Multicellular resistance: a paradigm for clinical resistance?

Research on resistance to cancer treatment was mainly focused for 20 years on multidrug resistance (MDR). No useful method of reversing MDR, suitable for clinical use, has yet emerged from this large quantity of work. The reason could be an inadequate evaluation of the target. When grown in spheroids, cancer cells exhibit a phenomenon known as 'multicellular resistance' (MCR). Tumours in patients seem to present the same characteristics. The mechanisms underlying MCR can be classified into two forms: contact resistance and resistance inherent in the spheroid structure. Mechanisms of MCR include: inhibition of apoptosis, high proportion of quiescent cells, modulation of protein expression (including topoisomerases and repair enzymes), potential permeability problems, presence of a hypoxic and necrotic centre and other possible mechanisms that remain to be discovered. A new therapeutic class of drugs is required to overcome MCR. Compounds, which are able to disrupt communication and binding between tumour cells and their microenvironment, seem to be able to circumvent MCR. Interesting results are obtained in vitro and in vivo in mice with specific antibodies or peptides recognised by cell binding proteins. Interestingly, these compounds also appear to be able to inhibit metastasis. Hyaluronidase has already been used with anticancer drugs in patients and was shown to increase drug potency. The explanation given is that it improves drug penetration into spheroids. We now hypothesise that hyaluronidase, in fact, decreases MCR and thus could be the first member of a new therapeutic class.

The effect of 12-alkoxy modification on the in vitro antileukaemic activity of N-methyl 2,3,8,9-tetramethoxybenzo[c]phenanthridinium salts.

Some members of a series of 12-alkyloxy benzo[c]phenanthridines are potent inhibitors of the growth of P388 tumour cells in vitro, with a strong dependence on the nature of the 12-substituent. Analogues with a quaternary nitrogen in the side chain bind strongly to DNA but are less active against the tumour cells. The multi-drug-resistant cell line Pr8/22 shows less sensitivity to the new compounds. K562 Human leukaemia cells undergo differentiation in the presence of the benzo[c]phenanthridine derivatives with a structure-activity relationship which does not correlate well with potency against the P388 cell line.

[Multicellular resistance: another mechanisms of pleiotropic resistance?]. / La résistance multicellulaire: un autre mécanisme de résistance pléiotropique?

Cells cultured as spheroids present an heterogeneity similar to that of tumours in vivo. In the spheroid peripheral layers, cells are proliferating, deeper cells are non-cycling, when in the aggregate centre, cells from often a necrotic core. A multicellular resistance appears in spheroids, it is a result of the cell contact to other cells (homogeneous or heterogeneous cells) and/or to the extracellular matrix. The mechanism of this resistance is not known, nevertheless, it can be hypothesised to be linked to the spheroid centre hypoxia, to the quiescence of a large fraction of the cell population and to the apoptosis inhibition due to the cell contact. The "classical" or "unicellular" mechanisms of resistance, as mdr1, MRP, can coexist with the multicellular resistance, but are not responsible for this resistance. The spheroid model of culture is a good opportunity to study a resistance type which looks close to the tumour resistance found in vivo in mice and in patients. A new class of therapeutic molecules appears that can reverse this multicellular resistance, inhibit tumours growth and preclude metastases. The principal mechanism of action of this new pharmacological class appears to be the disruption of the intercellular adhesion forces. Preliminary results obtained with these compounds in patients are promising.

Multicellular resistance: another mechanism for multidrug resistance?

Cells cultured as spheroids present an heterogeneity similar to that of tumours in vivo. In the spheroid peripheral layers, cells are proliferating, deeper cells are non-cycling, when in the aggregate centre, cells form often a necrotic core. A multicellular resistance appears in spheroids, it is a result of the cell contact to other cells (homogeneous or heterogeneous cells) and/or to the extracellular matrix. The mechanism of this resistance is not known, nevertheless, it can be hypothesised to be linked to the spheroid centre hypoxia, to the quiescence of a large fraction of the cell population and to the apoptose inhibition due to the cell contact. The classical or unicellular mechanisms of resistance, as mdr1, MRP, can coexist with the multicellular resistance, but are not responsible for this resistance. The spheroid model of culture is a good opportunity to study a resistance type which looks close to the tumour resistance found in vivo in mice and in patients. A new class of therapeutic molecules appears that can reverse this multicellular resistance, inhibit tumours growth and preclude metastases. The principal mechanism of action of this new pharmacological class appears to be the disruption of the intercellular adhesion forces. Preliminary results obtained with these compounds in patients are promising.

HL60 cells exhibit particular ultrastructural features during all-trans retinoic acid-induced apoptosis.

Ultrastructural features induced by 1 micromol/l all-trans retinoic acid (ATRA) treatment of HL60 cells were observed with transmission electron microscopy. Whilst some cells were unaffected, most of the others underwent granulocytic differentiation, starting point for apoptosis and then, possibly, secondary necrosis. First steps of apoptosis led to nuclear fragmentation into dense bodies. Then three different ways were observed: i) cells shrank and dense bodies were expelled, mostly associated with a fine lamellae of cytoplasm, ii) a secondary necrosis involved the release of apoptotic bodies without cytoplasm or iii) cells showed a dual or bipolar structure, not previously described.

Cofactors in in vitro induction of apoptosis in HL60 cells by all-trans retinoic acid (ATRA).

The aim of this study was to determine the culture conditions that could modulate the induction of apoptosis by all-trans retinoic acid (ATRA). Cell viability was evaluated by trypan blue test, differentiation by nitro blue tetrazolium test, and apoptosis by morphological analysis. ATRA induced apoptosis in HL60 cells only when more than 100,000 cells/mL were seeded, while differentiation was induced regardless of the seeded cell concentration. Reduction in the concentration of foetal calf serum or glutamine in the medium led to a weak increase in apoptosis. In contrast, a dramatic enhancement of apoptosis occurred when the culture medium was supplemented with glucose or when the culture pH was decreased. These characteristics were independent of the mechanism of action of ATRA, but the action of glucose could be of significance in diabetic patients. An exchange of supernatants after 3 days of culture showed that supernatants from control cultures seeded at high cell density were better apoptosis inducers than supernatants from cultures treated with ATRA, but seeded at low cell density. Factor(s) in this supernatant which induced apoptosis was (were) removed by ultrafiltration. In conclusion, our results showed that ATRA alone cannot induce apoptosis, but can do so in conjunction with cofactors. The depletion of some components of the medium and the appearance of secreted macromolecule(s) could be cofactor(s) in the induction of apoptosis.

Cell culture as spheroids: an approach to multicellular resistance.

Cells cultured as spheroids present an heterogeneity similar to that of tumours in vivo. In the spheroid peripheral layers, cells are proliferating, deeper cells are non-cycling, when in the aggregate centre, cells form often a necrotic core. A multicellular resistance is related on the cell contact to other cells or to the extracellular matrix. The mechanism of this resistance remains unknown. It seems to be linked to the spheroid centre hypoxia, quiescence of a large fraction of the cell population and to the apoptose inhibition. The "classical" or "unicellular" mechanisms of resistance, as mdr1, MRP, can coexist but are not responsible of this type of resistance. This culture model is a good opportunity to study a resistance which looks close to the patient tumour resistance. A new class of therapeutic molecules appears that can reverse multicellular resistance, inhibit tumours growth and preclude metastases. The mechanism of action of this new pharmacological class is the disruption of the cell adhesion forces.

Puzzles in the clinical pharmacokinetics of fluorouracil.

The pharmacokinetics of fluorouracil (5FU) were studied in two groups of patients, the administration of 105 i.v. as daily bolus (x5) or 5-day continuous infusions. The 5FU pharmacokinetics were extremely variable from day to day, i.e. from one bolus to the next or during the continuous infusion, especially in some patients. The variations were lower for the daily bolus, but still remained high. The pharmacokinetics of cisplatin, given simultaneously during continuous infusions did not show the same variability; therefore the variability could be specific for 5FU. The role of implantable subcutaneous ports as the most probable source of this extraordinary variability is discussed. We hypothesise that in some patients the implantable subcutaneous ports used for 5FU infusion, could cause transient and extremely high plasma concentrations, exacerbated by the very short half life of the drug and by saturation of its catabolism.

[In vivo evaluation of potential doubling time (Tpot) of tumors by flow cytometry: practical difficulties and possible solutions]. / Evaluation du temps potentiel de doublement des tumeurs in vivo (Tpot) par cytométrie en flux: difficultés pratiques et solutions possibles.

Kinetic parameters of tumor growth yield predictive values and allow an optimisation of the treatment schedule, especially for fractionation in radiotherapy. Among those parameters, the labelling index (LI) and the potential doubling time (Tpot) may be measured by flow cytometry, a semi-quantitative analysis, after in vivo administration of iododeoxyuridine (IdUrd) to humans. This Begg's recommended methodology needs the selection of thresholds and gates whose boundaries are arbitrary. They can be positioned on a more objective basis using a negative control (aspecific fixation). Moreover a previous identification of the different cell populations of the tumour samples according to the method of Vindelov allows a better determination of these cell populations processing IdUrd-DNA staining. This procedure was used with 11 tumour biopsies including mainly head and neck cancers. This method displayed results similar to the literature concerning LI and Tpot determinations as well as shortened Tpot when the patients recurred. One sample has no labelling at all. A small fraction, likewise up to 10% of cells exhibiting on S DNA content were not labelled by IdUrd. These cells leave the S phase or progress too slowly in order to display IdUrd uptake. Intra-tumoral hypoxia is a possible explanation of these findings. DNA ploidy and the percentage of cells in S phase could be worth while to precise the relationship between DNA index and tumour kinetic. The measurement of Tpot could also be used in other cancers and for optimisation of dose of chemotherapy.

Evaluation of a prediction model of cisplatin dose based on total platinum plasma concentration.

The aim of this study was to validate prospectively a model of cisplatin dose adjustment. 27 patients (63 courses) with lung cancer were treated by a 5 day continuous infusion of cisplatin and etoposide. The dose of cisplatin was adjusted in order to reach a target plasma concentration of total platinum (TP) of 2000 mu/l at the end of the infusion. The target concentration was reached with a mean bias of 2.7% and a precision of 7.8%. The results were compared with those of a population of 38 patients (97 courses) with lung cancer and treated with the same protocol of chemotherapy, but without dose adjustment. The average dose adjustment was an increase of cisplatin dose of 20.2%. This augmentation was most important during the first course, decreasing during the following courses. There was also an increase in the etoposide AUC, although its dose was not modified. Toxicity to polymorphonuclear cells was significantly increased and was linked to etoposide AUC.

Evaluation of two dose individualisation methods for carboplatin.

The pharmacokinetics of carboplatin are usually evaluated by measuring plasma concentrations of ultrafiltered platinum (UP). This approach, however may be less reliable than measuring the plasma concentration of total platinum (TP). In a group of 14 patients, which constituted a reference group, the clearance of TP was highly correlated with creatinine clearance, as estimated by the method of Cockroft and Gault. This relationship, together with only morphological and biological parameters, was used to estimate TP clearance, Vc and AUC, in a validation group of 8 patients. Estimated TP clearance was 97.9 +/- 18% of the actual value. The TP pharmacokinetic parameters of the reference group were used to estimate those of the validation group, using only two or three plasma concentration measurements (Bayesian approach). With the Bayesian approach, the estimated TP clearance was up to 99.9 +/- 2.7% of the actual value. In conclusion, estimation of TP pharmacokinetics may be reliably estimated as an alternative to UP in clinical practice.

Prognostic significance of routine clinical and laboratory data in advanced head and neck cancers.

Predictive factors for toxicity and response to chemotherapy in patients with advanced head and neck cancer are seldom reported. Therefore, from a short series of patients with a histologically proven cancer, who were treated by a neo-adjuvant protocol with cisplatin and fluorouracil, routine clinical and laboratory data were investigated. ALT (alanine aminotransferase) and Hb (hemoglobin) appeared to be predictive for efficacy. By multivariate analysis (principal component analysis), these laboratory data were involved in two independent axes: one which was considered as "inflammatory" and the other as "hepatic". Initial obesity indices were associated with the former. The predictive variables for toxicity (i.e. age, serum creatinine level, weight loss and plasma cisplatin) were probably biased in this series. Nevertheless cisplatin concentration regularly increased in each cycle. Hence it was dependent on the rank of the course. According to this preliminary study, it would be of interest to conduct future investigations on acquired protein-energy malnutrition, as well as on selected soluble mediators of cellular and humoral immune response.

Comparison of two dose prediction models for cisplatin.

Cisplatin toxicity could be decreased by adjusting its dosage to each patient. For this purpose, a limited sampling method was established and validated based on a Bayesian approach taken using the values of assays during a 5-day continuous infusion of cisplatin. Using this method, a dosing model to achieve a target plasma concentration of total platinum (Pt) was evaluated retrospectively; the calculated dose of cisplatin was 95.0 to 104.8% of the actual dose. This model was then studied prospectively and the actual plasma Pt concentration reached at the end of the infusion was 94.9% of the target concentration. A strong correlation was observed between the clearance of Pt and the calculated clearance of creatinine or Cockroft index (p = 1.7 x 10(-11), and this correlation was used to develop another cisplatin dosing model. With this model the actual concentration reached at the end of the infusion was 85.3% of the theoretical concentration. The Bayesian approach gave reliable results for most clinical uses, whereas the creatinine based model has to be improved.

Anticancer drug resistance and inhibition of apoptosis.

Apoptosis is a new concept which could be of great importance in the understanding and treatment of cancer. An important feature is the discovery of inhibitors of apoptosis, because they induce resistance to chemotherapeutic drugs and irradiation. Bcl-2 is the most well known of these apoptosis inhibitors. When it is overexpressed cells are less sensitive to cytotoxic drugs; on the contrary, when it is underexpressed they are more sensitive. Clinically, bcl-2 expression is associated with a poor prognosis in several cancers. Bcl-2 protein, p26-bcl-2, is located in the outer mitochondrial membrane, the nuclear envelope and the smooth endoplasmic reticulum. P26-bcl-2 is an antioxidant; this property could explain the anti-apoptotic activity since peroxides seem to be important mediators of apoptosis. Bcl-2 antisense oligonucleotides are able to reverse the apoptosis inhibition. New cancer treatments should take into account the expression of bcl-2.

Culture conditions modulate the effects of aclacinomycin A on growth, differentiation and apoptosis of HL60 cells.

The continuous incubation for several days of HL60 cells, in exponential growth, with aclacinomycin A (ACM) induces growth inhibition, necrosis, differentiation and apoptosis. Differentiation and apoptosis were assessed by optical microscopy (OM) and flow cytometry (FCM). ACM displayed dose-dependent effects, except for the differentiation induction, which was biphasic. Differentiation and apoptosis could also be induced after a 1 h ACM exposure only. The poor reproducibility of apoptosis induction led us to study the culture conditions described in the literature (without renewing the medium) where control cells are not growing exponentially during the 5 day incubation period. During kinetic studies with different ACM concentrations, the differentiation was detected earlier by FCM than by OM, while it was not the case for apoptosis. This induction appeared more reproducible when non optimal conditions of culture were used.