DEVELOPMENT OF A NOVEL CHEMOTHERAPY AGAINST SOLID TUMOURS

Koh T.H.¹, Cai J.² and Lee C.W.²

Department of Physiology

Contact me for a reprint with graphs and data

Adenocarcinoma of the pancreas is a virulent malignancy with a short survival. Despite a major advance in surgical procedures, radiation and chemotherapy, the prognosis of this cancer remains a dismal less-than-5% survival at 5 years, this is due to that the pancreas is an inaccessible gland and tumours remain silent till the later stage of disease. The aim of chemotherapy in pancreatic cancer is to improve symptoms and survival with low toxicity.

Tamoxifen is an anti-cancer drug widely used in the treatment of cancer. The anti tumor effect is believed to result from competitive interaction with the oestrogen receptor as a frank oestrogen as an agonist or as an antagonist, depending on the species studied, the target organ assessed and the amount of tamoxifen used.¹ The other effects include fluid modulation, inhibit nucleoside transporter and a differentiation inducer. It is used as a first line treatment against breast cancer as it is easily absorbed and metabolised. In pancreatic cancer, the anti cancer effect of tamoxifen is mediated by inhibiting the action of protein kinase C.

Dipyridamole is a well-known vasodilator, an anti platelet and antianginal agent with inhibitory effects on nucleoside transport. It also enhanced the anti proliferative activity in cell lines derived from various other kinds of tumor tissues. For example, dipyridamole is able to enhance the cytotoxicity of 5-fluorouracil by prolonging the retention of active metabolites of 5-flurouracil such as FdUMP.²

5-Fluorouracil (5-FU), a nucleobase analogue, is a rationally designed antineoplastic agent. For more than 30 years, this antimetabolite has been used in the treatment of advanced colorectal cancers, tumours of the gastrointestinal tract, breast cancers and head and neck cancers. Generally, antimetabolites are agents that interfere with several steps in normal metabolism due to their structural similarity with normal intermediates in the sxynthesis of RNA and DNA precursors. 5-FU is an analogue of uracil, it interferes with pathways involving these natural substrates too. It is one of the few drugs for which a limited clinical activity as a single agent has been shown in advanced colorectal cancer. It is frequently used in combination regimens as itself has only a modest anticancer activity.

In this study, we used these 3 drugs to form a novel combination to combat the growths both in vitro and in vivo. The significance of such an action is to give a better result in reducing the proliferative effect of the pancreatic adenocarcinoma (Aspc).

Pancreatic adenocarcinoma (Aspc) were maintained in continuous culture in RPMI 1640 medium (Gibco, Gaithersburg, MD) supplemented with 15% heat-inactivated (50^oC for 30 min) fetal bovine serum (Gibco) plus 50 units/ml of penicillin and 50ug/ml of streptomycin (Gibco).

Cells were harvested before reaching confluency or plateau growth phase and prepared for transport studies as follows. Monolayer cells were detached by trypsinization (0.05% trypsin). The cell suspension was centrifuged at 1000´ g for 5 min in a Sorvall GLC-4 centrifuge. The cell pellet was washed once with 50ml of Na⁺-free (choline replacement) Hanks balanced salt solution (HBSS) plus 5.5mM D-glucose and 4mM Hepes buffer, pH 7.4. The final cell pellet was resuspended with an appropriate volume of Na⁺-free HBSS. Cell numbers were determined with a electronic particle counter (sysmex). The equilibrative uptake of [³H]uridine (final concentration 50m M) was initiated by mixing 140m l of cell suspension (treated with or without tamoxifen) with 70m l of radioactive substrate in a 1.5ml Microfuge tube at 22-24^oC. After an appropriate interval the reactions were determined by transferring a 60m l aliquot of the cell mixture into an "oil-stop" tube consisting of a 400ul Microfuge tube containing 125m l of silicon oil (density 1.04g/ml) layered over 25m l of 15% (w/v) trichloroacetic acid. Cells were separated from the transport medium by pelleting into the trichloroacetic acid layer via centrifugation in a Beckman model E microcentrifuge. Microfuge tubes were then cut through the oil layer and both the supernatant and cell pellet were placed in separate mini-vials for scintillation counting. To each sample, 3.5ml of scintillation fluid (Amersham, Arlington Heights, IL, U.S.A.) was added and votex mixed vigorously. Radioactivity was determined with a liquid-scintillation counter. Approximate initial rates of uridine uptake were determined by using an uptake interval of 20s. "Time-zero" values for transport, attributed to radioactivity trapped in the cell pellet, were determined by centrifuging a cell suspension (20m l) through a layer of [³H]uridine (40m l) layered over the oil in an "oil-stop" tube.

Equilibrium nitrobenzylthioinosine ([³H]NBMPR) binding assays were initiated by incubating 110m l of cell suspensions which had been washed and resuspended with Hanks balanced salt solution (HBSS) with graded concentrations of tamoxifen (30m M – 0m M) in a total volume of 0.66ml at 22-24^oC, in the presence or absence of 20m M nitrobenzylthioguanosine (NBTGR) as a competing nonradioactive ligand. To ensure studies were performed under true equilibrium conditions, a long incubation time of 30 min was used. Reactions were stopped by the "oil-stop" method. Specific binding is defined as the difference in membrane content of [³H]NBMPR in the presence and absence of 20m M NBTGR.

To determine the percentage of viable cells within a population, the cell suspension was mixed with a vital dye and observed under the light microscope. Vital dyes are excluded from living cells but stained dead cells. The vital dye was a solution of 0.4% (w/v) trypan blue in phosphate buffer solution (PBS). When stored at room temperature, it is good for several years. A small amount of cell suspension (100m l) was mixed with a drop of the trypan blue. It was next transferred to a Neubauer counting chamber and counted. Only cells within the square or touching the top and left sides of the square were taken into account. Cell viability was expressed as a percentage of unstained cells versus the total number of cells within the same designated squares. Cell numbers of each well were determined with a particle counter at the end of 5 days. Viability was determined by immersing 20ul of the cells in 5ul of the 0.4% trypan blue dye using a haemocytometer under a light microscope.

3.0 Data Analysis

A single generalized method for analyzing dose effect relationships is applied using the median effect principle, which states f_a/f_b=(D/D_m)^m, where D is the dose, f_a anf f_b are the fractions of the system affected and unaffected respectively by the dose D. D_m is the dose required to produce a median effect. m is the Hill type coefficient signifying the sigmoidicity of the dose-effect curve. Combination index is a term used to quantify synergism, summation and antagonism. For mutually exclusive or non exclusive drugs when the CI is less than 1, synergism is indicated. When CI equals to 1, summation is indicated and when CI is more than 1, antagonism is indicated.³

Chemicals

[5-³H]Uridine (20 Ci/mmol), [³H]NBMPR (25 Ci/mmol) were purchased from New England Nuclear (Boston, MA). Nitrobenzylthioguanosine (NBTGR), tamoxifen, dipyridamole, 5-florouracil were obtained from Sigma Chemical Co. (St. Louis, MO, U.S.A.). All other reagents were of analytical grade.

Animal modeling

Nude mice 6 – 8 months of age were used. Aspc cells of 1.4 ´ 10⁶ concentration were injected into the back of the mice. Tumors were allowed to grow for 7 days without interference by drugs. At the respective days of 7, 14 and 21, drugs 5Fu, tamoxifen and dipyridamole were injected into the mice with concentration of 100mg/kg of the drugs. Tumors were measured using a micrometer, and tumor size were defined by the following; (shortest length² ´ longest length)/2. The lost of weight for the mice should not be more than 20% of its total weight before the start of the trials.

Effect of NBMPR and Dipyridamole on equilibrative [³H]uridine transport by ASPC cells.

The dosage response curves for the inhibition of [³H]uridine transport by the transporters are shown in figure 1. From the graphs, the nucleoside transport inhibitors dipyridamole, and NBMPR successfully inhibit the nucleoside transporters of the pancreatic adenocarcinoma up to 80%. Tamoxifen binds to the nuclear transport protreins and exhibiting the anti-estrogen action of the drug. By inhibiting the transport of the nucleoside into the cell, the growth and proliferation of the carcinoma might be significantly reduced.

Two components of equilibrative nucleoside transport which are distinguishable by NBMPR. The concentration-response curves for the inhibition of uridine transport by NBMPR and dipyridamole were compared under conditions as detailed in the legends. The forms of uridine transport inhibition were biphasic. 80% of the total transporters were inhibited by NBMPR and dipyridamole. IC₅₀ values of 1 and 0.03m M were detected. The wide plateau in the NBMPR inhibition curve suggests that the inhibition observed of NBMPR had occurred by a mechanism different from that involving lower concentrations of the inhibitor. Possess both the NBMPR-sensitive and NBMPR-insensitive components of uridine transport, denoted as es and ei, respectively in the literature. These two distinct forms of equilibrative nucleoside transport mechanisms were also distinguishable with dipyridamole.

One approach to investigate the properties of an equilibrative nucleoside transporter is to use NBMPR, a potent nucleoside transport inhibitor as a ligand probe for the protein. As shown in figure 2, Aspc cells were incubated with [³H]NBMPR in the presence of various concentrations of tamoxifen ranging from 0 to 30m M for 30 min. Results in figure 2 show that tamoxifen decreases the specific binding of [³H]NBMPR in Aspc with an IC₅₀ of 10m M without affecting the non-specific binding. Tamoxifen shows an effect of nearly 80% inhibition at the highest concentration of 30uM.

Nucleosides cross cell membranes via a family of equilibrative transport systems which accept a variety of purine and pyrimidine nucleosides as substrates. These transport systems can be distinguished by their differential sensitivities to inhibition by NBMPR and NBTGR. To date many cell types possess both NBMPR sensitive and insensitive transporters. Pancreatic adenocarcinoma is one that was identified having the es–ei transporters.

Tamoxifen binds to the nuclear transport proteins and exhibiting the anti-estrogen action of the drug. By inhibiting the transport of the nucleoside into the cell, the growth and proliferation of the carcinoma might be significantly reduced. The uptake of the Aspc cells was inhibited up to 80% at 30m M of tamoxifen as shown in figure 3. It exhibited an IC₅₀ of 3m M.

The quantitative relationship between the dose or concentration of a given ligand and its effect is a characteristic and important descriptor of many biological systems varying in complexity from isolated enzymes to intact animals. We can observe using the median effect principle to solve the problem of quantitating the effects of multiple inhibitors on such systems and provide definitions of summations of effects and consequently of synergism and antagonism. The CI at various f_a are given in figure 4. The results of 5-fluorouracil and dipyridamole indicate there is moderate antagonism at low f_a values and a marked synergism at high f_a vaules. This is comparable to that of tamoxifen and dipyridamole. However for 5-fluorouracil and tamoxifen there is marked synergism at low f_a levels and high antagonism at high f_a levels. Significance of such a result show that drugs have varying complexity of effect at different concentrations when mixed.

The effect of the drugs in vivo has a much similar effect as that in vitro. Drugs by themselves exhibit a much less effect than that in a combination. This show that there might be changes to the drugs chemically when they are mixed, resulting in increased toxicity to the carcinoma cells. Tumors in the 3-tier mixture shown in figure 6 exhibit the greatest reduction in growth over 35 days (~1000%). This far exceeds the percentage reduction in growth when the drugs are used individually on the mice.

In this paper we have demonstrated that tamoxifen, an anti oestrogen has an inhibitory effect on the NBMPR sensitive equilibrative nucleoside transport system. Prolonged exposure of pancreatic adenocarcinoma to tamoxifen and dipyridamole also cause down regulation of the NBMPR sensitive equilibrative nucleoside transporter on the cell membrane. Also exposure of the carcinoma cells to 5-fluorouracil, tamoxifen and dipyridamole over 5 days cause a reduction in growth of the cells, this may well pave the way to a similar experiment which is a time-based culture over 8 days incoperating the same drugs. My hypothesis is that the culture with the 3 drugs will exhibit minimal growth if not at all according to the results of the cultures above. This finding together with previous findings might support a new clinical role of these drugs for anti cancer regimes.

On the effect of the drugs on the tumors, this might be based on the hypothesis that the nucleoside inhibitor, dipyridamole which inhibits the efflux of the nucleosides but does not effect the influx of them. This allows the 5-fluorouracil, the analog of uracil, to enter the tumor cells thus increasing the effect of 5-Fu against the cells. The 3-tier effect will not be complete without tamoxifen, which might encourage differentiation of the carcinoma cells so that they might be "normal" again.

There is a presence of nucleoside transporters on Aspc cells. Tamoxifen has an inhibitory effect on nucleoside transporters therefore with this ability of tamoxifen to other effects such as increased cell differentiation, there is great possibility of applying tamoxifen as a chemotherapy regiment. Also tamoxifen with dipyridamole can enhance the 5-fluorouracil efficacy in Aspc cells. The objective of this paper is slightly met as the drugs are able to reduce the growth of carcinoma cells both in vitro and in vivo. Further studies could be made on the effect of drugs over longer periods of time in vivo to further test this hypothesis.

I thank Dr Lee Chee Wee for his academic expertise on this issue and Dr Cai Jun for her patience and technical knowledge which have been proven invaluable.

This project was supported by a grant from the National University of Singapore.

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