluable patients had received chemotherapy for metastatic disease. RESULTS: Among evaluable patients (74 in the epirubicin group and 65 in the epirubicin plus cisplatin group) there were 19% vs 29% complete responses, and 42% vs 37% partial responses, with no significant difference. In the epirubicin plus cisplatin group the response rate was significantly higher in previously untreated patients as compared with patients who had received adjuvant chemotherapy (74% vs 55%, P = 0.002). Median times to disease progression were 8.4 months in the epirubicin group and 15.3 months in the epirubicin plus cisplatin group (P = 0.045). Median survival times were 15.1 and 21.5 months, respectively (P = 0.41). In the epirubicin plus cisplatin group leukopenia and thrombocytopenia were significantly more frequent, 29% of the patients developed mild to moderate peripheral neurotoxicity, 34% reported tinnitus and hearing changes, 6 patients developed nephrotoxicity (one died due to nephrotic syndrome), and 3 patients developed leukaemia (two died of this cause). Congestive heart failure occurred in six patients in the epirubicin group and three patients in the epirubicin plus cisplatin group. CONCLUSION: Cisplatin plus epirubicin is an active, although highly toxic regimen when used as first-line therapy in advanced breast cancer. The time to disease progression was significantly longer in the cisplatin plus epirubicin group (increased by 82%). Due to toxicity, the combination regimen cannot be recommended. However, the study indicated a very high activity of cisplatin in advanced breast cancer. Studies of first-line therapy in advanced breast cancer including cisplatin or other platin derivatives in combination with, for example, the taxanes are suggested.

In vivo electroporation of skeletal muscle: threshold, efficacy and relation to electric field distribution.

In vivo electroporation is increasingly being used to deliver small molecules as well as DNA to tissues. The aim of this study was to quantitatively investigate in vivo electroporation of skeletal muscle, and to determine the threshold for permeabilization. We designed a quantitative method to study in vivo electroporation, by measuring uptake of (51)Cr-EDTA. As electrode configuration influences electric field (E-field) distribution, we developed a method to calculate this. Electroporation of mouse muscle tissue was investigated using either external plate electrodes or internal needle electrodes placed 4 mm apart, and eight pulses of 99 micros duration at a frequency of 1 Hz. The applied voltage to electrode distance ratio was varied from 0 to 2.0 kV/cm. We found that: (1) the threshold for permeabilization of skeletal muscle tissue using short duration pulses was at an applied voltage to electrode distance ratio of 0.53 kV/cm (+/-0.03 kV/cm), corresponding to an E-field of 0.45 kV/cm; (2) there were two phases in the uptake of (51)Cr-EDTA, the first indicating increasing permeabilization and the second indicating beginning irreversible membrane damage; and (3) the calculated E-field distribution was more homogeneous for plate than for needle electrodes, which was reflected in the experimental results.

Epirubicin cardiotoxicity: an analysis of 469 patients with metastatic breast cancer.

PURPOSE: To evaluate the influence of cumulative dose, dose-intensity, single-dose level, and schedule of epirubicin on the risk of developing congestive heart failure (CHF) in patients with advanced breast cancer. PATIENTS AND METHODS: Four hundred sixty-nine consecutive anthracyline-naive patients with metastatic breast cancer were included. Only patients with cardiac failure according to New York Heart Association (NYHA) function class II or more were recorded as having CHF. For each patient, the following were calculated: the cumulative dose of epirubicin, mean dose-intensity (cumulative dose of epirubicin/duration of treatment), and single-dose level (cumulative dose of epirubicin/number of injections). RESULTS: Thirty-four patients (7.2%) developed CHF. The cumulative risk of cardiotoxicity was 4% at 900 mg/m2 and increased exponentially to 15% at 1,000 mg/m2. Irradiation against the mediastinum and thoracic spine increased the risk of CHF (P=.025), but dose-intensity, single-dose level, and schedule had no influence on the risk of developing CHF. Age, previous adjuvant irradiation (to the left or right hemithorax), and previous chemotherapy (cyclophosphamide, methotrexate, and fluorouracil [CMF]) were not risk factors. The median time to onset of CHF following the last dose of epirubicin was 57 days (range, 0 to 853). Among patients with CHF, 13 (38.2%) died of cardiac failure. The median survival time for all patients with CHF was 162 days (range, 0 to +1,957). Previous irradiation directly against the heart increased the risk of death due to cardiac failure and decreased the median survival time to 125 days (range, 0 to 336). CONCLUSION: The present large retrospective study of 469 patients substantiates previous results concerning the cardiotoxicity of epirubicin. A significantly increasing risk of CHF in patients who receive cumulative doses greater than 950 mg/m2 was established. The future recommended maximum cumulative dose of epirubicin should be 900 mg/m2 in patients with metastatic breast cancer. Previous irradiation against the heart leads to an increased risk of developing CHF with an accelerated course to death, which indicates an additive cardiotoxic effect of irradiation and epirubicin.

P-glycoprotein expression in Ehrlich ascites tumour cells after in vitro and in vivo selection with daunorubicin.

Fluctuation analysis experiments were performed to assess whether selection or induction determines expression of P-glycoprotein and resistance in the murine Ehrlich ascites tumour cell line (EHR2) after exposure to daunorubicin. Thirteen expanded populations of EHR2 cells were exposed to daunorubicin 7.5 x 10(-9) M or 10(-8) M for 2 weeks. Surviving clones were scored and propagated. Only clones exposed to daunorubicin 7.5 x 10(-9) M could be expanded for investigation. Drug resistance was assessed by the tetrazolium dye (MTT) cytotoxicity assay. Western blot was used for determination of P-glycoprotein. Compared with EHR2, the variant cells were 2.5- to 5.2-fold resistant to daunorubicin (mean 3.6-fold). P-glycoprotein was significantly increased in 11 of 25 clones (44%). Analysis of variance supported the hypothesis that spontaneous mutations conferred drug resistance in EHR2 cells exposed to daunorubicin 7.5 x 10(-9) M. At this level (5 log cell killing) of drug exposure, the mutation rate was estimated at 4.1 x 10(-6) per cell generation. In contrast, induction seemed to determine resistance in EHR2 cells in vitro exposed to daunorubicin 10(-8) M. The revertant EHR2/0.8/R was treated in vivo with daunorubicin for 24 h. After treatment, P-glycoprotein increased in EHR2/0.8/R (sevenfold) and the cell line developed resistance to daunorubicin (12-fold), suggesting that in EHR2/0.8/R the mdr1 gene was activated by induction. In conclusion, our study demonstrates that P-glycoprotein expression and daunorubicin resistance are primarily acquired by selection of spontaneously arising mutants. However, under certain conditions the mdr1 gene may be activated by induction.

Challenging drug resistance in cancer therapy--review of the First Nordic Conference on Chemoresistance in Cancer Treatment, October 9th and 10th, 1997.

The First Nordic Conference on Chemoresistance in Cancer Treatment was held in the Danish town of Helsingør on October 9th and 10th, 1997, under the auspices of the Nordic Cancer Chemoresistance Group (NCCG). The meeting focused on biochemical chemoresistance in a multidisciplinary approach. There were 19 oral and 15 poster presentations documenting recent advances in experimental and clinical research of drug transport mechanisms, DNA repair systems, detoxifying enzymes, drug target regulation, in vitro sensitivity tests, apoptosis inhibition, and strategies to circumvent chemoresistance. In the present paper we review the main issues that were addressed and discuss the findings with reference to the current literature in the field. The meeting demonstrated the plurality and the complexity of chemoresistance, which is a major obstacle to successful chemotherapy in cancer patients. The new insights to mechanisms of drug resistance and sensitization represent a useful basis for further development of strategies to circumvent chemoresistance in clinical practice.

pH regulation in sensitive and multidrug resistant Ehrlich ascites tumor cells.

Maintenance and regulation of intracellular pH (pHi) was studied in wild-type Ehrlich ascites tumor cells (EHR2) and five progressively daunorubicin-resistant, P-glycoprotein (P-gp)-expressing strains, the maximally resistant of which is EHR2/1.3. Steady-state pHi was similar in cells expressing different amounts of P-gp, in the absence and presence of glucose. In EHR2/1.3, glucose-induced acidification was reduced, and proton efflux was increased, compared to the wild-type EHR2, differences which were not caused by increased activity of a Na+/H+ exchanger in the resistant cells. Comparing all six cell lines, no evidence was found for a correlation between the amount of P-gp in the membrane and pHi regulation, which was also unaffected by P-gp modulators. However, a correlation was seen between relative resistance/daunorubicin accumulation and acid extrusion rate, which is likely to be due to aspects of development of drug resistance other than P-gp.

Enhancement of cytotoxicity by electropermeabilization: an improved method for screening drugs.

Electropermeabilization (EPN), also termed electroporation, is a physical method to overcome the barrier of the cell membrane by applying short and intense electric pulses. It is the basis for a new cancer treatment modality, electrochemotherapy, where uptake of chemotherapeutics is enhanced by EPN. Preclinical and clinical trials have shown that application of electric pulses in vivo is feasible and that electrochemotherapy is highly efficient. The aim of this study was to develop an improved method of screening drugs on electropermeabilized versus non-electropermeabilized cells. In this study we describe an easy protocol which gives high cell viability, good reproducibility and a high rate of cell permeabilization. Cell cytotoxicity is simply determined by the MTT assay. Cell death due to the EPN procedure was less than 4% and more than 90% of cells were permeabilized. For daunorubicin, doxorubicin, etoposide and paclitaxel, no effect of EPN was found. For carboplatin and cisplatin the effect of EPN was a factor 3 and 2.3, respectively, on the IC50 (inhibitory concentration 50%). For bleomycin we found a dramatic effect of EPN of the magnitude of a factor 300 on the IC50. In conclusion, we have established a new, easy and reliable protocol to test new drugs for cytotoxicity with or without the limitations of the cell membrane. Our data support the role of bleomycin as the drug of choice for electrochemotherapy.

ATPase activity of P-glycoprotein related to emergence of drug resistance in Ehrlich ascites tumor cell lines.

We have characterized the ATPase activity of a sensitive and five progressively daunorubicin resistant Ehrlich ascites tumor cell lines passaged in mice. For the nine different modulators of drug resistance that we have studied, the ATPase activity first rose with the modulator concentration and then declined. We analyzed the ATPase activity profiles in terms of an activation constant and an inhibition constant for each of the nine drugs and six cell lines. In this series of cell lines, the drug-stimulatable ATPase activity was directly proportional to the amount of P-glycoprotein. Pumping of daunorubicin was also correlated with the amount of P-glycoprotein, except that, for a highly passaged line more daunorubicin was pumped than could be accounted for by the content of P-glycoprotein. Between the 12th and the 36th passage an additional source of resistance emerged, which was not correlated with P-glycoprotein. Pumping of daunorubicin was negatively correlated with the cell volume for the different lines.

Structure-activity relationships of P-glycoprotein interacting drugs: kinetic characterization of their effects on ATPase activity.

We have determined the kinetic parameters for stimulation and inhibition by 34 drugs of the P-glycoprotein ATPase in membranes derived from CR1R12 Chinese hamster ovary cells. The drugs chosen were sets of calmodulin antagonists, steroids, hydrophobic cations, hydrophobic peptides, chemotherapeutic substrates of P-glycoprotein, and some other drugs with lower affinity for P-glycoprotein. We studied how these kinetic parameters correlated with the partition coefficient and the Van der Waals surface area of the drugs. The maximum velocity of ATPase stimulation decreased with surface area and showed a suggestion of a maximum with increasing partition coefficient. The affinity of these drugs for P-glycoprotein showed no significant correlation with partition coefficient but was highly correlated with the surface area suggesting that binding between modulators and P-glycoprotein takes place across a wide interaction surface on the protein.

Competitive, non-competitive and cooperative interactions between substrates of P-glycoprotein as measured by its ATPase activity.

We have studied the interaction between verapamil and other modulators of the P-glycoprotein ATPase from membranes of CR1R12 Chinese hamster ovary cells. Four major categories of interaction were identified. (i) Non-competitive inhibition of verapamil's stimulation of enzyme activity was found with vanadate. (ii) Competitive inhibition of the ATPase was found for the pair verapamil and cyclosporin A. (iii) Allosteric inhibition with an increase in the Hill number for verapamil was found in the cases of daunorubicin, epirubicin, gramicidin S and D, vinblastine, amiodarone, and colchicine. (iv) Cooperative stimulation of verapamil-induced ATPase activity was found with progesterone, diltiazem, amitriptyline, and propranolol. At high levels, progesterone and verapamil mutually enhanced each other's inhibitory action on the ATPase. Our data show that the substrate binding behavior of P-glycoprotein is complex with more than one binding site being present. This information could form the basis for the development of improved modulators of P-glycoprotein.

Carcino-embryonic antigen in monitoring the growth of human colon adenocarcinoma tumour cells SK-CO-1 and HT-29 in vitro and in nude mice.

A set of experimental model systems were designed to investigate (a) the inter-relationship between growth of two human cancer cell lines (SK-CO-1, HT-29) and carcino-embryonic antigen (CEA) kinetics; and (b) whether neoplastic growth or CEA concentration is modulated by human growth hormone (hGH). We found that increasing CEA concentration depended on tumour burden. SK-CO-1 cells had the lowest growth rates but the highest rates of CEA production. The rate of CEA increase exceeded the growth rate of both SK-CO-1 and HT-29. hGH modulated neither neoplastic growth nor CEA production. In conclusion, our results suggest that experimental models may be useful for investigating the role of serological markers as monitors of increasing tumour burden. It will be of interest to investigate the performance of those model systems in examining the effect of cytotoxic agents in neoplastic growth.