D.J.T. Wagener

Significance of cell proliferation measurement in gastric cancer

Cell kinetic data may be important indicators of clinical behaviour in many types of cancer. Recently, several antibodies to cell-cycle associated antigens have been characterised. This overview summarises the advantages and disadvantages of different methods for the assessment of cell proliferation. Moreover, the prognostic value of proliferative activity in gastric cancer is discussed and suggestions for future research are given.

Pancreatic carcinoma with polyarthritis, fat necrosis, and high serum lipase and trypsin activity.

A 46 year old white man presented with subcutaneous and intramedullary fat necrosis, destructive polyarthritis, and osteolytic bone lesions, complicating a poorly differentiated adenocarcinoma of the tail of the pancreas with metastases in the liver and omentum. There was a 100-fold increase in serum lipase and trypsin activity. His condition deteriorated rapidly, was characterised by rapid tumour growth, formation of ascites, a 20 kg weight loss, extensive subcutaneous fat necrosis, and fistula formation in the left calf. Treatment with 5-fluorouracil 300 mg/m2 on days 1-5 and doxorubicin 50 mg/m2 and cisplatin 100 mg/m2 on day 1, every three weeks, was well tolerated and resulted in rapid clinical improvement. After three courses of treatment a partial remission was seen and after seven courses further improvement occurred with a return to normal of serum lipase and trypsin activity. One year after starting chemotherapy the tumour relapsed but responded again to chemotherapy (epirubicin 40 mg/m2 and carboplatin 300 mg/m2 on day 1, every three weeks).

Altered intracellular enzyme activity of monocytes and lymphocytes in Hodgkin's disease

To evaluate metabolic functionality of monocytes and lymphocytes in Hodgkins disease (HD) we studied 3 enzymes of the intermediary metabolism, G-6-PDH, PHI, ICDH, and the acid hydrolases, NAG and ACP. These enzymes were measured in purified cell fractions of 9 patients with advanced disease and 11 normal controls. The cells were isolated with cell scatter-monitored counterflow centrifugation. Enzymes were measured in the cell lysates by means of fluorimetric microassays. In the monocytes of HD patients a significantly increased G-6-PDH activity was found (P less than 0.01), indicating an enhanced activity of the hexose monophosphate shunt. The other enzymes showed no clear differences compared to normal controls. The lymphocytes of HD patients showed a significantly augmented activity of both G-6-PDH (P less than 0.001) and PHI (P less than 0.01), pointing to an increased HMPS and glycolytic activity. These findings are in support of an enhanced metabolic activity of both monocytes and lymphocytes in HD.

The effect of ethyldeshydroxy-sparsomycin and cisplatin on the intracellular glutathione level and glutathione S-transferase activity

Ethyldeshydroxy-sparsomycin (EdSm) is a ribosomal protein synthesis inhibitor which synergistically enhances the antitumor activity of cisplatin against L1210 leukemia in vivo. Because cellular glutathione (GSH) and glutathione S-transferases (GST) are reported to interfere with the antitumor activity of cisplatin, we analyzed the effect of EdSm and cisplatin on GSH and GST activity in selected tumor cells. For this purpose we used three murine leukemia tumors with different sensitivities towards EdSm and cisplatin: L1210-WT, sensitive to both drugs, L1210-Sm, resistant to EdSm, and L1210-CDDP, resistant to cisplatin. No significant differences were detectable between these three cell lines regarding the population doubling time, the cell size, and the cellular level of protein and glutathione. Neither of the resistant L1210 subclones showed P-glycoprotein expression. Drug exposure, however, changed the intracellular dynamics. Exposure to EdSm strongly decreased the amount of cellular protein, decreased the overall GST activity and led to GSH depletion, whereas exposure to cisplatin induced a rise in the amount of protein, in GSH, and in the total GST activity. These effects are dose-dependent and correlate well with the sensitivity of the tumor cells for EdSm or cisplatin. In addition, exposure to EdSm lowered the V(max) of GST in L1210-WT and L1210-Sm; however, in L1210-CDDP both the V(max) and the K(m) were increased. That this was not a direct effect of EdSm on GST was shown in a cell-free system, where EdSm did not influence the GST activity nor could it act as a substrate for GST. Our results suggest that the synergistic combination of EdSm and cisplatin might be explained by EdSm switching off the cellular detoxification mechanism for cisplatin, i.e. by inhibition of de novo synthesis and subsequent depletion of GSH and GST.

Preclinical antitumor activity of ethyldeshydroxysparsomycin in combination with cisplatin

SummaryThe efficacy of cisplatin (CDDP) in combination with the protein synthesis inhibitor ethyldeshydroxy-sparsomycin (EDSM) has been tested in two tumor models at various schedules. Mice with L1210 leukemia or B16 melanoma were treated with CDDP alone or in combination with EDSM. Against L1210 leukemia, which is sensitive to CDDP, combinations elicited increases in life-span for all treatment schedules compared to those achieved with the corresponding dose of CDDP. Moreover, the combination of EDSM with this platinum compound yielded a cure rate >80%, compared to <35% for single CDDP treatment. Although the B16 melanoma is rather resistant to both CDDP and EDSM, combinations of these agents against B16 melanoma showed schedule dependent efficacy and in certain schedules significant therapeutic advantage over individual drug treatment, but cures were not observed.Our results suggest that EDSM has significant synergistic capabilities in both animal tumor models, but strong therapeutic enhancement of cisplatin efficacy is only seen when the tumor is sensitive to CDDP.

ANTITUMOUR ACTIVITY AND RETINOTOXICITY OF ETHYLDESHYDROXY-SPARSOMYCIN IN MICE

The colony formation in agar of human tumour xenografts was used as a test system to study the cytostatic activity of ethyldeshydroxy-sparsomycin (EdSm) at the cellular level. EdSm was additionally studied in vivo in human tumour xenografts and murine tumour models. EdSm showed a clear dose-response effect in vitro. At continuous exposure with 0.01 micrograms/ml, 2 out of 11 of the tumours responded (a gastric and a small cell lung carcinoma). At 0.1 mu/ml EdSm, the tumour response was 5/11 tumours and at 1 microgram/ml the compound was active in all tumours. The maximal tolerable doses of EdSm in vivo have been determined in non-tumour bearing CDF1 mice. In the intraperitoneally (i.p.) given multiple dose schedules the respective LD10 doses indicated that the tolerable cumulative dose increases when lower doses are given more frequently. This also enhances the antitumour activity in L1210 leukaemia to 172% T/C. On the other hand, continuous infusion strongly diminished the tolerable dose as well as the antitumour activity. EdSm was also active against i.p. inoculated P388 leukaemia (150% T/C), B16 melanoma (156% T/C), and RC carcinoma (197% T/C), and the subcutaneously (s.c.) inoculated L1210 (139% T/C) and RC (138% T/C). Absence of tumour responses was found in the following s.c. implanted murine tumours: M5076 sarcoma, osteosarcomas C22LR and CP369, and the LL carcinoma, as well as in the human tumour xenografts: LXFG 529, a non-small cell lung carcinoma; GXF 251, a gastric carcinoma; and FMa, an ovary carcinoma. Possible long-range retinotoxic effects of EdSm were investigated in tumour-bearing mice, cured after surviving treatment with LD50 doses of EdSm, by assaying the protein biosynthetic capacity of the retinal by assaying the ocular rhodopsin and opsin levels as parameters. In none of these cases could a significant reduction in either opsin or rhodopsin levels be measured and no changes were seen histologically.

Schedule-dependent enhancement of antitumor activity of ethyldeshydroxy-sparsomycin in combination with classical antineoplastic agents

The efficacy of the protein synthesis inhibitor ethyldeshydroxy-sparsomycin (EDSM) as a biochemical response modifier of several antitumor agents against L1210 leukemia and B16 melanoma is described. Seven drugs with different intracellular targets were selected for this combination study. Tumor implantation and drug treatment were both i.p., and the time interval between the administration of EDSM and the cytostatic agent was varied. Our results show that in the B16 tumor model EDSM is not able to potentiate any of these drugs, whereas antagonism is seen in combination with doxo-rubicin (DX). In the L1210 tumor model, however, no loss of activity is seen for this specific combination. The effect of the combination of cytosar (Ara-C), 5-fluorouracil (5-FU) or vincristine (VCR) with EDSM in the L1210 model is strongly time interval dependent. Loss of 5-FU antitumor activity is seen when EDSM is given 3 or 24 h after 5-FU; however, no effect is observed when EDSM is given 6 h after 5-FU. Enhancement of the 5-FU activity is not noticed. The VCR activity is potentiated when EDSM is given at least 6 h after VCR administration, which increases the antitumor response from 32 to > 60 days and the percentage survivors from 33 to 83% (p = 0.04). In combination with Ara-C, potentiation of antitumor activity is seen only when EDSM is given 24 h after Ara-C, which increases the antitumor response from 32 to > 55 days and the percentage survivors from 11 to 50% (p = 0.008). No modulatory effects are found when EDSM is combined with carmustine or DX. Our results suggest that EDSM changes the antitumor efficacy of selected antitumor agents (Ara-C and VCR) in a schedule-dependent way and that potentiation is largely restricted to cell-cycle phase-specific cytostatic agents.

Correlation of the in vitro cytotoxicity of ethyldeshydroxysparsomycin and cisplatin with the in vivo antitumour activity in murine L1210 leukaemia and two resistant L1210 sublcones

SummaryThe cultured murine leukaemia L1210 cell populations used in the present study were derived from L1210 cells that had been grown in vivo. Subclones resistant to sparsomycin (L1210/Sm) or cisplatin (L1210/CDDP) were also developed in vivo. The doubling times of the cultured cell populations were identical. Fractions surviving after durg treatment in vitro were determined by colony formation in soft agar. The results, based on the differential sensitivity of the cell populations to ethyldeshydroxysparsomycin (EdSm) and CDDP, indicated that after a short exposure, cultured L1210/CDDP cells were cross-resistant to EdSm. L1210/Sm cells, however, were not cross-resistant to CDDP. The results obtained in cultured cell populations were confirmed in vivo. CD2f1 mice bearing i.p. implants of 1×105 tumour cells were given EdSm or CDDP and a combination of the two agents. Drugs were given once daily every 4 days for 3 doses starting at 24 h after tumour implantation. Treatment of mice bearing L1210/wt leukaemia with combined EdSm and CDDP caused strongly synergistic amtitumour activity. In animals bearing the two resistant subclones, however, combined drug treatment did not improve the antitumour activity. The corresponding median survival of mice receiving combined drug treatment was 60 days in each group containing 6 mice bearing L1210/wt, with 4–6 cures being noted; 19 days in animals harbouring L1210/Sm, with 2 cures being recorded among 6 mice; and 11 days in mice bearing L1210/CDDP, with no cure being obtained. The results of this study indicate that the synergism resulting from combined treatment with CDDP and EdSm is a function of the cellular properties of the target tumour-cell populations and is independent of host factors.