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Tamoxifen and trifluoroperazine (Stelazine) potentiate cytostatic/cytotoxic effects of P-30 protein, a novel protein possessing anti-tumour activity

Tamoxifen and trifluoroperazine (Stelazine) potentiate cytostatic/cytotoxic effects of P-30... Alfacell Corporation, Bloomfield, New Jersey 07003 and *Department of Pharmacology, Thomas Jeferson University, Philadelphia, Pennsylvania 19107, U.S.A. (Received 18 December 1989; revision accepted 22 January 1990) Abstract. P-30 protein, a novel protein isolated in our laboratory from fertilized Rana pipiens eggs, has been shown to possess significant anti-proliferative and cytotoxic activity against a variety of human tumour cell lines. This protein also shows a potent anti-tumour activity in uiuo in animal tumour models and is currently undergoing Phase I human clinical trials in cancer patient volunteers. The present study describes the in uitro effects of the concerted action of this protein and two other agents which affect the cell proliferative cycle. A significant potentiation of the P-30 protein-induced cell growth inhibition by tamoxifen as well as trifluoroperazine (Stelazine) in both the human A-549 lung carcinoma and the ASPC-1 pancreatic adenocarcinoma systems at wide ranges of drug concentrations was observed. The effect was apparently due to the synergistic action of P-30 protein and the agents tested. This data may provide clues that can be useful in explaining the mechanism of its anti-tumour activity. The results are also helpful for the designing in uiuo animal and, perhaps eventually, human studies, whereby the combination therapies utilizing P-30 protein with agents of relatively low toxicity such as tamoxifen and/or Stelazine could offer a promising treatment(s) for these notoriously refractory types of human cancer. P-30 protein is a novel protein isolated in our laboratory from Rana pipiens eggs subjected to fertilization (purified from the original extract by a three-step procedure involving cationic exchange and molecular sieving chromatographies). It has a molecular weight of 12,000daltons and an isoelectric point in the range of 9.5-105 and has been shown to exhibit a significant antiproliferative and cytotoxic activity against a variety of human tumour cell lines (Darzynkiewicz et al., 1988). Although the exact mechanism of action leading to cell death has not yet been determined, P-30 protein exerts its effect(s) on the cell proliferative cycle, resulting in the accumulation of cells in G I phase with concomitant decrease of the S (DNA synthesis) and G 2 M (mitosis) fractions, as measured by flow cytometry. The effects observed were dosedependent in both flow cytometry and clonogenicity experiments in HL-60 leukaemia, COLO Correspondence: Dr Stanislaw M. Mikulski, Alfacell Corporation, 225 Belleville Avenue, Bloomfield, NJ 07003, U.S.A. S. M. Mikulski et al. 320DM carcinoma and A-253 squamous cell carcinoma lines (Darzynkiewicz et al., 1988). The in vitro anti-tumour activity of P-30 protein has been corroborated most recently by the results of an in uiuo animal study, whereby a striking increase of survival of mice bearing M109 Madison carcinoma (with significant proportion of long-term survivors), has been observed (Mikulski et al., 1990). Currently, P-30 protein is undergoing Phase I human clinical trial in the United States. In view of these results and our previous experience regarding P-30 protein activity against several human tumour cell lines using the MTT colorimetric assay (Mosmann, 1983; Alley et al., 1986; Czirbik et al., 1987), we decided to study the effects of P-30 protein alone and in combination with other agents known to affect the cell proliferation regulatory networks. In this study, we have investigated the in uitro effects of P-30 protein, the anti-oestrogen tamoxifen and the calmodulin inhibitor trifluoroperazine (Stelazine) alone ; and the combinations of P-30 protein with tamoxifen and P-30 protein with Stelazine. The use of these combinations was based on the rationale that the oestrogen-irreversible activity of anti-oestrogens (Musgrove, Wakeling & Sutherland, 1989) coincides with the early G , phase inhibitory effect(s) of calmodulin and protein kinase C (PK-C) inhibitors (Mori et al., 1980; Goyns & Hopkins, 1981; Schatzman, Wise & Kuo, 1981) and that either type of agent, or both, may act synergistically with P-30 protein's cell cycle inhibitory effect. MATERIALS A N D METHODS Cell limes The two cell lines, ASPC-1 human pancreatic adenocarcinoma and A-549 human lung carcinoma, were selected as representing human solid tumours resistant to treatment, and, at the same time, being relatively resistant to P-30 protein in our assay system. The human pancreatic adenocarcinoma ASPC-1 (ATCC CRL 1682) and human lung carcinoma A-549 (ATCC CCL 185) cell lines were obtained from American Type Culture Collection. Both cell lines were cultured in RPMI 1640 medium (Hazleton Research Products, Lenexa, KS) and supplemented with 20% (ASPC-1) or 10% (A-549) heat-inactivated fetal bovine serum (Hazleton Research Products), 200 m L-glutamine (Gibco Life Technologies, M Grand Island, NY) and antibiotic-antimycotic solution composed of : 10,000 units ml-' penicillin, 10 mg ml-' streptomycin and 25 pg ml-' fungizone (complete growth medium). Determination of cell number The number of cells was determined by a direct count in a AO-Spencer Brightline haemocytometer (Reichert Scientific Instruments, Buffalo, NY) with a Neubauer ruling. All solutions used for this purpose were products of Hazleton Research Products. Attached cells were washed three times with Hanks' Balanced Salt Solution and treated with 2 ml of a 0.25% Trypsin-O.02%EDTA solution in buffered saline for about 30 s. The solution was removed and the cells were left at 37°C for 10 min, then suspended in 10 ml of the complete growth medium. Then 0.25 ml of the cell suspension was diluted with 0.75 ml of the complete growth medium, 1 ml of 0.5% trypan blue solution was added and viable cells were counted. Drugs P-30 protein (Alfacell Corporation, Bloomfield, NJ) was dissolved in phosphate buffered saline (PBS) to obtain 1 mg ml-I stock solution. Tamoxifen (Z-1-p-dimethylaminoethoxyphenyl-1,2-diphenyl-l-butene), citrate salt (Sigma Tamoxifen and trzjluoroperazine (Stelazine) Chemical Co., St. Louis, MO), was dissolved in absolute ethanol and diluted with RPMI 1640 medium, to obtain 1 m stock solution (final concentration of ethanol 11%). M Stelazine, trifluoperazine-lO-[3-(4-methylpiperazin-1-yl)-propyl]-2-trifluoromethylphenothiazine-(SK&F Co., subsidiary of SmithKline Beckman Co., Carolina, Puerto Rico), was M diluted with RPMI 1640 medium, to obtain 1 m stock solution. Assay system The cells from stock cultures were seeded into 96-well tissue culture plate (Falcon, Oxnard, CA) at a density of 2000 viable cells (50 pl) per well for the A-549 cell line and 4000 viable cells (50 pl) per well for the ASPC-1 cell line. The cell number was based on the previously determined growth curve characteristics for 7 days of culture. The cells were allowed to settle for 24 h and then 50 p1 of appropriate solutions of P-30 protein and/or 50 p1 of tamoxifen or Stelazine solutions were added per well. The following final concentrations were used: a P-30 protein 20 ng ml-l to 10 pg ml-l; b tamoxifen 10 p~ for ASPC-1 cells, and 3.3 p~ for A-549 cells; c Stelazine 5 p~ for both A-549 and ASPC-1 cells. The plates were incubated for an additional 6 days at 37°C and 5% carbon dioxide atmosphere. The total assay time was, therefore, 7 days. Percentages of viable cells were then determined by MTT colourimetric assay (Mosmann, 1983), using the Bio-Rad EIA microtiter plate reader and an Apple IIE computer (Analysis of Variance, ANOVA Program of Human System Dynamics) for computation. Statistical analysis In order to determine statistical significance of differences between various treatment regimens, the Newman-Keuls method was applied. Using this aproach, the degree of tumour cell growth inhibition induced by P-30 protein alone was compared to the combinations of P-30 protein with tamoxifen and P-30 protein with Stelazine. The activities of the combination treatments was compared. In order to determine the type of interactions between different agents used in combination(s), the interaction index has been used (Berenbaum, 1981) according to the following formula for zero (0) interaction (i.e. complete lack of interaction) : A, B C -+c+>+.. . = 1.0; Aa B, Ca where A,, B,, C, etc., represent an equi-effective dose (e.g. ED50value = 50% of decrease of cell viability as compared with untreated control) of each of the interacting drugs in combination, and A,, B,, C, etc., represent the same equi-effective dose of each drug used alone. The sum of the ratios of these equi-effective doses equal to 1.0 represents zero (0) interaction, i.e. lack of interaction(s); value above 1.0 represents antagonism; and below 1.0 synergism. It must be remembered that even though the interaction may indicate antagonism, the net absolute antitumour effect may be greater using an antagonistic combination than either of the interacting agents alone. RESULTS The growth inhibitory activity of P-30 protein alone and in combination with tamoxifen and Stelazine were tested in two human tumour cell lines, ASPC-1 and A-549. Results are shown in Tables 1-6 and they represent mean percentages of inhibition of tumour cell growth as compared with untreated control. S. M. Mikulski et al. Table 1. ASPC-I human pancreatic carcinoma: Mean values of % of growth inhibition for varying doses of P-30 protein alone, with tamoxifen and with Stelazine ~~ ~~~~~~ Mean % growth inhibition Final conc. P-30 (pg m1-I) 0 0.02 0.1 0.2 1.o 2.0 10.0 (SD) P-30 alone (A) P-30 tamoxifen* (B) 27.4 (15.8) 52.6 (5.8) 74.5 (3.5) 82.9 (3.4) 84.3 (1.8) 87.8 (2.0) 93.0 (1.1) P-30 St e1a zin et (C) 4.6 (10.3) 14.3 (2.7) 29.3 (8.5) 33.3 (1.4) 30.3 (5.6) 38.2 (7.4) 75.0 (8.5) A v. B A v. C NS 0.00 1 0.001 0.001 0.001 0.001 NS B v C 0 -5.4 (0) (6.6) 1.1 (11.9) -5.1 (9.5) 8.8 (4.6) 20.0 (10.3) 72.0 (4.0) P = significance of difference using Newman-Keuls method. * Constant final concentration = 10 P M ; tamoxifen t Constant final concentration = 5 PM; Stelazine alone induced 4.6% growth inhibition. alone induced 27.4% growth inhibition. The experiment was repeated three times; in all experiments statistically significant differences noted with P value in the range of 0.001 when A v. B , A v. C and B v. C were compared. The negative values of the %growth inhibition represent the not significantly increased cell growth in these samples. Table 2. A-549 human lung adenocarcinoma: Mean values of growth inhibition for varying doses of P-30 protein alone, with tamoxifen and with Stelazine ~ ~ Mean % growth inhibition Final conc. P-30 (pg m1-0 0 0.02 0.1 0.2 1.o 2.0 (SD) P-30 alone (A) P-30 tamoxifen* (B) 14.7 19.1 24.8 20.5 30.2 35.7 84.6 (7.0) (4.4) (1.3) (2.8) (3.1) (2.3) (3.4) P-30 Stelazinet (C) 52.5 64.5 63.0 68-6 78.1 82.9 95.8 (6.2) (7.9) (6.7) (9-5) (2.8) (5.0) A v. B 0.001 0.01 0.00 1 0-0 1 0.001 0.001 0.001 A v. C B v. C (0) 5.4 (4.8) 4.7 (6.3) 7.2 (12.5) 11.1 (2.9) 18.8 (3.8) 59.5 (8.5) (0.6) P = significance of difference using Newman-Keuls method. * Constant t Constant final concentration = 5 P M ; Stelazine alone induced 52.5% growth inhibition. final concentration = 3.3 p ~ tamoxifen alone induced 14.7% growth inhibition. ; The experiment was repeated three times; in all experiments statistically significant differences noted with P values in the range of 0.001 when A v. B, A v. C and B v. C were compared. In addition, the equi-effective doses, i.e. ED5,,values, were calculated for P-30 protein alone, tamoxifen and Stelazine alone and the combinations of P-30 protein with tamoxifen and P-30 protein with Stelazine, in order to determine whether any significant interaction(s) occurred within these treatment combinations. Tables 1 and 2 present mean percentages of growth inhibition whereby varying doses of P-30 protein were used either alone or in combination with constant concentrations of tamoxifen and Stelazine. These concentrations were selected based on our previous titration experiments to Tamoxijen and trfluoroperazine (Stelazine) 24 I Table 3. Mean % of growth inhibition of ASPC-1 human pancreatic carcinoma cells and ED5, values for varying doses of P-30 protein alone, tamoxifen alone and their combination Dose of tamoxifen ( PM) Dose of P-30 ( p g ml-I) 10.0 52.0 86.5 (S) 96.8 (S) 98.6 (S) 0.89 1 20.0 77.9 94.2 (S) 97.6 (S) 99.0 (S) 0.682 ED,,/P-30 6.8 18 0.337 0.035 0.026 ED,,/tamoxifen 8.1 30.1 (S) 50.0 (S) 96.0 (S) 5.734 11.8 48.8 (S) 85.0 (S) 97.9 (S) 3.625 -~ 34.8 79.5 (S) 96.9 (S) 96.8 (S) 1,876 (S) = synergism, based upon ED,, values, as per Interaction Index (see Statistical analysis, in Materials and methods). Table 4. Mean % of growth inhibition of ASPC-1 human pancreatic carcinoma cells and ED5, values for varying doses of P-30 protein alone, Stelazine alone and their combination Dose of Stelazine (PM) Dose of P-30 ( p g ml-l) 0.05 8.1 1.1 (A) 9.9 (A) 58.4 (A) 8.562 5.0 34.8 14.6 (A) 29.5 (A) 97.2 (S) 3.738 10.0 52.0 53.8 (A) 62.7 (0) 97.8 (S) 1.419 20.0 17.9 79.0 (A) 83.8 (S) 98.8 (S) 0.605 ED,o/P-30 6.8 18 13.118 5.656 0.025 11.8 0 (A) 6.5 (A) 84.9 (S) ED,,/Stelazine (A) = antagonism; (S) = synergism (see footnote Table 3). choose suboptimal doses, appropriate for detecting drug interactions. The mean values were derived from quadruplicate tests for each data point. As can be seen in Table 1, the increased ASPC-1 tumour cell growth inhibitory activity of the P-30 protein with tamoxifen combination, when compared with P-30 protein alone, was highly significantly different with P value of 0.001 across varying concentrations of P-30 protein, and highly reproducible (in three different experiments per cell line). The same P-30 protein with tamoxifen combination was also significantly more active than P-30 protein with Stelazine, and P-30 protein with Stelazine was significantly more active than P-30 protein alone. Table 2 represents the pattern of activity of the same types of treatment in A-549 lung carcinoma system. Here, the results are, again, very reproducible in three different experiments and, interestingly, differ consistently from those in ASPC-1 tumour system. Namely, the combination of P-30 protein with Stelazine is the most active, P-30 protein with tamoxifen significantly less active, and, at the same time, the latter is significantly more active than P-30 protein alone. Tables 3-6 represent our approach to the assessment of the type of interactions between various treatment regimens. As can be seen in Table 3, the interactions between P-30 protein and three different concentrations of tamoxifen, i.e. 5, 10 and 15 PM,were clearly synergistic. On the other hand, the interaction between P-30 protein and Stelazine (Table 4) demonstrates synergism only at the highest concentration of the latter agent. Table 5 shows the pattern of interactions between P-30 protein and tamoxifen in A-549 lung S. M. Mikulski et al. Table 5. Mean % of growth inhibition of A-549 human lung adenocarcinoma cells and EDs0 values for varying doses of P-30 protein alone, tamoxifen alone and their combination ~~~~ ~~ ~ ~ Dose of tamoxifen (PM) Dose of P-30 (pg ml-l) 0.05 0 3.9 (A) 0 (S) 99.2 (S) 0 4.7 (A) 0.2 (S) 99.7 (S) 10.0 34.6 37.0 (A) 62.7 (S) 99.8 (S) 1.090 20.0 53.4 60.7 (A) 76.0 (S) 100.0 (S) 0.626 ED,o/P-30 9.0 27.3 (A) 33.3 (S) 99.9 (S) 1.480 ED,,/tamoxifen (A) = antagonism; (S) = synergism (see footnote Table 3). Table 6. Mean % of growth inhibition of A-549 human lung adenocarcinoma cells and EDSovalues for varying doses of P-30 protein alone, Stelazine alone and their combination Dose of Stelazine (PM) Dose of P-30 (pg ml-l) 0.05 0 0 (S) 1.1 (S) 90.8 (S) 0 0 (S) 2.3 (S) 90.8 (S) 5.0 9.0 14.7 (S) 24.0 (S) 92.3 (S) 4.980 20.0 53.4 60.4 (S) 70.2 (S) 93.8 (S) ED,o/P-30 18,720 14.490 12.330 0.027 34.6 39.4 (S) 50.2 (S) 93.6 (S) 2.530 ED,,/Stelazine (S) = synergism (see footnote Table 3). caicinoma. The antagonism is noted at 1 PM level of tamoxifen, whereas clear synergism occurs with higher doses of the same agent (3.3 and 10 PM). The clear synergism is observed across the spectrum of Stelazine and P-30 protein doses (Table 6). The combination of P-30 protein with tamoxifen is clearly and consistently the most effective against ASPC-1 pancreatic adenocarcinoma, and P-30 protein with Stelazine is consistently the most effective in A-549 lung carcinoma system. These consistent patterns of activity, demonstrated in three separate experiments per tumour cell line, strongly suggest a specific biological difference between these two tumour cell lines. DISCUSSION We decided to study the effect(s) of combination(s) of P-30 protein with tamoxifen and Stelazine, in an attempt to shed some light on the possible mechanism of action of P-30 protein and its interaction with other agents. Specifically, we hypothesized that since P-30 protein consistently blocks various proliferating cells in the G , phase of the cell cycle (Darzynkiewicz et al., 1988), it would be interesting to combine it with agent(s) known to affect specific molecular targets of the cellular regulatory networks, such as protein kinase C (PK-C) and/or calmodulin/Ca+2,the inhibition of which can also prevent cells from entering the S phase of the cell cycle. Tamoxifen and trguoroperazine (Stelazine) Table 7. Mean %of growth inhibition of ASPC-1 cells for varying doses of P-30 protein alone and in various combinations with 17-beta-oestradiol (17-ES), tamoxifen and trioxifene (values in parentheses represent SD) Dose of P-30 (pg ml-I) Growth inhibitor Experiment 1 P-30 alone tamoxifen 17-ES tamoxifen 1 7 4 3 0 0.02 0.2 2.0 54.0 (76) 4.3 (3.9) 48.0 (10.9) 8.4 80.8 7.6 74.0 14.2 18.5 78.5 -4.1 4.3 91.2 (2.7) (2.1) (4.2) (2.9) 8.1 90.4 8.2 86.0 12.4 17.8 99.4 -12.9 -0.9 99.7 (2.4) (2.2) (3.1) (2.0) 17.8 89-8 13.6 85.9 (1.4) (1.7) (4.0) (1.3) Experiment 2 P-30 alone trioxifene tamoxifen 17-ES trioxifene + 17-ES tamoxifen 17-ES 13.1 (5.5) 63.0 (9.1) - 15.4 (15.1) 1.7 (10.8) 72.1 (5.5) (3.7) (4.4) (11.9) (9.6) (5.5) (9.5) (7.2) (1.8) (0.5) (12.5) (12.5) (0.6) 13.5 (5.1) 12.3 (9.7) 100.0 (0) - 1.9 (10.4) 7.5 (4.6) 100.0 (0) The negative values of the % growth inhibition represent the not significantly increased cell growth in these samples. Tamoxifen may affect the cell proliferative cycle in several ways. The best known is its antioestrogen activity, via modulation of the oestrogen receptor (ER) conformation (Nelson et al., 1988); as a result, the inhibition of tumour cell growth may occur (Osborne, Hobbs & Clark, 1985; Lippman, Bolan & Huff, 1983; Sutherland, Hall & Taylor, 1983). In addition, tamoxifen can induce the biosynthesis of beta-transforming growth factor in certain tumour cell lines (Knabbe et al., 1987), which, in turn, may affect tumour cell growth (Roberts et al., 1985). In micromolar (PM) concentrations, tamoxifen can interfere with other mechanisms involved in the regulation of cellular proliferation, such as the PK-C and inositol triphosphate (IP3)-calmodulin/Ca+2 systems (Berridge & Irvine, 1984; Gulino et al., 1986; Jondal et al., 1986; O’Brien et al., 1986) and/or via interaction with the anti-oestrogen binding site (AEBS)/ histamine (H,) receptor (Brandes et al., 1987). The early G , phase inhibition of the cell cycle by the oestrogen-irreversible activity of antioestrogens (Musgrove, Wakeling & Sutherland, 1989), coincides with the early G I phase inhibitory effect(s) of calmodulin inhibitors (Goyns & Hopkins, 1981). This is consistent with the calmodulin-inhibitory effect@)of tamoxifen. Stelazine (trifluoroperazine) has been known to interfere with the function of calmodulin/Ca+2system (Musgrove et al., 1989; Mori et al., 1980; Goyns & Hopkins, 1981; Schatzman et al., 1981) and PK-C (Mori et al., 1980; Goyns & Hopkins, 1981; Schatzman, Wise & Kuo, 1981). The interactive effect of tamoxifen does not seem to be hormonally-mediated, since it is totally unaffected by equimolar concentrations of 17-beta-oestradiol(Table 7). Moreover, trioxifene (kindly provided by Eli Lilly & Co.), a more potent anti-oestrogen than tamoxifen, does not demonstrate similar synergistic interactions with P-30 protein (Table 7). Also, the range of tamoxifen concentrations (phi) suggests non-hormonal interaction (Furr & Jordan, 1984). We feel that the previously documented effect of P-30 protein in blocking susceptible cells in G I phase (Darzynkiewicz et al., 1988) and the additional effect of tamoxifen and/or Stelazine inhibiting calmodulin/Ca+2,with or without concomitant PK-C inhibition, and with or without S. M. Mikulski et al. Table 8. Mean % of growth inhibition of A-549 and ASPC-1 cells for varying doses of P-30 protein alone and P-30 protein with H7* in MTT assay. Three different experiments were performed per cell line (values in parentheses represent SD) Dose of P-30 (pg ml-*) Experiment ~~~~ ot A-549 P-30 P-30 H7 P-30 P-30 H7 P-30 P-30 H7 + + + -0.1 (1.3) 2.6 (2.0) - 11.0 (18.5) -3.7 9.6 5.6 12.3 (4.0) (7.9) (4.8) (7.6) 5.0 (7.6) - 1.6 (11.7) 4.5 0.9 -5.4 11.0 8.4 5.8 (9.1) (3.3) (6.6) (5.7) (2.7) (2.0) 1.0 (8.1) 11.4 (6.8) 7.2 (6.8) 2.9 (11.2) 1.8 (10.5) -2.7 (11.9) 23.5 (6.7) 32.8 (0.1) 18.8 (3.8) 21.8 (2.0) 11.7 (10.3) 7.1 (11.4) 20.1 (22.2) 30-9 (12-0) 20.0 (10.3) 13.4 (5.0) 17.8 (1.4) 108 (5.2) ASPC-1 P-30 P-30 H7 P-30 P-30 H7 P-30 P-30 H7 + + + -6.3 (3.6) 9.1 (3.1) 1.7 (6.9) 3.5 (7.6) 0-7 (4.8) -5.1 (10.9) 11.7 (8.0) 8.1 (2.4) 3.3 (5.0) 10 p ~ . * H7 used in both cell lines at final concentration of t H7 alone. The negative values of the % growth inhibition represent the not significantly increased cell growth in these samples. concomitant negative interaction with the AEBS/H, receptor, could together produce a synergistic net anti-tumour growth effect, as has been observed in our present study. Particularly interesting, in this context, is the dependence of certain genes’ expression, such as c-myc, on the permissive intracellular levels of pH and Ca+* (Berridge, 1986). It was found (V. Allfrey, personal communication), that 0.1 p~ P-30 protein decreased expression of c-myc by SO% or more in intact COLO 320DM cells at 2 and 24 h, and at 2 h in isolated nuclei of the same cells. A 15-20% inhibition of the expression of ribosomal DNA was also observed in the intact COLO 320DM cells. The decrease of ribosomal DNA expression could result in inhibiting protein synthesis which precedes initiation of DNA synthesis (S phase of the cell cycle). At higher concentration of P-30 protein (20 pg ml-*), there was an increased expression of c-fos gene observed which, in combination with the decreased c-myc expression, is consistent with the differentiation inducing effect. It appears, therefore, that the synergistic interaction observed in our study may, at least partly, be due to the combination of the inhibitory effects of P-30 protein on c-myc expression and the same effect resulting from tamoxifen and/or Stelazine-induced PK-C and/or calmodulin/Ca+*system inhibition. Such concerted effects could be manifested by a decreased expression of certain genes, such as c-myc. The level of expression of c-myc has recently been shown to correlate inversely with the duration of GI to S phase transition in the cell cycle (Karn et al., 1989). The lack of any detectable interaction between P-30 protein and the known PK-C inhibitor H7 (Table 8), further favours calmodulin, rather than PK-C mediated effect(s). The consistent differences regarding tamoxifen and Stelazine interactions in these two cell lines point to the possibility of a more complex interaction, which does not necessarily depend only on a calmodulin-inhibitory effect, but may also involve interaction with AEBS/H3 receptor. Tamoxifen and try7uoroperazine (Stelazine) The present data suggests that the specific combinations of P-30 protein with tamoxifen in human pancreatic ASPC-1 adenocarcinoma and P-30 protein with Stelazine in human lung A-549 carcinoma systems, should be studied in vivo in animals and, subsequently, introduced to human trials. So far, we have been able to find a correlation between our in vitro and in vivo P-30 protein activity (Darzynkiewicz et al., 1988; Mikulski et al., 1990). Thus, a possibility of developing combination therapy regimens with greater efficacy and/or lower toxicity, as compared with other treatments or P-30 protein alone, should be explored. Such an approach seems especially promising in the case of human tumours notorious for being refractory to all available therapies. The potential similarities in molecular signal(s) between early embryonal and malignant cells may conceivably constitute the basis for a relative tumour cell specificity of P-30 protein. This is consistent with previous findings of tumour growth regulatory effects exhibited by early embryonal tissues (Mintz, 1985). It should be stressed that concentrations of tamoxifen used in our present study are within the pharmacological range that was achieved in vivo by other investigators in both animal (DeGregorio, Coronado & Osborne, 1989) and human clinical studies (Furr & Jordan, 1984). We feel that we have identified promising therapeutic combinations in tumour types where, most importantly, the treatment with either agent alone, might not be considered to produce significant results. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Cell Proliferation Wiley

Tamoxifen and trifluoroperazine (Stelazine) potentiate cytostatic/cytotoxic effects of P-30 protein, a novel protein possessing anti-tumour activity

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References (30)

Publisher
Wiley
Copyright
1990 Blackwell Science Limited
ISSN
0960-7722
eISSN
1365-2184
DOI
10.1111/j.1365-2184.1990.tb01119.x
Publisher site
See Article on Publisher Site

Abstract

Alfacell Corporation, Bloomfield, New Jersey 07003 and *Department of Pharmacology, Thomas Jeferson University, Philadelphia, Pennsylvania 19107, U.S.A. (Received 18 December 1989; revision accepted 22 January 1990) Abstract. P-30 protein, a novel protein isolated in our laboratory from fertilized Rana pipiens eggs, has been shown to possess significant anti-proliferative and cytotoxic activity against a variety of human tumour cell lines. This protein also shows a potent anti-tumour activity in uiuo in animal tumour models and is currently undergoing Phase I human clinical trials in cancer patient volunteers. The present study describes the in uitro effects of the concerted action of this protein and two other agents which affect the cell proliferative cycle. A significant potentiation of the P-30 protein-induced cell growth inhibition by tamoxifen as well as trifluoroperazine (Stelazine) in both the human A-549 lung carcinoma and the ASPC-1 pancreatic adenocarcinoma systems at wide ranges of drug concentrations was observed. The effect was apparently due to the synergistic action of P-30 protein and the agents tested. This data may provide clues that can be useful in explaining the mechanism of its anti-tumour activity. The results are also helpful for the designing in uiuo animal and, perhaps eventually, human studies, whereby the combination therapies utilizing P-30 protein with agents of relatively low toxicity such as tamoxifen and/or Stelazine could offer a promising treatment(s) for these notoriously refractory types of human cancer. P-30 protein is a novel protein isolated in our laboratory from Rana pipiens eggs subjected to fertilization (purified from the original extract by a three-step procedure involving cationic exchange and molecular sieving chromatographies). It has a molecular weight of 12,000daltons and an isoelectric point in the range of 9.5-105 and has been shown to exhibit a significant antiproliferative and cytotoxic activity against a variety of human tumour cell lines (Darzynkiewicz et al., 1988). Although the exact mechanism of action leading to cell death has not yet been determined, P-30 protein exerts its effect(s) on the cell proliferative cycle, resulting in the accumulation of cells in G I phase with concomitant decrease of the S (DNA synthesis) and G 2 M (mitosis) fractions, as measured by flow cytometry. The effects observed were dosedependent in both flow cytometry and clonogenicity experiments in HL-60 leukaemia, COLO Correspondence: Dr Stanislaw M. Mikulski, Alfacell Corporation, 225 Belleville Avenue, Bloomfield, NJ 07003, U.S.A. S. M. Mikulski et al. 320DM carcinoma and A-253 squamous cell carcinoma lines (Darzynkiewicz et al., 1988). The in vitro anti-tumour activity of P-30 protein has been corroborated most recently by the results of an in uiuo animal study, whereby a striking increase of survival of mice bearing M109 Madison carcinoma (with significant proportion of long-term survivors), has been observed (Mikulski et al., 1990). Currently, P-30 protein is undergoing Phase I human clinical trial in the United States. In view of these results and our previous experience regarding P-30 protein activity against several human tumour cell lines using the MTT colorimetric assay (Mosmann, 1983; Alley et al., 1986; Czirbik et al., 1987), we decided to study the effects of P-30 protein alone and in combination with other agents known to affect the cell proliferation regulatory networks. In this study, we have investigated the in uitro effects of P-30 protein, the anti-oestrogen tamoxifen and the calmodulin inhibitor trifluoroperazine (Stelazine) alone ; and the combinations of P-30 protein with tamoxifen and P-30 protein with Stelazine. The use of these combinations was based on the rationale that the oestrogen-irreversible activity of anti-oestrogens (Musgrove, Wakeling & Sutherland, 1989) coincides with the early G , phase inhibitory effect(s) of calmodulin and protein kinase C (PK-C) inhibitors (Mori et al., 1980; Goyns & Hopkins, 1981; Schatzman, Wise & Kuo, 1981) and that either type of agent, or both, may act synergistically with P-30 protein's cell cycle inhibitory effect. MATERIALS A N D METHODS Cell limes The two cell lines, ASPC-1 human pancreatic adenocarcinoma and A-549 human lung carcinoma, were selected as representing human solid tumours resistant to treatment, and, at the same time, being relatively resistant to P-30 protein in our assay system. The human pancreatic adenocarcinoma ASPC-1 (ATCC CRL 1682) and human lung carcinoma A-549 (ATCC CCL 185) cell lines were obtained from American Type Culture Collection. Both cell lines were cultured in RPMI 1640 medium (Hazleton Research Products, Lenexa, KS) and supplemented with 20% (ASPC-1) or 10% (A-549) heat-inactivated fetal bovine serum (Hazleton Research Products), 200 m L-glutamine (Gibco Life Technologies, M Grand Island, NY) and antibiotic-antimycotic solution composed of : 10,000 units ml-' penicillin, 10 mg ml-' streptomycin and 25 pg ml-' fungizone (complete growth medium). Determination of cell number The number of cells was determined by a direct count in a AO-Spencer Brightline haemocytometer (Reichert Scientific Instruments, Buffalo, NY) with a Neubauer ruling. All solutions used for this purpose were products of Hazleton Research Products. Attached cells were washed three times with Hanks' Balanced Salt Solution and treated with 2 ml of a 0.25% Trypsin-O.02%EDTA solution in buffered saline for about 30 s. The solution was removed and the cells were left at 37°C for 10 min, then suspended in 10 ml of the complete growth medium. Then 0.25 ml of the cell suspension was diluted with 0.75 ml of the complete growth medium, 1 ml of 0.5% trypan blue solution was added and viable cells were counted. Drugs P-30 protein (Alfacell Corporation, Bloomfield, NJ) was dissolved in phosphate buffered saline (PBS) to obtain 1 mg ml-I stock solution. Tamoxifen (Z-1-p-dimethylaminoethoxyphenyl-1,2-diphenyl-l-butene), citrate salt (Sigma Tamoxifen and trzjluoroperazine (Stelazine) Chemical Co., St. Louis, MO), was dissolved in absolute ethanol and diluted with RPMI 1640 medium, to obtain 1 m stock solution (final concentration of ethanol 11%). M Stelazine, trifluoperazine-lO-[3-(4-methylpiperazin-1-yl)-propyl]-2-trifluoromethylphenothiazine-(SK&F Co., subsidiary of SmithKline Beckman Co., Carolina, Puerto Rico), was M diluted with RPMI 1640 medium, to obtain 1 m stock solution. Assay system The cells from stock cultures were seeded into 96-well tissue culture plate (Falcon, Oxnard, CA) at a density of 2000 viable cells (50 pl) per well for the A-549 cell line and 4000 viable cells (50 pl) per well for the ASPC-1 cell line. The cell number was based on the previously determined growth curve characteristics for 7 days of culture. The cells were allowed to settle for 24 h and then 50 p1 of appropriate solutions of P-30 protein and/or 50 p1 of tamoxifen or Stelazine solutions were added per well. The following final concentrations were used: a P-30 protein 20 ng ml-l to 10 pg ml-l; b tamoxifen 10 p~ for ASPC-1 cells, and 3.3 p~ for A-549 cells; c Stelazine 5 p~ for both A-549 and ASPC-1 cells. The plates were incubated for an additional 6 days at 37°C and 5% carbon dioxide atmosphere. The total assay time was, therefore, 7 days. Percentages of viable cells were then determined by MTT colourimetric assay (Mosmann, 1983), using the Bio-Rad EIA microtiter plate reader and an Apple IIE computer (Analysis of Variance, ANOVA Program of Human System Dynamics) for computation. Statistical analysis In order to determine statistical significance of differences between various treatment regimens, the Newman-Keuls method was applied. Using this aproach, the degree of tumour cell growth inhibition induced by P-30 protein alone was compared to the combinations of P-30 protein with tamoxifen and P-30 protein with Stelazine. The activities of the combination treatments was compared. In order to determine the type of interactions between different agents used in combination(s), the interaction index has been used (Berenbaum, 1981) according to the following formula for zero (0) interaction (i.e. complete lack of interaction) : A, B C -+c+>+.. . = 1.0; Aa B, Ca where A,, B,, C, etc., represent an equi-effective dose (e.g. ED50value = 50% of decrease of cell viability as compared with untreated control) of each of the interacting drugs in combination, and A,, B,, C, etc., represent the same equi-effective dose of each drug used alone. The sum of the ratios of these equi-effective doses equal to 1.0 represents zero (0) interaction, i.e. lack of interaction(s); value above 1.0 represents antagonism; and below 1.0 synergism. It must be remembered that even though the interaction may indicate antagonism, the net absolute antitumour effect may be greater using an antagonistic combination than either of the interacting agents alone. RESULTS The growth inhibitory activity of P-30 protein alone and in combination with tamoxifen and Stelazine were tested in two human tumour cell lines, ASPC-1 and A-549. Results are shown in Tables 1-6 and they represent mean percentages of inhibition of tumour cell growth as compared with untreated control. S. M. Mikulski et al. Table 1. ASPC-I human pancreatic carcinoma: Mean values of % of growth inhibition for varying doses of P-30 protein alone, with tamoxifen and with Stelazine ~~ ~~~~~~ Mean % growth inhibition Final conc. P-30 (pg m1-I) 0 0.02 0.1 0.2 1.o 2.0 10.0 (SD) P-30 alone (A) P-30 tamoxifen* (B) 27.4 (15.8) 52.6 (5.8) 74.5 (3.5) 82.9 (3.4) 84.3 (1.8) 87.8 (2.0) 93.0 (1.1) P-30 St e1a zin et (C) 4.6 (10.3) 14.3 (2.7) 29.3 (8.5) 33.3 (1.4) 30.3 (5.6) 38.2 (7.4) 75.0 (8.5) A v. B A v. C NS 0.00 1 0.001 0.001 0.001 0.001 NS B v C 0 -5.4 (0) (6.6) 1.1 (11.9) -5.1 (9.5) 8.8 (4.6) 20.0 (10.3) 72.0 (4.0) P = significance of difference using Newman-Keuls method. * Constant final concentration = 10 P M ; tamoxifen t Constant final concentration = 5 PM; Stelazine alone induced 4.6% growth inhibition. alone induced 27.4% growth inhibition. The experiment was repeated three times; in all experiments statistically significant differences noted with P value in the range of 0.001 when A v. B , A v. C and B v. C were compared. The negative values of the %growth inhibition represent the not significantly increased cell growth in these samples. Table 2. A-549 human lung adenocarcinoma: Mean values of growth inhibition for varying doses of P-30 protein alone, with tamoxifen and with Stelazine ~ ~ Mean % growth inhibition Final conc. P-30 (pg m1-0 0 0.02 0.1 0.2 1.o 2.0 (SD) P-30 alone (A) P-30 tamoxifen* (B) 14.7 19.1 24.8 20.5 30.2 35.7 84.6 (7.0) (4.4) (1.3) (2.8) (3.1) (2.3) (3.4) P-30 Stelazinet (C) 52.5 64.5 63.0 68-6 78.1 82.9 95.8 (6.2) (7.9) (6.7) (9-5) (2.8) (5.0) A v. B 0.001 0.01 0.00 1 0-0 1 0.001 0.001 0.001 A v. C B v. C (0) 5.4 (4.8) 4.7 (6.3) 7.2 (12.5) 11.1 (2.9) 18.8 (3.8) 59.5 (8.5) (0.6) P = significance of difference using Newman-Keuls method. * Constant t Constant final concentration = 5 P M ; Stelazine alone induced 52.5% growth inhibition. final concentration = 3.3 p ~ tamoxifen alone induced 14.7% growth inhibition. ; The experiment was repeated three times; in all experiments statistically significant differences noted with P values in the range of 0.001 when A v. B, A v. C and B v. C were compared. In addition, the equi-effective doses, i.e. ED5,,values, were calculated for P-30 protein alone, tamoxifen and Stelazine alone and the combinations of P-30 protein with tamoxifen and P-30 protein with Stelazine, in order to determine whether any significant interaction(s) occurred within these treatment combinations. Tables 1 and 2 present mean percentages of growth inhibition whereby varying doses of P-30 protein were used either alone or in combination with constant concentrations of tamoxifen and Stelazine. These concentrations were selected based on our previous titration experiments to Tamoxijen and trfluoroperazine (Stelazine) 24 I Table 3. Mean % of growth inhibition of ASPC-1 human pancreatic carcinoma cells and ED5, values for varying doses of P-30 protein alone, tamoxifen alone and their combination Dose of tamoxifen ( PM) Dose of P-30 ( p g ml-I) 10.0 52.0 86.5 (S) 96.8 (S) 98.6 (S) 0.89 1 20.0 77.9 94.2 (S) 97.6 (S) 99.0 (S) 0.682 ED,,/P-30 6.8 18 0.337 0.035 0.026 ED,,/tamoxifen 8.1 30.1 (S) 50.0 (S) 96.0 (S) 5.734 11.8 48.8 (S) 85.0 (S) 97.9 (S) 3.625 -~ 34.8 79.5 (S) 96.9 (S) 96.8 (S) 1,876 (S) = synergism, based upon ED,, values, as per Interaction Index (see Statistical analysis, in Materials and methods). Table 4. Mean % of growth inhibition of ASPC-1 human pancreatic carcinoma cells and ED5, values for varying doses of P-30 protein alone, Stelazine alone and their combination Dose of Stelazine (PM) Dose of P-30 ( p g ml-l) 0.05 8.1 1.1 (A) 9.9 (A) 58.4 (A) 8.562 5.0 34.8 14.6 (A) 29.5 (A) 97.2 (S) 3.738 10.0 52.0 53.8 (A) 62.7 (0) 97.8 (S) 1.419 20.0 17.9 79.0 (A) 83.8 (S) 98.8 (S) 0.605 ED,o/P-30 6.8 18 13.118 5.656 0.025 11.8 0 (A) 6.5 (A) 84.9 (S) ED,,/Stelazine (A) = antagonism; (S) = synergism (see footnote Table 3). choose suboptimal doses, appropriate for detecting drug interactions. The mean values were derived from quadruplicate tests for each data point. As can be seen in Table 1, the increased ASPC-1 tumour cell growth inhibitory activity of the P-30 protein with tamoxifen combination, when compared with P-30 protein alone, was highly significantly different with P value of 0.001 across varying concentrations of P-30 protein, and highly reproducible (in three different experiments per cell line). The same P-30 protein with tamoxifen combination was also significantly more active than P-30 protein with Stelazine, and P-30 protein with Stelazine was significantly more active than P-30 protein alone. Table 2 represents the pattern of activity of the same types of treatment in A-549 lung carcinoma system. Here, the results are, again, very reproducible in three different experiments and, interestingly, differ consistently from those in ASPC-1 tumour system. Namely, the combination of P-30 protein with Stelazine is the most active, P-30 protein with tamoxifen significantly less active, and, at the same time, the latter is significantly more active than P-30 protein alone. Tables 3-6 represent our approach to the assessment of the type of interactions between various treatment regimens. As can be seen in Table 3, the interactions between P-30 protein and three different concentrations of tamoxifen, i.e. 5, 10 and 15 PM,were clearly synergistic. On the other hand, the interaction between P-30 protein and Stelazine (Table 4) demonstrates synergism only at the highest concentration of the latter agent. Table 5 shows the pattern of interactions between P-30 protein and tamoxifen in A-549 lung S. M. Mikulski et al. Table 5. Mean % of growth inhibition of A-549 human lung adenocarcinoma cells and EDs0 values for varying doses of P-30 protein alone, tamoxifen alone and their combination ~~~~ ~~ ~ ~ Dose of tamoxifen (PM) Dose of P-30 (pg ml-l) 0.05 0 3.9 (A) 0 (S) 99.2 (S) 0 4.7 (A) 0.2 (S) 99.7 (S) 10.0 34.6 37.0 (A) 62.7 (S) 99.8 (S) 1.090 20.0 53.4 60.7 (A) 76.0 (S) 100.0 (S) 0.626 ED,o/P-30 9.0 27.3 (A) 33.3 (S) 99.9 (S) 1.480 ED,,/tamoxifen (A) = antagonism; (S) = synergism (see footnote Table 3). Table 6. Mean % of growth inhibition of A-549 human lung adenocarcinoma cells and EDSovalues for varying doses of P-30 protein alone, Stelazine alone and their combination Dose of Stelazine (PM) Dose of P-30 (pg ml-l) 0.05 0 0 (S) 1.1 (S) 90.8 (S) 0 0 (S) 2.3 (S) 90.8 (S) 5.0 9.0 14.7 (S) 24.0 (S) 92.3 (S) 4.980 20.0 53.4 60.4 (S) 70.2 (S) 93.8 (S) ED,o/P-30 18,720 14.490 12.330 0.027 34.6 39.4 (S) 50.2 (S) 93.6 (S) 2.530 ED,,/Stelazine (S) = synergism (see footnote Table 3). caicinoma. The antagonism is noted at 1 PM level of tamoxifen, whereas clear synergism occurs with higher doses of the same agent (3.3 and 10 PM). The clear synergism is observed across the spectrum of Stelazine and P-30 protein doses (Table 6). The combination of P-30 protein with tamoxifen is clearly and consistently the most effective against ASPC-1 pancreatic adenocarcinoma, and P-30 protein with Stelazine is consistently the most effective in A-549 lung carcinoma system. These consistent patterns of activity, demonstrated in three separate experiments per tumour cell line, strongly suggest a specific biological difference between these two tumour cell lines. DISCUSSION We decided to study the effect(s) of combination(s) of P-30 protein with tamoxifen and Stelazine, in an attempt to shed some light on the possible mechanism of action of P-30 protein and its interaction with other agents. Specifically, we hypothesized that since P-30 protein consistently blocks various proliferating cells in the G , phase of the cell cycle (Darzynkiewicz et al., 1988), it would be interesting to combine it with agent(s) known to affect specific molecular targets of the cellular regulatory networks, such as protein kinase C (PK-C) and/or calmodulin/Ca+2,the inhibition of which can also prevent cells from entering the S phase of the cell cycle. Tamoxifen and trguoroperazine (Stelazine) Table 7. Mean %of growth inhibition of ASPC-1 cells for varying doses of P-30 protein alone and in various combinations with 17-beta-oestradiol (17-ES), tamoxifen and trioxifene (values in parentheses represent SD) Dose of P-30 (pg ml-I) Growth inhibitor Experiment 1 P-30 alone tamoxifen 17-ES tamoxifen 1 7 4 3 0 0.02 0.2 2.0 54.0 (76) 4.3 (3.9) 48.0 (10.9) 8.4 80.8 7.6 74.0 14.2 18.5 78.5 -4.1 4.3 91.2 (2.7) (2.1) (4.2) (2.9) 8.1 90.4 8.2 86.0 12.4 17.8 99.4 -12.9 -0.9 99.7 (2.4) (2.2) (3.1) (2.0) 17.8 89-8 13.6 85.9 (1.4) (1.7) (4.0) (1.3) Experiment 2 P-30 alone trioxifene tamoxifen 17-ES trioxifene + 17-ES tamoxifen 17-ES 13.1 (5.5) 63.0 (9.1) - 15.4 (15.1) 1.7 (10.8) 72.1 (5.5) (3.7) (4.4) (11.9) (9.6) (5.5) (9.5) (7.2) (1.8) (0.5) (12.5) (12.5) (0.6) 13.5 (5.1) 12.3 (9.7) 100.0 (0) - 1.9 (10.4) 7.5 (4.6) 100.0 (0) The negative values of the % growth inhibition represent the not significantly increased cell growth in these samples. Tamoxifen may affect the cell proliferative cycle in several ways. The best known is its antioestrogen activity, via modulation of the oestrogen receptor (ER) conformation (Nelson et al., 1988); as a result, the inhibition of tumour cell growth may occur (Osborne, Hobbs & Clark, 1985; Lippman, Bolan & Huff, 1983; Sutherland, Hall & Taylor, 1983). In addition, tamoxifen can induce the biosynthesis of beta-transforming growth factor in certain tumour cell lines (Knabbe et al., 1987), which, in turn, may affect tumour cell growth (Roberts et al., 1985). In micromolar (PM) concentrations, tamoxifen can interfere with other mechanisms involved in the regulation of cellular proliferation, such as the PK-C and inositol triphosphate (IP3)-calmodulin/Ca+2 systems (Berridge & Irvine, 1984; Gulino et al., 1986; Jondal et al., 1986; O’Brien et al., 1986) and/or via interaction with the anti-oestrogen binding site (AEBS)/ histamine (H,) receptor (Brandes et al., 1987). The early G , phase inhibition of the cell cycle by the oestrogen-irreversible activity of antioestrogens (Musgrove, Wakeling & Sutherland, 1989), coincides with the early G I phase inhibitory effect(s) of calmodulin inhibitors (Goyns & Hopkins, 1981). This is consistent with the calmodulin-inhibitory effect@)of tamoxifen. Stelazine (trifluoroperazine) has been known to interfere with the function of calmodulin/Ca+2system (Musgrove et al., 1989; Mori et al., 1980; Goyns & Hopkins, 1981; Schatzman et al., 1981) and PK-C (Mori et al., 1980; Goyns & Hopkins, 1981; Schatzman, Wise & Kuo, 1981). The interactive effect of tamoxifen does not seem to be hormonally-mediated, since it is totally unaffected by equimolar concentrations of 17-beta-oestradiol(Table 7). Moreover, trioxifene (kindly provided by Eli Lilly & Co.), a more potent anti-oestrogen than tamoxifen, does not demonstrate similar synergistic interactions with P-30 protein (Table 7). Also, the range of tamoxifen concentrations (phi) suggests non-hormonal interaction (Furr & Jordan, 1984). We feel that the previously documented effect of P-30 protein in blocking susceptible cells in G I phase (Darzynkiewicz et al., 1988) and the additional effect of tamoxifen and/or Stelazine inhibiting calmodulin/Ca+2,with or without concomitant PK-C inhibition, and with or without S. M. Mikulski et al. Table 8. Mean % of growth inhibition of A-549 and ASPC-1 cells for varying doses of P-30 protein alone and P-30 protein with H7* in MTT assay. Three different experiments were performed per cell line (values in parentheses represent SD) Dose of P-30 (pg ml-*) Experiment ~~~~ ot A-549 P-30 P-30 H7 P-30 P-30 H7 P-30 P-30 H7 + + + -0.1 (1.3) 2.6 (2.0) - 11.0 (18.5) -3.7 9.6 5.6 12.3 (4.0) (7.9) (4.8) (7.6) 5.0 (7.6) - 1.6 (11.7) 4.5 0.9 -5.4 11.0 8.4 5.8 (9.1) (3.3) (6.6) (5.7) (2.7) (2.0) 1.0 (8.1) 11.4 (6.8) 7.2 (6.8) 2.9 (11.2) 1.8 (10.5) -2.7 (11.9) 23.5 (6.7) 32.8 (0.1) 18.8 (3.8) 21.8 (2.0) 11.7 (10.3) 7.1 (11.4) 20.1 (22.2) 30-9 (12-0) 20.0 (10.3) 13.4 (5.0) 17.8 (1.4) 108 (5.2) ASPC-1 P-30 P-30 H7 P-30 P-30 H7 P-30 P-30 H7 + + + -6.3 (3.6) 9.1 (3.1) 1.7 (6.9) 3.5 (7.6) 0-7 (4.8) -5.1 (10.9) 11.7 (8.0) 8.1 (2.4) 3.3 (5.0) 10 p ~ . * H7 used in both cell lines at final concentration of t H7 alone. The negative values of the % growth inhibition represent the not significantly increased cell growth in these samples. concomitant negative interaction with the AEBS/H, receptor, could together produce a synergistic net anti-tumour growth effect, as has been observed in our present study. Particularly interesting, in this context, is the dependence of certain genes’ expression, such as c-myc, on the permissive intracellular levels of pH and Ca+* (Berridge, 1986). It was found (V. Allfrey, personal communication), that 0.1 p~ P-30 protein decreased expression of c-myc by SO% or more in intact COLO 320DM cells at 2 and 24 h, and at 2 h in isolated nuclei of the same cells. A 15-20% inhibition of the expression of ribosomal DNA was also observed in the intact COLO 320DM cells. The decrease of ribosomal DNA expression could result in inhibiting protein synthesis which precedes initiation of DNA synthesis (S phase of the cell cycle). At higher concentration of P-30 protein (20 pg ml-*), there was an increased expression of c-fos gene observed which, in combination with the decreased c-myc expression, is consistent with the differentiation inducing effect. It appears, therefore, that the synergistic interaction observed in our study may, at least partly, be due to the combination of the inhibitory effects of P-30 protein on c-myc expression and the same effect resulting from tamoxifen and/or Stelazine-induced PK-C and/or calmodulin/Ca+*system inhibition. Such concerted effects could be manifested by a decreased expression of certain genes, such as c-myc. The level of expression of c-myc has recently been shown to correlate inversely with the duration of GI to S phase transition in the cell cycle (Karn et al., 1989). The lack of any detectable interaction between P-30 protein and the known PK-C inhibitor H7 (Table 8), further favours calmodulin, rather than PK-C mediated effect(s). The consistent differences regarding tamoxifen and Stelazine interactions in these two cell lines point to the possibility of a more complex interaction, which does not necessarily depend only on a calmodulin-inhibitory effect, but may also involve interaction with AEBS/H3 receptor. Tamoxifen and try7uoroperazine (Stelazine) The present data suggests that the specific combinations of P-30 protein with tamoxifen in human pancreatic ASPC-1 adenocarcinoma and P-30 protein with Stelazine in human lung A-549 carcinoma systems, should be studied in vivo in animals and, subsequently, introduced to human trials. So far, we have been able to find a correlation between our in vitro and in vivo P-30 protein activity (Darzynkiewicz et al., 1988; Mikulski et al., 1990). Thus, a possibility of developing combination therapy regimens with greater efficacy and/or lower toxicity, as compared with other treatments or P-30 protein alone, should be explored. Such an approach seems especially promising in the case of human tumours notorious for being refractory to all available therapies. The potential similarities in molecular signal(s) between early embryonal and malignant cells may conceivably constitute the basis for a relative tumour cell specificity of P-30 protein. This is consistent with previous findings of tumour growth regulatory effects exhibited by early embryonal tissues (Mintz, 1985). It should be stressed that concentrations of tamoxifen used in our present study are within the pharmacological range that was achieved in vivo by other investigators in both animal (DeGregorio, Coronado & Osborne, 1989) and human clinical studies (Furr & Jordan, 1984). We feel that we have identified promising therapeutic combinations in tumour types where, most importantly, the treatment with either agent alone, might not be considered to produce significant results.

Journal

Cell ProliferationWiley

Published: May 1, 1990

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