Cytotoxicity of Poly(Phenolic)Sulfonates and Their Sodium Salts in L1210 Lymphoid Leukemia Cells

Poly(phenolic)-sulfonates demonstrated very good cytotoxicity against the growth of tumor cell lines (L1210, Tmolt-3, HeLa-S3) and are comparable in potency with typical clinically used anticancer drugs. Four of the most active compounds, i.e. GL-2021, GL-2029, GL-2041 and GL-2063, were selected for a mode of action study in L1210 lymphoid leukemia cells at concentration of 25μM to 100μM for 60 min. The agents did not alkylate bases of ct-DNA, cause intercalation between base pairs, produce cross linking of ct-DNA strands or generate free radicals although L1210 DNA fragmentation was observed after 24 hr incubation. L1210 DNA synthesis was preferentially inhibited which was achieved by (1) suppressing DNA polymerase α activity which reduced the synthesis of new strands of DNA, (2) reducing of de novo purine synthesis at the regulatory enzyme PRPP amido transferase which reduced d(GMP) levels, and (3) inhibiting of nucleoside kinase activities which further reduced DNA synthesis. DNA template activity was altered by the poly(phenolic)sulfonates since they reduced DNA polymerase α and m-RNA and t-RNA polymerase activities. The kinetic studies at 50 μM over 2 hr demonstrated that the agents’ effect on PRPP-amido transferase activity is probably a major target of the compounds.


INTRODUCTION
Large molecular weight polymers, e.g. sulfated polysaccharide dextran sulfate, pentosan polysulfate, have demonstrated anti-HIV-1 and anti-HIV-2 activity by interfering with binding of the gp 120 protein to CD4 and blocking reverse transcriptase activity 1]. Hepsulfam, a new antineoplastic alkanesulfonate agent, has demonstrated a broad preclinical activity in human tumor xenografts [2,3] and oat cell lung cancer; however, this agent exhibited in vitro toxicity in human bone marrow cells [3]. Suramin, a known antitrypanosomal agent, was found to exert a strong inhibitory effect on RNA-directed DNA polymerase (reverse transcriptase) activity of several oncornaviruses such as Moloney murine leukemia virus, murine Rauscher leukemia viruses, Moloney murine sarcoma virus and avian myeloblastosis viruses [4][5][6][7]. Related to suramin is a class of sulfonic acid azo dyes, for example, Evans blue, which has been shown to be active against replication of the AIDS virus [6]. Recently a series of poly(phenolic)sulfonated compounds were shown to have potent anti-inflammatory activity and protection against induced endotoxic shock in mice at 8 and 16 mg/kg, I.P [8]. These agents prove to be potent elastase and prostaglandin cyclooxygenase inhibitors, blocking the release of TNFt and IL-1 release. The binding of these cytokines to high affinity receptors on their target cells were also competitively suppressed by the agents. These compounds blocked the adhesion of leukocytes and macrophages to L929 fibroblasts grown in tissue culture [5]. Because of these properties it was surmised that these poly(phenolic)sulfonated compounds may have antineoplastic activity.   [10] utilizing crystal violeeOH and read at 562 nm (Molecular Devices). Values for cytotoxicity were expressed as EDs0 g/ml, i.e. the concentration of the compound inhibiting 50% of cell growth. A value of less than 4 g/ml was required for significant activity of growth inhibition.

Incorporation Studies
Incorporation of labeled precursors into 3H-DNA, 3H -RNA and 3H -protein for 10 L1210 cells was obtained [11]. The concentration response for inhibition of DNA, RNA and protein synthesis of the compounds at 25, 50 and 100 l.tM was determined for 60 min incubations. Incorporation of 14C -glycine (53.0 mCi/mmol) into purines was obtained by the method of Cadman et al. [12]. Incorporation of 4Cformate (53.0 mCi/mmol) into pyrimidines was determined by the method of Christopherson et al. [ 13].
Enzyme Assays Inhibition of various enzyme activities was performed by first preparing the appropriate L1210 cell homogenates or subcellular fractions, then adding the drug to be tested during the enzyme assay. For the concentration response studies, inhibition of enzyme activity was determined at 25, 50 and 100 l.tM of compounds G1-2021, G1-2029, GI-2041 and G1-2063 after 60 min incubations. DNA polymerase Ot activity was determined in cytoplasmic extracts isolated by Eichler et aL's method [14]. The DNA polymerase assay was described by Sedwick et al. [15] using 3H-TTP. Messenger-, ribosomal-and transfer-RNA polymerase enzymes were isolated with different concentrations of ammonium sulfate; individual RNA polymerase activities were determined using 3H - UTP 16,17]. Ribonucleoside reductase activity was measured using 4C -CDP with dithioerythritol [18]. The deoxyribonucleotides 4C -dCDP were separated from the ribonucleotides by TLC on PEI plates. Thymidine, TMP and TDP kinase activities were determined using 3H -thymidine (58.3 mCi/mmol) in the medium of Maley and Ochoa [19].
Carbamyl phosphate synthetase activity was determined with the method of Kalman et al. [20] and citrulline was determined colorimetrically [21]. Aspartate transcarbamylase activity was measured using the incubation medium of Kalman et al. [20], carbamyl aspartate was determined colorimetrically by the method of Koritz et aL [22]. Thymidylate synthetase activity was analyzed by Kampf et al. 's method [23].

DNA Studies
After deoxyribonucleoside triphosphates were extracted [27], pool levels were determined by the method of Hunting and Henderson [28] with calf thymus DNA, E. coli DNA polymerase I, non-limiting amounts of the three deoxyribonucleoside triphosphates not being assayed, and either 0.4 mCi of (3H-methyl)-dTTP or (5-3H)-dCTP. The effects of poly(phenolic)sulfonates on L1210 DNA strand scission was determined by the methods of Suzuki et al. [29], Pera et al. [30], and Woynarowski et al. [31]. L1210 [33]. Superoxide scavenger activity was determined in an analogous manner with 0.4 mM ferricytochrome C in 20 mM phosphate buffer pH 7.4 at 37 C at 550 nm. [34]. Standards, i.e. 3% hydrogen peroxide and zymosan at 5mg/ml [Sigma] were used to generate free radicals in the assays for comparison to the poly(phenolic)sulfonates.

Statistical Test
In Tables 3-6, the data is represented as the average of the percent of control and the standard deviations. In Fig 2-12, all standard deviations are within 5.2% of the value. The probable significant different [p] between the treated raw data and the control raw data for each assay was determined using the Student's "t" test.

RESULTS
The poly(phenolic)sulfonates demonstrated potent cytotoxic activity [Table2] with EDso values of less than 4 t.tg/ml for significant activity. Mouse L 1210 lymphocytic leukemia, human Tmolt3 T cell leukemia and human HeLa-S uterine carcinoma growth were significantly reduced by all of the agents including the standard pentosan sulfate with EDs0 values < 3 lag/ml. G1-2157 and G1-2021 demonstrated the best activity in the L1210 lymphoid leukemia screen. G1-2063 was very potent in suppressing growth of human Tmolt3 T cell leukemia cells. G1-2029 demonstrated the best activity against the growth of HeLa-S uterine carcinoma. Human solid HeLa uterine carcinoma growth was significantly reduced by G1-2021 with an EDs0 value of 1.97 lag/ml. KB nasopharynx growth was reduced by G1-2041 [EDs0 2.60 g/ml] and moderately reduced by G1-2158 and G1-2063 with values of 3.24 and 3.42 lag/ml, respectively. Human SW480 adenocarcinoma colon growth was significantly reduced by G1-2030, and GI-2021 with EDs0 values less than 2 g/ml. Human lung MB9812 bronchogenic growth was suppressed moderately by G1-2063 and pentosan sulfate. Human HCT-8 ileum adenocarcinoma, lung A549, glioma HS683 and rat UMR-106 osteosarcoma growth was not affected by the test compounds. Compounds G1-2021, G1-2029, G1-2041 and G1-2063 demonstrated the most potent activity in the mouse L 1210 lymphocytic leukemia screen and consequently were selected for the mode of action study. These four agents preferentially affected L 1210 lymphoid leukemia DNA synthesis with reduction of greater than 60% at 100 l.tM over 60 min [Tables 3-6]. RNA and protein syntheses were more moderately affected by the agents with 13 -22% reduction of RNA synthesis over 60 min at 100 M and 29% to 43% reduction of protein synthesis over hr. DNA polymerase x activity was reduced 33% to 56% after hr at 100 I.tM. m-RNA polymerase activity was reduced 50% to 60% and t-RNA polymerase activity was reduced 24% to 41% at 100 laM over hr. r-RNA polymerase activity was elevated slightly by G1-2021 20% to 23% after hr from 25 to 100 ktM, but the other three agents had no significant effect on this enzyme activity. 6.94 pmol Ribonucleoside reductase activity was not reduced by any of the four poly(phenolic)sulfonates over hr but rather it was stimulated significantly particularly at the lower concentrations after this time period. Dihydrofolate reductase activity was reduced only by compounds G1-2021 by 24% and G1-2063 by 46% at 100 t.tM after hr. L1210 de novo purine synthesis was suppressed 18% to 46% at 100 ktM of poly(phenolic)sulfonates over hr with the activities of regulatory enzyme PRPP amido transferase activity being suppressed 61% to 90% and IMP dehydrogenase activity being reduced 23% to 34% at 100 tM over hr. In all three of these assays the agents demonstrated a concentration dependent suppression of activity. De novo pyrimidine synthesis was not significantly reduced by the agents at these concentrations for hr. Carbamylate phosphate synthetase activity was reduced 27% to 38% at 100 M after hr. But aspartate transcarbamylase activity was not affected by the compounds, significantly. Thymidylate synthetase activity was reduced marginally 8% to 18% at 100 txM after hr. Thymidine kinase activity was suppressed 12% to 24%, TMP kinase activity was reduced 63% to 68%, TDP kinase activity was inhibited 46% to 74% after hr at 100 laM. Again, the effects of the agents on these latter two enzyme activities was concentration dependent, d[NTP] pool levels were only marginally affected by the compounds, d[ATP] levels were reduced 32% to 38% after hr at 100 M; whereas, d[GTP] pools were reduced21% to 28% by the poly(phenolic)sulfonates, d[CTP] pool levels were lowered 4% to 33% and d[TTP] levels 2% to 17% after hr incubation at 100 l,tM. ct-DNA studies demonstrated that the agents did not affect DNA denaturation since the Tm values for the control was 82.8 C, G1-2021 was 81.5 C, G1-2029 was 82.3 C, G1-2041 was 83 C and G1-2063 was 83C suggesting no cross linking of the strands of DNA. ct-DNA U.V. absorption from 220 to 340 nm did not demonstrate a hyperchromic shift to a higher wavelength [ Fig. 1] suggesting that the agents did not interact with the individual bases of DNA, i.e. alkylation of purines and pyrimidines. which are usually multiples of the ED50 values, in order to identify the targets of the agents. L 1210 DNA synthesis over 2 hours in the presence of agents at 50 tM was reduced significantly after 60 min and continued to be reduced in higher magnitude throughout the 2 hr. [Fig 6] following a time dependent effect. DNA polymerase activity was reduced after 30 min with agents at 50 l.tM but with G1-2041 and G1-2063 between 60 and 120 min the activity recovered. With two of the compounds, i.e. GI-2021 and G1-2029, activity remained suppressed at 60 and 90 min. [Fig 7] while with GI-2041 and G1-2063 the DNA polymerase activity returned to normal at 90 min. L1210 m-RNA polymerase activity was significantly reduced after 30 min, but with increasing times of incubation achieved the lowest value, e.g. 50% to 60% at 120 min. [Fig 8 ]. PRPP-amido transferase activity was significantly reduced greater tha 60% aider 60 min and remained consistently suppressed over the next 2 hr. [Fig 9]. Thymidine kinase activity was only marginally inhibited at 90 min and required 2 hr to observe at least 40% inhibition of activity [Fig 10]. TMP kinase activity demonstrated marked reduction at 60 min but the activity recovered at 90 and 120 min [Fig 11 ]. TDP kinase activity was erratic also with the highest reduction at 60 min but again the activity recovered after 90 min [Fig 12].

DISCUSSION
The poly(phenolic)sulfonates like many polymeric agents, e.g. suramin, demonstrated cytotoxic activity against mouse, rat and human tumor cell lines with EDs0 values in the range of standard clinical agents. These derivatives were very effective inhibitors in suspended mouse and human tumor cells, e.g. mouse L1210 lymphoid leukemia, human Tmolt3 T cell leukemia, and HeLa-S uterine carcinoma. The compounds were not as active against the growth of solid rat and human tumor cultured cells. Selective activity was demonstrated by some of the agents. GI-2021 was effective against human solid HeLa uterine carcinoma, GI-2041 was active against human KB nasopharynx carcinoma, GI-2158 was effective against lung MB9812 bronchogenic carcinoma and GI-2030 and GI-2021 were active against growth of human colon adenocarcinoma growth. The agents were not active against the growth of HCT-8 ileum adenocarcinoma, lung A549, nor glioma HS683 growth.
Lymphoid Leukemia Cells  The type of selectivity demonstrated by the poly(phenolic)sulfonates, was typical of antineoplastic agents which interfere with cellular metabolism. Suramin has been shown to be an effective antineoplastic agent against Kaposi's sarcoma, non-Hodgkin lymphomas, lung carcinoma, metastatic prostate, adrenal adenocarcinoma and kidney adenocarcinoma [35][36][37]. Kinetic growth studies demonstrated that the poly(phenolic)sulfonate agents were cytostatic as opposed to cytocidal and they did not follow a concentration dependent inhibition of growth of L1210 cells [from  M] at higher concentrations of the agents. This could be due to saturation of cellular transport processes by the agent or saturation of the key target receptor by the agent for inducing cell death. It is interesting to note that suramin, a hexasulfated naphthylurea, required a concentration of 250 l.tg/ml or 1.75 mM in HeLa cells to cause death and required 40 laM to inhibit SV40 replication in HeLa cells [38]. The growth of human lung cancers PC-9, PC-14 and H69 and their resistant strains to cisplatin or etoposide was suppressed by suramin at 60 to 400 ktg/ml [39]. In Chinese hamster growth DC-3F and DC-3F/9-OH-E (resistant 9-hydroellipticine subline) EDs0 values for suramin were 31 ktM and 203 ktM [41 ]. The poly(phenolic)sulfonates in the HeLa solid tumor screen afforded EDs0 values between 1.97 l.tg/ml and 8.74 ktg/ml and the A549 and MB98 lung tumor were suppressed between 3.49 gg/ml and 10.3 ktg/ml by the agents suggesting that they probably would be more potent than suramin in vivo in suppressing cancer growth. L1210 lymphoid leukemia mode of action studies with four of the more potent agents, i.e. G1-2021, G1-2029, G1-2041, and G1-2063 from 25-100 ktM over 60 min demonstrated that DNA synthesis appears to be the major target of the agents.That is not to exclude the moderate inhibition of protein synthesis that occurred with some of the derivatives but certainly not by all four of the compounds. DNA synthesis inhibition by the poly(phenolic)sulfonates is probably the result of multiple inhibitory effects of the compounds which are additive to induce cell death. All four agents suppressed the activities of DNA polymerase ix, PRPP-amido tmnsfemse and TMP and TDP kinases significantly in a concentration dependent manner. Moderate inhibition was observed by the agents for dihydrofolate reductase and IMP dehydrogenase activities. The magnitude of inhibition of either one of these enzyme activities was not sufficient to account for the observed reduction of DNA synthesis.Nevertheless, the summation of the suppression of all of these metabolic events would be sufficient to account for the observed DNA synthesis suppression in 60 min. The four agents generally followed a concentration dependent response for both the inhibition of DNA synthesis and suppression of the DNA polymerase tx, PRPP-amido transferase and TMP and TDP kinases enzyme activities from 25-100 laM over 60 min. values ofthis enzyme for GI-2021 since the only difference between GI-2021 and G1-2063 is that the former has a methyl group in R' and G1-2063 has a methoxy group. The IC50 values [21-42 l.tM] for TDP kinase inhibition were lower for the two compounds which were substituted in the R' position with a methyl or methoxy group. The "n" number of the compound being 6 or 8 did not seem to explain the difference observed for the IC50 values for enzyme inhibition. The IC50 values [78-90 mM] for mRNA polymerase inhibition was slightly better with compounds possessing an "n" of 8. The ability of the agents to suppress multiple enzymatic sites in nucleic acid metabolism is not surprising since 6-MP, araC and 5-FU inhibit more than one metabolic process in cancer cells. Inhibition of DNA polymerase x activity by the agents would suppress the synthesis of a new strand of DNA in the S phase of the cell cycle. Ordinarily this would lead to the accumulation of d[NTP] pool levels. However, this was not observed after the 60 min of this study, i.e. the d[NTP] remained approximately normal or were slightly reduced in concentration. Since the poly(phenolic)sulfonates also inhibited L 1210 PRPP-amido transferase activity markedly from 25 to 100 l.tM over 60 min, this should result in the reduction of over all de novo purine synthesis lowering the pool levels of AMP and GMP. These studies reflected the inhibition of purine synthesis in 60 min but the magnitude of reduction by the agent was not as great as the inhibition of PRPP-amido transferase activity at this time. This may be due simply to a time delay in the effects of the agent and eventually the overall reduction of de novo purine synthesis would be of the same magnitude of reduction as the suppression of the regulatory enzyme and also the effects will be further reflected in the cellular d[NTP] pools. Certainly even after 60 min the purine deoxyribonucleotides were generally reduced but the effects on pyrimidine deoxyribonucleotides were not in evidence at this time of incubation. L1210 ribonucleoside reductase activity was not affected by the agents; thus, the conversion from ribonucleotides to deoxyribonucleotides was not a factor in the mode of action of these agents.  [7]. Since L1210 RNA synthesis was marginally reduced by the poly(phenolic)sulfonates over the 60 min period at 100 l.tM, this was probably the result of the reduction in ribonucleotides due to the reduction in de novo purine synthesis by these agents. This does not appear to be a major site of action of the derivatives which would be responsible for cell death or apoptosis. These studies also indicated that the compounds suppressed m-RNA [II] and t-RNA [III] polymerase activities after 60 min. Since in these assays all of the ribonucleotides were added to the reaction medium, this inhibition with the agents appears to be due to interference with template activity of the RNA polymerases by the compounds. Even the inhibition of DNA polymerase ct activity by the agents may reflect interference with the use of the template by the poly(phenolic)sulfonates. The polyanionic nature of the agents may allow binding of some type to the DNA molecule since the histones are basic so that polymerase may be having difficulty in using the template to copy a new DNA or RNA molecules. Suramin has also been reported to block KB III nasopharynx RNA polymerase activities, E coli DNA polymerase and RNA polymerase and Rauscher murine leukemia virus reverse transcriptase activity by competing for the DNA template binding site on the polymerase enzyme [42]. It was postulated that suramin binds to the basic amino acids of the enzyme perhaps via the sulfonic groups. A similar argument could be made for the observed inhibition of polymerase activities with the poly(phenolic)sulfonates. The poly(phenolic)sulfonates did not directly affect the DNA molecule nor does suramin [41]. L1210 lymphoid leukemia DNA fragmentation did occur after 24 hours incubation using whole cells. Suramin has been shown to be a DNA topoisomerase II inhibitor in PC-9 lung cancer cells and Chinese hamster fibrosarcoma cells [41]. It is possible that poly(phenolic)sulfonates function by this method also to afford DNA fragmentation. Evidence would suggest from the findings of the studies on poly(phenolic)sulfonates that they have a dual mechanism of action on cancer cells. These studies have already demonstrated that these derivatives suppress polymerase and PRPP-amido transferase activities in L 1210 lymphoid leukemia cells. These studies have demonstrated that poly(phenolic)sulfonates have characteristics of the antineoplastic agents similar to suramin which has been used in advanced human malignant cancers that are refractory to standard cancer chemotherapy [43]. Whereas the studies with poly(phenolic)sulfonates are in their initial stages and certainly additional studies are warranted, and since they have demonstrated more potency in a number of these biochemical assays than suramin, they may have potential in the future as clinical agents to treat cancer patients.