Antoinette Koolemans-Beynen

Growth suppression of human breast carcinoma cells in culture by N-(4-hydroxyphenyl)retinamide and its glucuronide and through synergism with glucarate

The inhibitory effects of N-(4-hydroxyphenyl)retinamide (HPR) and its glucuronide derivative on the growth of MCF-7 human breast cancer cells in vitro were compared. The results indicate that the glucuronide had slightly greater potency and much less cytotoxicity than the free retinoid. At a concentration of 10(-6) M, HPR inhibited MCF-7 cell growth by approximately 25%, whereas an equimolar concentration of the glucuronide caused a 40% growth inhibition. Higher concentrations of HPR were highly cytotoxic. At a 10(-5) M concentration of the glucuronide, cell viability was 77%, and 65% of the cells were able to resume growth. On the other hand, at 10(-5) M HPR, cell viability dropped to 49%, and only 15% of the cells were capable of resuming growth. The lower cytotoxicity and higher potency of the retinoid glucuronide compared to the parent retinamide suggest that the conjugate may have a chemotherapeutic advantage over the parent compound. The apparent higher efficacy of HPR in combination with glucarate (GT) compared to the single agents could be due to increased net formation of HPR glucuronide conjugate following conversion of GT to the beta-glucuronidase inhibitor, D-glucaro-1,4-lactone. However, HPLC analysis of the cell metabolites did not show any detectable levels of the retinoid glucuronide upon treatment of MCF-7 cells with HPR and GT.

Synergistic interaction between 13-cis-retinoic acid and glucarate: activity against rat mammary tumor induction and MCF-7 cells

At high dietary levels in vivo, both 13-cis-retinoic acid and calcium glucarate inhibit the induction of rat mammary tumors by 7,12-dimethylbenz(a)anthracene. The present study shows that sub-optimal dietary levels of each, which individually have no effect on tumor induction, when combined together in the diet, significantly increases tumor latency and suppresses tumor frequency in the rat system. Weight gain of animals was similar in control and experimental groups. Furthermore, ineffective sub-optimal dosages of glucarate and 13-cis-retinoic acid interacted synergistically to inhibit the growth in vitro of the MCF-7 human breast cancer cells. By varying the concentrations of glucarate and 13-cis-retinoic acid independently, evidence was obtained that in combination glucarate may play an adjuvant role, with the retinoid as the effector. Thus, the results of this experimental animal study demonstrate for the first time the potential use in synergistic combination of 2 normal metabolites in non-toxic chemoprevention and chemotherapy.

Histocultures of patient head and neck tumors for pharmacodynamics studies.

This investigation was to establish a clinically relevant experimental model to evaluate the pharmacodynamics of drugs used for head and neck cancers. A total of 83 surgical samples of primary and lymph nodal metastatic tumors was obtained from 66 patients. Fragments of these tumors were cultured on a collagen gel matrix. The tumor cell labeling index (LI) was determined by [3H]thymidine incorporation and autoradiography. Seventeen tumors (20%) were contaminated. About 80% of the remaining 65 tumors were successfully cultured for at least 2 weeks. The cultured tumor fragments retained the morphology and architecture of the freshly removed specimens; both tumor and stromal cells were present. The tumor cell LI after 2–3 weeks in culture, determined from the most proliferative area of the tissue, averaged 77 ± 12% for primary tumors and 78 ± 12% for nodal metastases. The activity of three clinically active agents, 5-fluorouracil (FU), cisplatin (DDP), and mitomycin C (MMC), was evaluated in 47 tumors. All three drugs inhibited the tumor LI. The concentrations needed to produce a 50% inhibition of the tumor LI (IC50) were within the clinically achievable concentration range. The intertumor variation in the IC50 for FU (60-fold) was considerably greater than that for DDP and MMC (7- to 8-fold). The nodal metastatic tumors appeared to be less sensitive to FU than the primary tumors, while there were no apparent differences for DDP or MMC. Tumors from patients previously treated with chemotherapy and/or radiotherapy appeared less sensitive to FU and DDP than tumors from untreated patients, but the differences were not statistically significant. Interestingly, tumors from previously treated patients were significantly more sensitive to MMC than tumors from untreated patients. The maintenance of the morphology of the fresh tumor and the observed intertumor variability in IC50 values indicate the preservation of intra- and intertumor heterogeneity in the histocultures. In summary the viability and high success rate of growth of human head and neck tumors in organ-like culture and the chemosensitivity of the cultured tumors to clinically active agents at clinically achievable concentrations support the use of this experimental model for pharmacodynamic evaluation.

Expression of an oncofetal protein (OFP) in rat and human leukemia cells

A unique oncofetal protein (OFP) previously identified in rat fetal tissue and rat and human solid tumors, is now shown to be present in rat and human leukemia cells by use of a monoclonal antibody-based assay. Using a highly specific anti-rat OFP monoclonal antibody OFP has been unquivocally immunolocalized to the cytoplasm of the rat leukemia cells. The factor is rapidly released to the circulation as 50 and 55 kD species which share the immunological determinants. When leukemia cells are transplanted to normal rats, OFP increases in the circulation in a biphasic manner which may be due to immune clearance since circulating anti-OFP antibodies have been demonstrated. Induction of differentiation in the human HL-60 leukemia cell line by 13-cis-retinoic acid caused a down regulation of OFP synthesis, both intra- and extra-cellular levels dropping to essentially zero. Induction of differentiation with dibutyryl cyclic AMP caused a cessation of secretion of OFP, with a marked increase in its intracellular concentration, a condition resembling the retention in fetal cells. Leukemia cells add to a growing list of tumors previously shown to produce OFP, suggesting that OFP is intimately involved in some facet of tumorigenesis.

Impact of metastases on nodal immunoreactivity in head and neck cancer.

It has been previously demonstrated by the authors that lymph nodes from patients with head and neck cancer are capable of regional immunoreactivity and that this immunoreactivity could be enhanced with certain nonspecific immunostimulants. However, it is unknown how metastases to the neck nodes would affect this immunoreactivity.

Antitumour synergism between non-toxic dietary combinations of isotretinoin and glucarate

Dietary calcium glucarate (CGT) increased the activity of non-toxic levels of dietary isotretinoin against pre-established tumors in the chemically-induced rat mammary tumour model. In the range of 1.0-1.5 mmol/kg diet, isotretinoin enhanced tumour growth by 20% over a 4 week course of treatment. Tumour growth inhibition not exceeding 15% was observed only at dosages as high as 2.0 mmol/kg, i.e. in the cumulative toxicity range. Growth inhibition by 64 mmol/kg diet of CGT alone was marginal, varying from zero to 8%. In contrast, the combination of 1.0 mmol/kg of isotretinoin and 64 mmol/kg of CGT caused a reversible inhibition of tumour growth, culminating in a net decrease in tumour volume of 20%. This study documents the marginal enhancement of tumour growth by high sub-optimal concentrations of isotretinoin alone, and describes conditions for inhibition of tumour growth by sub-optimal concentrations of the natural retinoid. Related in vitro studies on retinoid sensitive and insensitive cell lines suggest that the anticancer activity of the combination is dependent on sensitivity of the cells to retinoids.

An improved human tumor stem cell assay in ovarian cancer

To evaluate the effect of improved growth rates of ovarian cancers in the human tumor stem cell assay and its value in predicting clinical chemotherapy response, we studied 59 assays in 54 patients. A total of 81.6% of solid specimens and 85.7% of ascites specimens were successfully cultured and yielded an overall growth rate of 82.9%. Simultaneous primary and metastatic cultures were concordant for chemosensitivity in 80% (n = 16). The patients were evaluated for previous chemotherapy, residual volume of tumor, histologic type, and grade, and these were not statistically different between clinical responders and nonresponders. In vivo-in vitro correlations were made in 27 patients and yielded a predictive response of 13% and predictive resistance of 86% at 70% colony inhibition and 31% and 71% at 50% colony inhibition. Improved growth rates therefore did not result in better predictive correlations. The reported experience in ovarian cancer is summarized and the current status of the human tumor stem cell assay is reviewed.

Effects of 5-fluorouracil, leucovorin, and glucarate in rat colon-tumor explants

SummaryIn a previous study, we showed that 5-fluorouracil (FU) is active against the dimethylhydrazine-induced colon tumor in rats; a 7-day infusion of FU at 30 mg/kg daily produced 85% tumor-free cures. The present study examined the effects of FU alone and in combination with leucovorin (LV) ord-glucarate (GT) using an ex vivo system that maintained the growth of the rat colon-tumor explants on collagen gels. The labeling index (LI) was determined by the incorporation of [3H]-thymidine and autoradiography. The mean LI of the untreated control was 64.8%±19.8%. The IC50, IC90, and IC95 values following a 7-day exposure to FU were 0.36, 0.75, and 1.22 μm, respectively. In comparison, the steady-state FU concentration required to produce 67% tumor-free cures in rats following a 7-day infusion is 1.54 μm. LV alone did not produce any antiproliferative effect at concentrations as high as 10 μm. The addition of LV at concentrations of 0.001–10 μm did not significantly reduce the IC50 of FU. The lack of effect of LV may have been due to tissue saturation with folate provided in the culture medium. GT alone reduced the tumor LI by 20%–30% at concentrations of 0.1–10 μm. GT enhanced the effect of FU. As compared with FU alone, the addition of GT at concentrations of 0.1 and 1.0 μm reduced the IC50 of FU by 47% and 60% to 0.21 and 0.16 μm, respectively. Assessment of the potentiation of the inhibitory effect of FU by GT using two-way analysis of variance and the isobologram method indicated a significant synergistic interaction between FU and GT. This interaction occurred within the FU concentration range of 0.08 and 0.4 μm. In summary, these data indicate that (a) the IC values for FU are comparable in tumor explants and in rats, suggesting that the effects in cultured tumors reflect those in intact animals; (b) GT alone showed antitumor activity, albeit relatively minor as compared with FU; (c) FU and GT exhibited synergistic activity, which was most pronounced at FU concentrations that produced submaximal activity (<30% inhibition of tumor LI); and (d) GT and LV had different effects on the growth inhibition by FU, suggesting that GT acts by a mechanism different from the thymidylate synthase-directed effect of FU and LV.