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Inhibition of DNA Synthesis in P388 Lymphocytic Leukemia Cells of BDF1 Mice by cis-Diamminedichloroplatinum(II) and Its Derivatives
Inhibition of DNA Synthesis in P388 Lymphocytic Leukemia Cells of BDF, Mice by cisDiamminedichloroplatinum(ll) and Its Derivatives
c. BRENTOSWALD*, STEPHEN G.CHANEY*, AND IRIS H. HALL*' Received August 30, 1988, from the 'Division of Medicinal Chemisty and Natural Products, School pf Pharmacy C6#7360,and the *Department of B/ochemisfry and Nutrition, School of Medicine CB#7260, University of North Carolina, Chapel Hill, NC 27599. Accepted for publication November 29, 1989. Abstract 0 ci+Diamminedichloroplatinurn(ll) (cisplatin; cDDP) derivatives were found to afford T/C% values >200 against the growth of P388 lymphocytic leukemia cells in vivo. The parent compound, cDDP, preferentially inhibited DNA synthesis. The RNA synthesis was elevated, whereas protein synthesis was unaffected after two or three daily ip doses. Radiolabeled drug studies demonstrated cellular uptake and binding of cDDP derivatives to the DNA molecule. cis-Diamminedichloroplatinum(l1) (cDDP) treatment resulted in DNA strand scission after a single dose, but caused cross-linking of DNA strands after two or three ip doses. There was an accumulation of deoxynucleoside triphosphates [d(NTP)s]on day 2 and 3, indicating that incorporation of nucleotides into the DNA strand had been blocked. Thymidine kinase, thymidine monophosphate kinase, carbamoyl phosphate synthetase, and aspartate transcarbarnoylase activities were inhibited in vivo after three doses of cDDP at 1.5 mglkglday. However, only the inhibition of a cytoplasmic preparation of DNA polymerase a by cDDP appeared to be directly related to the inhibition of DNA synthesis and the accumulation of d(NTP) pool levels. Thus, the primary target for cDDP appears to be DNA itself, although direct inhibition of DNA polymerase a may play a minor role in the inhibition of DNA replication by cDDP.
cis-Diamminedichloroplatinum(I1) (cisplatin; cDDP) has been demonstrated to have antineoplastic activity against the growth of Sarcoma 180 and L-1210 lymphocytic leukemia in mice14 and positive results against Dunning ascitic leukemia and intramuscular Walker 256 carcinosarcoma.4 In humans, the agent has been effective in causing regression of testicular, colon, bladder, breast, a n d thyroid cancer, and neuroblastoma, malignant thymoma, lymphomas, endometrial cancer, and squamous cell carcinoma.l-6 Harder and Rosenberg" have shown that cDDP preferentially inhibited DNA synthesis in human amnion AV3 cells, as has been observed in mouse Ehrlich ascites t u m o r cells8 and human Hela cells.9 The inhibition of DNA synthesis by cDDP was not immediate. The purine bases of DNA (primarily G-6 and A-6) appeared t o be the major target of the drug.S.9 Intrastrand and interstrand cross-linking of the bases of DNA h a v e been proposed as a mode of action for the drug.10 However, the inhibition of DNA polymerase a activity b y cDDP has also been observed by Harder et al.11 a n d cannot be excluded as possibly contributi n g to the overall cell killing effect of the drug. Thus, the effect of cDDP on P388 lymphocytic leukemia cells i n vivo was investigated to determine if inhibition of DNA polymerase a or o t h e r enzyme activities might be contributing factors in tumor cell death.
Nuclear, Boston, MA. Radioactive samples were placed in Fisher Scintiverse and counted using a Packard Tricarb scintillation counter correcting for quenching by a n internal standardization technique. cis-Diamminedichloroplatinum(I1) (cisplatin;cDDP) was eynthesized by the method of Dhara,', cis-diamminemalonatoplatinum(II) [Pt(mal)(NH,),l was synthesized as reported by Speer et al.,14 (1,2diaminocyclohexane)dichloroplatinum(II) [PtCl,(dach)] was synthesized by the method of It0 e t al.,13 and (1,2-diaminocylohexane)malonatoplatinum(I1) [Pt(mal)(dach)]was synthesized by the method of Speer et al.14 Radioactive syntheses of the latter two compounds have previously been published.15 All compounds were satisfactorily analyzed for carbon and hydrogen (i.e., <0.4% difference between calculated and analyzed values).l6J7 Determination of T/C%-All experiments were performed with BDF, male mice (25 g) inoculated with 1 x lo6 P388 lymphocytic leukemia cells18 suspended in sterile isotonic saline on day 0. The drugs cDDP, Pt(mal)(NH,),, PtCl,(dach), and Pt(mal)(dach),and the internal standard 5-fluorouracil were suspended in 0.05% polysorbate 80-water by homogenation. Drug solutions were administered ip a t a dose of 0.25 to 25.0 mg/kg/day. The TIC% values were obtained according to the NCI protocol.16 The average life span of the treated group was divided by the control group's life span and then multiplied by 100 to obtain a percent. 5-Fluorouracil was used as a standard for the in vivo screen. Cytotoxic activity18 was determined by incubating P388 cells (5 x lo4) with &-platinum drugs at final concentrations of 0-25 pg/mL in Eagle's MEM 10% fetal calf serum plus streptomycidpenicillin. The number of viable cells was determined on Day 3 using a Levy cell counting chamber and trypan blue exclusion. The concentration of drug necessary to achieve 50% inhibition of cell growth (ED,,) was determined by plotting the log of drug concentration versus the cell number.18 Etoposide VP-16 was used as an internal standard for this assay. In Vivo Studies-All in vivo studies were performed by treating test animals on days 7,8, and 9 after tumor inoculation. Three hours after the last dose (noted as day 1,2, and 3, respectively) the animals were sacrificed, the ascites fluid was harvested, and assays were performed on the appropriate cell preparations. In Vivo DNA, RNA, and Protein S y n t h e s i s o n e hour prior to sacrifice, the animals were injected ip with 10 pCi of [methyl'Hlthymidine (84 Ci/mmol), 10 pCi of [6-'H]uridine (22.4 Ci/mmol), or 10 pCi of L-[4,5-3H(N)]leucine(56.5 Ci/mmol). Following sacrifice,
cDDP
,.O
Experimental Section Materials-All biochemicals, 5-fluorouracil, and etoposide were purchased from Sigma Chemical, St. Louis, MO. [8-14C]Inosine-5'monophosphate was purchased from Amersham, Arlington Heights, IL. All other radiochemicals were purchased from New England OO22-3~9/90/1000-0875$0 1.OO/O 0 1990, American Pharmaceutical Association
MAL
DACH-Pt-CI
DACH-Pt-MAL
Journal of Pharmaceutical Sciences I 875 Vol. 79, No. 10, October 1990
the P388 cells were collected. Incorporation of 13Hlthymidine into DNA was determined by the modified method of Chae e t al.19 Results are reported a~ disintegrations per minute (dpms) of l3H1thymidine incorporated into DNA per milligram of DNA is6lated'g as determined by the diphenylamine reaction. Incoiporation of [3Hluridine into RNA was determined according to the method of Wilson et al.2" The concentration of hydrolyzed RNA was assayed by the orcinol method.*] Results were determined as dpms of [3Hluridine incorporated into RNA per milligram of RNA isolated. Incorporation of I3Hlleucine into protein was determined by the method of Booth and Sartbrelli.22 Results were calculated as dpms of ["Hlleucine incorporated per milligram of protein isolated as determined by the Lowry technique.23 Enzyme Assays-The DNA polymerase (Iactivity was determined using a cytoplasmic extract isolated by the method of Eichler et al." The nuclear supernatant DNA polymerase activity was determined in a sample prepared by centrifuging isolated washed nuclei a t 800 x g for 10 min and using the nuclei supernatant to assay DNA polymerase. The DNA polymerase assay was that of Sawadaa using 13H]lTP. Messenger. ribosomal, and transfer RNA polymerase enzymes were isolated using different concentrations of ammonium sulfatez6 and the individual RNA polymerase activities were determined using I3H1UTP.27 Ribonucleotide reductase activity was measured by the method of Moore and Hurlbertm using 114C1CDPwith dithioerythriol. Thymidine and thymidine mono- and diphosphate kinase activities were measured by a spectrophotometric assay based on the disappearance of NADH a t 340 11111.29 Carbamoyl phosphate synthetase (CAP) activity was measured by the method of Kalman e t aL.30 using a colorimetric determination of citrulline.31 Aspartate transcarbamoylase (ATC) activity was measured by the method of Kalman e t aI.,31 with carbamoyl aspartate being determined colorimetrically.32 The OMP decarboxylase activity was assayed with a 16,300 x g for 20 min supernatant and [l4CJ0MP.33Thymidylate synthetase activity was analyzed by the method of Kampf e t al."4 Tritiated water (3H,0) was measured as being proportional to the amount of TMP formed from 13H]dUMP. Dihydrofolate reductase activity was determined by the spectrophotometric method of Ho et a1.s Formate incorporation into purines was determined using ["C]formic acid. Adenine ahd guanine were separated by TLC.36 The PRPP amidotransferase activity was determined by the method of Spaseova et a1.,37 and inosinic acid monophosphate dehydrogenase activity wa6 determined using I"CIIMP, where XMP was separated on PEI plates by TLC.m Protein was determined for all of the enzymatic assays by the Lowry technique.23 Deoxyribonucleotide triphosphates were extracted by the method of Bagnara and Finch.39 Deoxyribonucleotides were determined by the method of Hunting and Henderson40 using calf thymus DNA, E . coli DNA polymerase 1, nonlimiting amounts of the three deoxyribonucleoeide triphosphates that were not being assayed, and 0.4 pCi of either [3H-methyl]d?TP or [5'H]dCTP. T h e effects of cDDP, Pt(mal)(NH3),, PtCl,(dach), a n d Pt(mal)(dach) on DNA were measured on alkaline sucrose gradients aa described by Suzuki e t al.,41 Pera et al.,42 and Woynarowski e t a1.d3 The BDF, mice bearing P388 lymphocytic leukemia cells were injected ip with 10 KCi of thymidine ( m e t h ~ l - ~84.0 H , Ci/mmol) 24 h prior to sacrifice. After harvesting the P388 cells from the peritoneal cavity, 5 x lo6 cells were centrifuged at 600 x g for 10 min in PBS, washed, and suspended in 1 mL of PBS. Lysis buffer (0.5 mL of O.5M NaOH, 0.02 M EDTA. 0.01% triton X-100, and 2.5% sucrose) was layered onto a 5 2 0 % alkaline sucrose gradient (5mL of 0.3 M NaOH, 0.7 KCI. and 0.01 M EDTA) followed by 0.2 mL of cell preparation. After incubating for 30 min at room temperature, the gradient was centrifuged a t 25 000 rpm and 20'C for 60 min (Beckman rotor SW 60). Fractions were collected (0.2mL) from the top of the gradient, neutralized with 0.2 mL of 0.3 M HCI, and counted. P388 Cell Uptake and Binding of DNA, RNA. and Protein-The BDF, tumor bearing mice were treated with [3HlPtCLZ(dach)(0.75 mg/kg/day, ip) or I'HH]Pt(mal)(dach) (17.5 mglkglday. ip) on days 7.8, and 9.15 Three hours after each treatment, the ascites fluid was collected and centrifuged at 800 x g for 5 min. The cells were washed six times and counted. The cells were sonicated to break the cell membranes and the aliquots were counted. Cells were chemically extractedfor DNA, RNA, and protein as previously described, and the aliquota were counted. Results are expressed as d p d m g of isolated macromolecule. 876 / Journal of Pharmaceutical Sciences Vol. 79, No. 10, October 7990
Results cis-Diamminedichloroplatinum(I1)demonstrated potent antineoplastic activity against P388 lymphocytic leukemia growth in BDF, mice. The dose response study demonstrated that the optimum dose for cDDP was 1.5 mg/kg/day ip, affording a T/C% of 219 which compares favorably with that for 5-fluorouracil (5FU) (Table I). The optimum doses for Pt(mal)(NH,),, PtClJdach), and Pt(mal)(dach) were found to be 10, 0.75, and 17.5 mgkglday ip, respectively. Since radiolabeled PtCl,(dach) and Pt(mal)(dach) were available, the uptake and distribution of these two compounds in P388 cells in vivo were measured over a 3-day period. A greater percentage of the [3H]F'tCl,(dach) entered the cell on each day than did L'H]Pt(mal)(dach) (p 5 0.001; Figure 1).After two doses of the I3H1PtCl,(dach) at 0.75 mgkg, >200 pmol of drug/106 P388 cells was associated with the cells. This level remained essentially unchanged after three doses. The T'H]Pt(mal)(dach) a t 17.5 mg/kg exhibited a cell uptake of only 15 pmol of drug/106 cells after three doses (Figure 1). At the intracellular level, 34% of the ['H]F't(mal)(dach) was bound to DNA after one dose, 48% after two doses (p 5 0.005 analysis of variance), and 38% after three doses (Figure 2). For l'HlPtCl,(dach), 5.4% was bound to DNA after one dose, and this was significantly different (p 5 0.05 analysis of variance) from the 1.88 after two doses and 1.7% after three doses. Binding of the 3H-labeled drugs to isolated DNA, RNA, and protein from P388 cells showed that both drugs bound preferentially to DNA (Figure 3). The ["H]PtCl,(dach), a t 0.75 mg/kg, afforded >600 pmol of drug/mg of DNA (p 5 0.001) after two doses and the third daily dose produced no further binding to DNA. The RNA binding peaked after two daily doses at -200 pmol of drug/mg of RNA. The ['H]Pt(mal)(dach), at 17.5 mg/kg, produced a dose-dependent binding to DNA with -900 pmol of drug/mg of DNA after three doses (p 5 0.001).The RNA binding reached a peak of -600 pmol of drug/mg of RNA after two doses (p 5 0.001), with no further binding with three doses. Protein binding was -50 pmol of drug/mg of protein after 1, 2, or 3 days of treatment with either drug. Mechanistic studies were carried out primarily with cDDP at a dose of 1.5 mg/kg/day since that dose gave the best survival time for this strain of mice. Doses of 1.5 mg:kg cDDP on days 7, 8, and 9 after P388 innoculation led to 54% reduction in the tumor cell numberlml of ascites on day 9 (Tables I1 and 111). Thymidine incorporation into DNA after a single dose of cDDP was elevated 37% (day 71, but after the second dose was reduced 64% (day €9, and by the third dose Table CCytotoxiclty and Antlneoplastic Activity of cDDP and R r Derlvatlves againrt P388 Lymphocytic Leukemia Growth In BDF, Male Mlce
Antitumor Screen Compound (n = 5) cDDP Pt(mal)(NH,), PtCl,(dach) Pt(mal)(dach) 5-Fiuorouracil VP-16 (Etoposide) Control
Optimum Avg. Days dose' Survived mglkglday 1.5 10 0.75 17.5 12.5
-
22.8 2 23.4 ? 29.8 -t 22.6 2 21.7 2
-
3.4' 3.8' 7.6' 4.3' 3.5'
10.4 2 1.1
Cytotoxicity Yo T/C
Value' 219 225 287 217 209
-
MImLb 2.4 11.0 10.5 13.6 3.7 2.5
-
a ?/oT/C= [average days survived by treated animalslaverage days survived by control animals] x 100; %TIC > 125 are considered significant according to NCI protocol (ref 16). Values < 4 M/mL are considered significant according to NCI protocol (ref 16). p = 5 0.001 r test.
3H-OACH-Pt-MAL (17.5mg/kg/dose)
3H-DACH-Pt-CI, (0.75rng/kg/dosd
200 1000
160
! t
j120
\
W 0
a
0 \
3 40
m
W 0
E
o
-
400
E"a
(3
0
600
a
80
U
5a / , ,
*----ADACH-Pt-MAL
I
6
200
0
I
2
3
# DAILY DOSES
(1
4 2 0
vivo binding of 3H-labeled platinum compounds to P388 macromolecules after one, two, and three daily ip doses of [3H]Pt(mal)(dach)or [3H]PtC12(dach).Key: (0-0) binding to DNA; (0-0) binding to RNA; (A-A) binding to protein. Standard deviations for all values were <8 pmol/mg for each data point given (n = 6). Binding of each drug to DNA was significantlydifferent (p 5 0.001)than RNA and protein on each day. Figure &In
2
I
3
# OF DAILY DOSES Figure 1-In vivo P388 leukemic cell uptake and DNA binding of (17.5 rng/kg/ [3H]PtC12(dach)(0.75mg/kg/day) and [3H]Pt(mal)(dach) day) after one, two, and three daily ip doses. Standard deviations for all values were <6 pmol for the cell uptake and 0.5 pmol for DNA binding of drugs (n = 6). A t test demonstrated a p 5 0.001 for the cell uptake of drugs on each day. Only on day 3 was the pmoles of drug bound to DNA significantly different (p 5 0.001) between the two drugs.
2
D 40
'ADACH-Pt-MAL
0
i2 30 zj 20
*.f"---
........ .........9 DACH-Pt -C12
I 2 3 X OF DAILY DOSES Figure 2-Percentage bound to DNA of intracellular [3H]PtCl,(dach) (0.75rngikglday) and [3H]Pt(mal)(dach)(17.5mg/kg/day) P388 after one, two, and three daily ip doses. The percent drug bound to DNA between drugs was significantly different (p s 0.001) on each day. Standard deviation for all values was 4%(n = 6). (day 9) was inhibited 82% (Tables I1 and 111).Incorporation of uridine into RNA was increased, but incorporation of leucine into protein was unchanged after three daily doses of cDDP (data not shown). The effect of cDDP treatment on a number of enzyme activities, required for nucleic acid synthesis, was then determined. Many of these enzyme activities were not significantly altered by cDDP treatment for three days at the therapeutic dose, including mRNA, rRNA, and tRNA polymerase, ribonucleotide reductase, PRPP amidotransferase, OMP decarboxylase, and thymidylate synthetase activities. Some enzyme activities were slightly elevated after cDDP treatment [e.g., dihydrofolate reductase (44%) and IMP dehydrogenase (25%), with an overall increase of formate incorporation into purines (50%;p 5 0.001). Several other enzyme activities did appear to be significantly inhibited by cDPP (Tables I1 and 111).Thymidine, thymidine monophosphate, and thymidine diphosphate kinase activities showed progressively greater inhibition with sequential doses of
cDDP over the 3-day period. Carbamoyl synthetase and aspartate transcarbamoylase activities were not inhibited by a single dose. However, carbamoyl phosphate synthetase activity was inhibited significantly after two doses and aspartate transcarbamoylase activity after three doses. Since DNA synthesis was decreased preferential by the drug and some of the enzymes required to synthesize nucleotide precursors for DNA synthesis were also inhibited, the location of the metabolic block was studied further by examining deoxynucleoside triphosphate (dNTP) levels after exposure to cDDP. Deoxynucleoside triphosphate pool levels were elevated in a dose-dependent manner by cDDP treatment (Tables I1and 111).One, two, and three daily doses of cDDP caused elevations in all four dNTP pools. The effect of repeated cDDP doses on DNA polymerase activity was studied in several subcellular fractions. A single dose of cDDP increased DNA polymerase activity in the cell homogenate. The second dose caused a 38% decrease in activity which was essentially the same decrease seen at the end of day 3 (Table IV). The cDPP in the 600-g pellet was significantly inhibited after all three doses. However, the inhibition could have been due to either inhibition of the enzyme or damage to the DNA template. Since DNA polymerase activity in the cell extract is a composite of repair and replicative activities,24 DNA polymerase activity was also measured in the nuclear supernatant and in a cytoplasmic DNA polymerase a preparation. Both of these assays were strongly dependent on exogenous DNA, so damage to the DNA template was not a factor. The nuclear supernatant DNA polymerase activity was elevated 334% by a single dose of cDDP, but then declined to only a 90% stimulation following the third dose (Table IV). Cytoplasmic DNA polymerase a showed little inhibition following the first and second doses, but was inhibited 24% after the third dose (Table IV). The effects on DNA of cDDP and the other platinum drugs in this study were examined on alkaline sucrose gradients (Figure 4). A single dose of cDDP caused a shift of the DNA molecule t o a lower molecular weight compared with the control DNA, suggesting strand scission (Figure 4A). Two and three daily doses of cDDP led to a shift of the DNA to higher molecular weight DNA, but with no prominent peaks, suggesting cross-linking of the DNA. Three daily doses of PtCl,(dach) and Pt(mal)(dach) also shifted the DNA to higher molecular weight fractions (Figure 4B). Journal of Pharmaceutical Sciences / 877 Vol. 79, No. 10, October 7990
Table ll-In Vlvo Effect0 of cDDP (1.5 mg/kg/day Ip) on Enzyme Activities and d(NTP) Pool Levels ofP388 Lymphocytlc Leukemic Cells after One, Two, or Three Daya of Dosing
Treatment
Control (n = 5) Day 1 2 3
a
p
5
Thymidine
Thymidine
Carbamoyl Phosphate
Aspartate Transcarbamoylase, mole of N carbamoyl aspartateihlmg protein
density unit/h/mg protein
Monophosphate Kinase, optical density unitlhlmg protein
Diphosphate Kinase, optical density unit/h/mg protein
136332 2 14990
0.062 f 0.009
0.039 f 0.006
0.150 f 0.014
0.316 f 0.02
3.989 f 0.0485
186775 2 3891 49079 2 5674' 25107 5 982'
0.047 f 0.008' 0.044 f 0.006' 0.037 f 0.006'
0.031 f 0.010 0.024 f 0.005' 0.015 f 0.003'
0.138 f 0.012 0.125 f 0.015 0.110 f 0.007'
0.349 f 0.022 0.226 f 0.019' 0.250 f 0.017'
3.916 f 0.367 4.029 f 0.425 2.526 f 0.231"
Thymidine
DNA Synthesis, dpm/h/mg DNA
0.001. p
5
Optical
'ynthetase* mole of
citrulline/h/mg protein
0.05 ( t test).
Table Ill-Deoxyrlbonucleoslde Trlphosphate Levels'
Treatment Control (n Day 1
dATP
= 5)
7.56 f 0.73 16.08 f O M b 19.60 2 1.98' 24.41 2 O.8lb
2 3 a Expressed as
pmol/lO6cells. p
5
dCTP
dlTP 12.34 2 13.88 -t 29.65 2 51.62 2
0.001. p
5
1.29
dGTP
0.48 f 0.44 9.06 f 0.78 16.51 f 2.31' 30.43 f 0.91
0.83
'
0.61 1.84'
5.73 2 7.23 8.03 2 12.47 2
0.56
* 0.59: 0.61 3.51
Cell Number (10*/mL) 2.71 f 0.34 2.61 f 0.37 2.12 f 0.36= 1.25 f 0.36b
0.05 ( f test).
Discussion Cisplatin (cDDP) and its derivatives proved to be effective antineoplastic agents against the growth of P388 lymphocytic leukemia cells in vivo in mice as reported previously.4~6The TIC% values for the four compounds compare favorably with those for standard commercially available antineoplastic agents. Although previous studies have implicated DNA as the target of platinum anticancer agents, this is the first study to quantitate the effect of cDDP on the intracellular enzyme activities and d(NTP) pool levels required of cDDP on the intracellular enzyme activities and d(NTP) pool levels required for DNA synthesis. Our data are certainly consistent with the role of DNA as the major target of the cDDP derivatives employed in this study. There was little or no inhibition of RNA polymerases and RNA synthesis was actually elevated following cDDP treatment. Elevation of RNA synthesis following cDDP treatment has been reported previously.'~~The inhibition of the activity of enzymes involved in de novo synthesis of purines (e.g., PRPP amidotransferase and IMP dehydrogenase), orpyrimidines (e.g., OMP decarboxylase and thymidylate synthetase), or of both (e.g., dihydrofolate reductase), was not sufficient to account for the magnitude of inhibition of DNA synthesis or cell kill observed with cDDP. Thymidine, thymidine monophosphate, diphosphate kinase, CAP, and ATC enzyme activities did appear to be significantly inhibited by cDPP treatment. However, the fact that d(NTP) pool levels were elevated after drug treatment indicated that sufficient precursors were
available to maintain DNA synthesis for cell survival and growth, and that DNA synthesis was inhibited at the step involving the incorporation of deoxyribonucleotidebases into the DNA. The observed inhibition of DNA polymerase a by cDDP would appear to be a n effect of cDDP on the enzyme rather than the DNA template since the assay used was largely dependent on exogenous DNA. The addition of calf thymus DNA in the assay resulted in a threefold increase in enzyme activity, but little change in the extent of inhibition of the polymerase a activity in cDDP-treated cells.44 Additional studies have demonstrated that cDDP treatment in vivo inhibits a P388 lymphocytic leukemia cell cytoplasmic DNA polymerase a preparation by 24% and a purified DNA polymerase a by 348.45 Harder et al.11 have previously noted that DNA polymerase a was inhibited by cDDP, although they reported significant inhibition only at nonphysiological cDDP levels. Our data show that inhibition of DNA polymerase a probably makes a small, but significant contribution to the inhibition of DNA synthesis at therapeutic levels of cDDP. The elevation of the nuclear supernatant DNA polymerase activity most likely reflects increased DNA repair activity in response to the formation of platinum-DNA adducts. Harder et al.11 have previously reported that cDDP elevates DNA polymerase p activity. Although platinum-induced DNA cross-linking has been widely studied in cell culture, it has seldom been examined in vivo. The DNA strand scission observed after the first day of
Table IV-In Vlvo Effects of cDDP (1.5 mglkglday Ip) on the DNA Polymerase Actlvlty from Vsrlour Cell Fractlons of P388 Lymphocytlc Leukemic Cells ~
dpm dlTP incorporated/h/mg/protein
Cell Fraction (n
=
6)
Cell homogenate
600-9 Pellet
Nuclear supernatant Cytoplasmic a preparation a p _c
0.001. p < 0.05 (t test).
878 / Journal of Pharmaceutical Sciences Vol. 79, No. 10, Ocfober 1990
Control 7181 f 363 8161 f 655 4438 2 391 4166 2 2414
1 Day
9177
f 1011'
5060 f 321' 19257 f 4102' 42719 f 6635
Treatment 2 Day
3 Day
4452 f 502' 6689 f 546' 13935 f 843' 39588 f 1268
4883 f 359" 2367 f 95" 8432 f 668' 31657 f 2068'
333r
-
22
A
261t18
/I iI
-
-
10
II
A
-
I
14
interesting to note that the extent of interaction with DNA was very similar a t comparable therapeutic doses. The maximum binding of the [SHlPtC12(dach)and [3HlPt(mal)(dach) to P388 DNA after three doses was calculated to be approximately one platinum atod100 base pairs for in vitro studies, with a binding ratio of 0.01 (1:100, to 1platinum a t o d l . 5 x lo6 base pairs.45>61The values for drug binding to DNA for this in vivo P388 leukemia study lie between these two extremes.
9
References and Notes
62
It
-_ 1
2
1
I
4
I
I
6
I
I
8
I
I
10
Y
I
1
12
I
14
1
1 1 -
16
18
FRACTION NUMBER
Bollom
20 Top
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1. Rosenber , B.; Van Camp, L.; Trosko, J. E.; Mausour, V. H. Nature 1869,222,385. 2. Rosenberg, B.; Van Camp, L. Cancer Res. 1970,30,1799. 3. Leh, F. K. V.; Wolf, W. J . Phurm. Sci. 1976,65,323. 4. Kociba, R. T.; Sleight, S. P.; Rosenberg, B. Cancer Chemother. Rep. 1970,54,325. 5. Wolpert-DeFillippes, M. K. Cancer Treat. Rep. 1979,63, 1453. 6. Hill, J. M.; Loeb, E.; MacLellan, A.; Hill, N. 0.;Kahn, A.; King, J. J. Can. Chemother. Rep. 1975,59,647. 7. Harder, H. C.; Rosenberg, B. Znt. J . Cancer 1970,6,207. 8. Howle, J. A.; Gale, G. R. Biochem. Phummcol. 1970,19,2757. 9. Pascoe, J. M.; Roberts, J. J. Biochem. Pharmacol. 1974,23, 1345. 10. Kelman, A. D.; Peresie, H. J. Cancer Treat. Rep. 1979,63, 1445. 11. Harder, H. S.;Smith, R. G.; LeRoy, A. F. Cancer Res. 1976,36, 3821. 12. Dhara, S.C. Znd. J . Chem. 1970,8,193. 13. Ito, H.; Fuamita, J.; Saito, K. Bull Chem. Soc. Japan 1967,40, 2584. 14. S eer, R. J.; Ridway, H.; Hall, L. M.; Newman, A. D.; Howe, E.; Stewart, D. P.; Edwards, G. R.; Hall, J. M. J . Clin. Hematol. Oncol. 1975,5,335. 15. Oswald, C. B.; Wyrick, S. D.; Chane , S. G.; Shrewsbu R. 0.; Hall, I. H. Res. Comm. Chem. P a t h . h m n a c o l . 1989,6?141-58. 16. Wyrick, S . D.; Chaney, S. G. J . Labeled Compds. Radiopharmceut. 1988,25,349-357. 17. Maudlin, S. K.; Richard, F. A,; Plescia, M.; Wyrick, S. D.; Sancar, A.; Chaney, S. G. Anal. Biochem. 1988,157,129-143. 18. Geran, R. I.; Greenberg, N. H: MacDonald, M. M.; Schumacher, A. M.; Abbott, B. J. Cancer Ckemother. Rep. 1972,3,9-24. 19. Chae, C. B.; Williams, A,; Krasny, H.; Irvin, J. L.; Piantadosi, C. Cancer Res. 1970,30,2652-2660. 20. Wilson, R. G.; Bodner, R. H.; MacWhorter, G. E. Biochem. Biophys. Acta 1975,378,260-268. 21. Ashwell, G. Meth. Enzymol. 1957,3,87-90. 22. Booth, B. A.; Sartorelli, A. C. J.Biol. Chem. 1961,236,203-206. 23. Lowry, O.H.; Rosebrough, J.; Farr, A. L.; Randall, R. J. J . Bwl. Chem. 1951,193,265-275. 24. Eichler, D. C.; Fisher, P. A.; Korn, D. J . B i d . Chem. 1977,252, 40114014. 25. Sawada, H.; Tatsumi, K.; Sasada, M.; Shirakawa, S.; Nakamura, T.; Wakisaka, G. Cancer Res. 1974,34,3341-3346. 26. Hall, I. H.; Carlson, G. L.; Abernathy, G. S.; Piantadosi, C. J . Med. Chem. 1974,17,1253-1257. 27. Anderson, K. M.; Mendelson, I. S.; Guzik, G. Bwchem. Biophys. Acta 1975,383,56-66. 28. Moore, E. C.; Hurlbert, R. B. J . B i d . Chem. 1966,241,48024809. 29. Malev. F.;Ochoa, S. J . Biol. Chem. 1958,233,153f3-1543. 30. Kalman, S. M.; D a e l d , P. H.; Brzozwski, T. J . B i d . Chem. 1966, 241,1871-1877. 31. Archibald, R. M. J . Biol. Chem. 1944,156,121-142. 32. Koritz, S. B.; Cohen, P. P. J . Biol. Chem. 1954,209,145-150. 33. Appel, S.H. J . Biol. Chem. 1968,243,3024-3029. 34. Kampf, A.; Barfltnecht, R. L.; Shaffer, P. J.; Osaki, S.; Metes, M. P. J . Med. Chem. 1976,19,903-908. 35. Ho, Y . K.; Hakala, T.; Zakrezewski, S. F. Cancer Res. 1972,32, 1023-1028. - - _- - - --. 36. Woynarowski, J. M.; Konopa, J. Molec. Pharmucol. 1981, 19, 97-102. 37. S assova, M. K.; Russev, G. C.; Bolovinsku, E. V. Biochem. P i armacol. 1976,25,923-924. 38. Becker, H. J.; Lohr, G. W. Klin. Wochenschr. 1979,57, 11091115. 39. Bagnara, A. S.; Finch, L. R. Anal. Biochem. 1972,45,24. 40. Hunting, D.; Henderson, J. F. Can J . Biochem. 59,723-727.
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Figure H A ) In vivo effects of cDDP on DNA after one, two, and three daily ip doses at 1.5 mg/kg/day.Key: ( C B ) control P388 DNA; (0-0) one day of treatment with cDDP at 1.5 mg/kg/day;(A-A) two days of three days of treatment treatment with cDDP at 1.5 mg/kg/day;(0-0) with cDDP at 1.5 mg/kg/day. Standard deviations were
treatment was unexpected, but probably reflects the fact that a larger percentage of the cells present a t that time had been irreversibly damaged and were in the process of dying. Sorenson and Eastman46 have reported that strand scission is the first detectable change indicative of cell death. The DNA cross-linking observed on days 2 and 3 of the treatment is consistent with previous reports.4749 The studies with 3H-labeledPtCl,(dach) and Pt(mal)(dach) offer an interesting contrast to previous studies in cell culture. Mauldin et also have reported that PtClJdach) was more efficiently taken up by cultured L-1210 cells than Pt(mal)(dach), and that it was more efficiently incorporated into DNA. The difference between the previous in vitro experiments and these results in vivo may very well reflect differences in the biodistribution andlor bioactivation of these two compounds in the BDF, mouse. While the uptake and incorporation into DNA varied significantly for these drugs, it is
Journal of Pharmaceutical Sciences I 879 Vol. 79, No. 10, October 1990
41. Suzuki, H: Niehimura, T.; Muto, K.; Tanaka, N. J. Antibiot.
1978,32, 8?5-883. 42. Pera M.F., Jr.; Rawlin B, C J.; Shackleton, J.; Roberts, J. J. Biochirn. Bwphys. Actcr f981,655, 152-166. 43. Woynarowski, J. W.; Beerman, T. A.; Konopa, J. Biochem. Pharmacol. 1981,30, 30053007. 44. Shooter, K. V.;House, R.; Merrifield, R, K.; Robin, A. B. Chem. Bwl. Zntl. 1972,5, 289. 45. Oswald, C. B.; Hall, I. H. Anticancer Res., 1989, 9, 915. 46. Sorenson, C. M.; Eaetman, A. Cancer Res. 1988,48,4484. 47. Stone, P. J: Kelman, A. D.; Sinex, F. M.; Bhargava, M. M.; Halvereon, k. 0. J. Molec. Bwl. 1976,104, 793.
880 I Journal of Pharmaceutical Sciences Vol. 79, No. 10, October 1990
48. Zwelling, L. A.; Anderson, F.; Kohn, K. W. CancerRes. 1979,39, 365. 49. Zwellin , L. A . Michaels, S.; Schwartz, H.; Dobson, P. P.; Kohn, K. W. Ckancer i i e s . 1981,41, 640. 50. Maudlin, S. K.; Husain, I.; Sancar, A.; Chaney, S.G. CancerRes. 1986,46,28762882. 51. Fravel, H. N. A,; Roberts, J. J. Cancer Res. 1979,39, 1793.
Acknowledgments This work was supported by a grant from NIH - CA34082.
c. BRENTOSWALD*, STEPHEN G.CHANEY*, AND IRIS H. HALL*' Received August 30, 1988, from the 'Division of Medicinal Chemisty and Natural Products, School pf Pharmacy C6#7360,and the *Department of B/ochemisfry and Nutrition, School of Medicine CB#7260, University of North Carolina, Chapel Hill, NC 27599. Accepted for publication November 29, 1989. Abstract 0 ci+Diamminedichloroplatinurn(ll) (cisplatin; cDDP) derivatives were found to afford T/C% values >200 against the growth of P388 lymphocytic leukemia cells in vivo. The parent compound, cDDP, preferentially inhibited DNA synthesis. The RNA synthesis was elevated, whereas protein synthesis was unaffected after two or three daily ip doses. Radiolabeled drug studies demonstrated cellular uptake and binding of cDDP derivatives to the DNA molecule. cis-Diamminedichloroplatinum(l1) (cDDP) treatment resulted in DNA strand scission after a single dose, but caused cross-linking of DNA strands after two or three ip doses. There was an accumulation of deoxynucleoside triphosphates [d(NTP)s]on day 2 and 3, indicating that incorporation of nucleotides into the DNA strand had been blocked. Thymidine kinase, thymidine monophosphate kinase, carbamoyl phosphate synthetase, and aspartate transcarbarnoylase activities were inhibited in vivo after three doses of cDDP at 1.5 mglkglday. However, only the inhibition of a cytoplasmic preparation of DNA polymerase a by cDDP appeared to be directly related to the inhibition of DNA synthesis and the accumulation of d(NTP) pool levels. Thus, the primary target for cDDP appears to be DNA itself, although direct inhibition of DNA polymerase a may play a minor role in the inhibition of DNA replication by cDDP.
cis-Diamminedichloroplatinum(I1) (cisplatin; cDDP) has been demonstrated to have antineoplastic activity against the growth of Sarcoma 180 and L-1210 lymphocytic leukemia in mice14 and positive results against Dunning ascitic leukemia and intramuscular Walker 256 carcinosarcoma.4 In humans, the agent has been effective in causing regression of testicular, colon, bladder, breast, a n d thyroid cancer, and neuroblastoma, malignant thymoma, lymphomas, endometrial cancer, and squamous cell carcinoma.l-6 Harder and Rosenberg" have shown that cDDP preferentially inhibited DNA synthesis in human amnion AV3 cells, as has been observed in mouse Ehrlich ascites t u m o r cells8 and human Hela cells.9 The inhibition of DNA synthesis by cDDP was not immediate. The purine bases of DNA (primarily G-6 and A-6) appeared t o be the major target of the drug.S.9 Intrastrand and interstrand cross-linking of the bases of DNA h a v e been proposed as a mode of action for the drug.10 However, the inhibition of DNA polymerase a activity b y cDDP has also been observed by Harder et al.11 a n d cannot be excluded as possibly contributi n g to the overall cell killing effect of the drug. Thus, the effect of cDDP on P388 lymphocytic leukemia cells i n vivo was investigated to determine if inhibition of DNA polymerase a or o t h e r enzyme activities might be contributing factors in tumor cell death.
Nuclear, Boston, MA. Radioactive samples were placed in Fisher Scintiverse and counted using a Packard Tricarb scintillation counter correcting for quenching by a n internal standardization technique. cis-Diamminedichloroplatinum(I1) (cisplatin;cDDP) was eynthesized by the method of Dhara,', cis-diamminemalonatoplatinum(II) [Pt(mal)(NH,),l was synthesized as reported by Speer et al.,14 (1,2diaminocyclohexane)dichloroplatinum(II) [PtCl,(dach)] was synthesized by the method of It0 e t al.,13 and (1,2-diaminocylohexane)malonatoplatinum(I1) [Pt(mal)(dach)]was synthesized by the method of Speer et al.14 Radioactive syntheses of the latter two compounds have previously been published.15 All compounds were satisfactorily analyzed for carbon and hydrogen (i.e., <0.4% difference between calculated and analyzed values).l6J7 Determination of T/C%-All experiments were performed with BDF, male mice (25 g) inoculated with 1 x lo6 P388 lymphocytic leukemia cells18 suspended in sterile isotonic saline on day 0. The drugs cDDP, Pt(mal)(NH,),, PtCl,(dach), and Pt(mal)(dach),and the internal standard 5-fluorouracil were suspended in 0.05% polysorbate 80-water by homogenation. Drug solutions were administered ip a t a dose of 0.25 to 25.0 mg/kg/day. The TIC% values were obtained according to the NCI protocol.16 The average life span of the treated group was divided by the control group's life span and then multiplied by 100 to obtain a percent. 5-Fluorouracil was used as a standard for the in vivo screen. Cytotoxic activity18 was determined by incubating P388 cells (5 x lo4) with &-platinum drugs at final concentrations of 0-25 pg/mL in Eagle's MEM 10% fetal calf serum plus streptomycidpenicillin. The number of viable cells was determined on Day 3 using a Levy cell counting chamber and trypan blue exclusion. The concentration of drug necessary to achieve 50% inhibition of cell growth (ED,,) was determined by plotting the log of drug concentration versus the cell number.18 Etoposide VP-16 was used as an internal standard for this assay. In Vivo Studies-All in vivo studies were performed by treating test animals on days 7,8, and 9 after tumor inoculation. Three hours after the last dose (noted as day 1,2, and 3, respectively) the animals were sacrificed, the ascites fluid was harvested, and assays were performed on the appropriate cell preparations. In Vivo DNA, RNA, and Protein S y n t h e s i s o n e hour prior to sacrifice, the animals were injected ip with 10 pCi of [methyl'Hlthymidine (84 Ci/mmol), 10 pCi of [6-'H]uridine (22.4 Ci/mmol), or 10 pCi of L-[4,5-3H(N)]leucine(56.5 Ci/mmol). Following sacrifice,
cDDP
,.O
Experimental Section Materials-All biochemicals, 5-fluorouracil, and etoposide were purchased from Sigma Chemical, St. Louis, MO. [8-14C]Inosine-5'monophosphate was purchased from Amersham, Arlington Heights, IL. All other radiochemicals were purchased from New England OO22-3~9/90/1000-0875$0 1.OO/O 0 1990, American Pharmaceutical Association
MAL
DACH-Pt-CI
DACH-Pt-MAL
Journal of Pharmaceutical Sciences I 875 Vol. 79, No. 10, October 1990
the P388 cells were collected. Incorporation of 13Hlthymidine into DNA was determined by the modified method of Chae e t al.19 Results are reported a~ disintegrations per minute (dpms) of l3H1thymidine incorporated into DNA per milligram of DNA is6lated'g as determined by the diphenylamine reaction. Incoiporation of [3Hluridine into RNA was determined according to the method of Wilson et al.2" The concentration of hydrolyzed RNA was assayed by the orcinol method.*] Results were determined as dpms of [3Hluridine incorporated into RNA per milligram of RNA isolated. Incorporation of I3Hlleucine into protein was determined by the method of Booth and Sartbrelli.22 Results were calculated as dpms of ["Hlleucine incorporated per milligram of protein isolated as determined by the Lowry technique.23 Enzyme Assays-The DNA polymerase (Iactivity was determined using a cytoplasmic extract isolated by the method of Eichler et al." The nuclear supernatant DNA polymerase activity was determined in a sample prepared by centrifuging isolated washed nuclei a t 800 x g for 10 min and using the nuclei supernatant to assay DNA polymerase. The DNA polymerase assay was that of Sawadaa using 13H]lTP. Messenger. ribosomal, and transfer RNA polymerase enzymes were isolated using different concentrations of ammonium sulfatez6 and the individual RNA polymerase activities were determined using I3H1UTP.27 Ribonucleotide reductase activity was measured by the method of Moore and Hurlbertm using 114C1CDPwith dithioerythriol. Thymidine and thymidine mono- and diphosphate kinase activities were measured by a spectrophotometric assay based on the disappearance of NADH a t 340 11111.29 Carbamoyl phosphate synthetase (CAP) activity was measured by the method of Kalman e t aL.30 using a colorimetric determination of citrulline.31 Aspartate transcarbamoylase (ATC) activity was measured by the method of Kalman e t aI.,31 with carbamoyl aspartate being determined colorimetrically.32 The OMP decarboxylase activity was assayed with a 16,300 x g for 20 min supernatant and [l4CJ0MP.33Thymidylate synthetase activity was analyzed by the method of Kampf e t al."4 Tritiated water (3H,0) was measured as being proportional to the amount of TMP formed from 13H]dUMP. Dihydrofolate reductase activity was determined by the spectrophotometric method of Ho et a1.s Formate incorporation into purines was determined using ["C]formic acid. Adenine ahd guanine were separated by TLC.36 The PRPP amidotransferase activity was determined by the method of Spaseova et a1.,37 and inosinic acid monophosphate dehydrogenase activity wa6 determined using I"CIIMP, where XMP was separated on PEI plates by TLC.m Protein was determined for all of the enzymatic assays by the Lowry technique.23 Deoxyribonucleotide triphosphates were extracted by the method of Bagnara and Finch.39 Deoxyribonucleotides were determined by the method of Hunting and Henderson40 using calf thymus DNA, E . coli DNA polymerase 1, nonlimiting amounts of the three deoxyribonucleoeide triphosphates that were not being assayed, and 0.4 pCi of either [3H-methyl]d?TP or [5'H]dCTP. T h e effects of cDDP, Pt(mal)(NH3),, PtCl,(dach), a n d Pt(mal)(dach) on DNA were measured on alkaline sucrose gradients aa described by Suzuki e t al.,41 Pera et al.,42 and Woynarowski e t a1.d3 The BDF, mice bearing P388 lymphocytic leukemia cells were injected ip with 10 KCi of thymidine ( m e t h ~ l - ~84.0 H , Ci/mmol) 24 h prior to sacrifice. After harvesting the P388 cells from the peritoneal cavity, 5 x lo6 cells were centrifuged at 600 x g for 10 min in PBS, washed, and suspended in 1 mL of PBS. Lysis buffer (0.5 mL of O.5M NaOH, 0.02 M EDTA. 0.01% triton X-100, and 2.5% sucrose) was layered onto a 5 2 0 % alkaline sucrose gradient (5mL of 0.3 M NaOH, 0.7 KCI. and 0.01 M EDTA) followed by 0.2 mL of cell preparation. After incubating for 30 min at room temperature, the gradient was centrifuged a t 25 000 rpm and 20'C for 60 min (Beckman rotor SW 60). Fractions were collected (0.2mL) from the top of the gradient, neutralized with 0.2 mL of 0.3 M HCI, and counted. P388 Cell Uptake and Binding of DNA, RNA. and Protein-The BDF, tumor bearing mice were treated with [3HlPtCLZ(dach)(0.75 mg/kg/day, ip) or I'HH]Pt(mal)(dach) (17.5 mglkglday. ip) on days 7.8, and 9.15 Three hours after each treatment, the ascites fluid was collected and centrifuged at 800 x g for 5 min. The cells were washed six times and counted. The cells were sonicated to break the cell membranes and the aliquots were counted. Cells were chemically extractedfor DNA, RNA, and protein as previously described, and the aliquota were counted. Results are expressed as d p d m g of isolated macromolecule. 876 / Journal of Pharmaceutical Sciences Vol. 79, No. 10, October 7990
Results cis-Diamminedichloroplatinum(I1)demonstrated potent antineoplastic activity against P388 lymphocytic leukemia growth in BDF, mice. The dose response study demonstrated that the optimum dose for cDDP was 1.5 mg/kg/day ip, affording a T/C% of 219 which compares favorably with that for 5-fluorouracil (5FU) (Table I). The optimum doses for Pt(mal)(NH,),, PtClJdach), and Pt(mal)(dach) were found to be 10, 0.75, and 17.5 mgkglday ip, respectively. Since radiolabeled PtCl,(dach) and Pt(mal)(dach) were available, the uptake and distribution of these two compounds in P388 cells in vivo were measured over a 3-day period. A greater percentage of the [3H]F'tCl,(dach) entered the cell on each day than did L'H]Pt(mal)(dach) (p 5 0.001; Figure 1).After two doses of the I3H1PtCl,(dach) at 0.75 mgkg, >200 pmol of drug/106 P388 cells was associated with the cells. This level remained essentially unchanged after three doses. The T'H]Pt(mal)(dach) a t 17.5 mg/kg exhibited a cell uptake of only 15 pmol of drug/106 cells after three doses (Figure 1). At the intracellular level, 34% of the ['H]F't(mal)(dach) was bound to DNA after one dose, 48% after two doses (p 5 0.005 analysis of variance), and 38% after three doses (Figure 2). For l'HlPtCl,(dach), 5.4% was bound to DNA after one dose, and this was significantly different (p 5 0.05 analysis of variance) from the 1.88 after two doses and 1.7% after three doses. Binding of the 3H-labeled drugs to isolated DNA, RNA, and protein from P388 cells showed that both drugs bound preferentially to DNA (Figure 3). The ["H]PtCl,(dach), a t 0.75 mg/kg, afforded >600 pmol of drug/mg of DNA (p 5 0.001) after two doses and the third daily dose produced no further binding to DNA. The RNA binding peaked after two daily doses at -200 pmol of drug/mg of RNA. The ['H]Pt(mal)(dach), at 17.5 mg/kg, produced a dose-dependent binding to DNA with -900 pmol of drug/mg of DNA after three doses (p 5 0.001).The RNA binding reached a peak of -600 pmol of drug/mg of RNA after two doses (p 5 0.001), with no further binding with three doses. Protein binding was -50 pmol of drug/mg of protein after 1, 2, or 3 days of treatment with either drug. Mechanistic studies were carried out primarily with cDDP at a dose of 1.5 mg/kg/day since that dose gave the best survival time for this strain of mice. Doses of 1.5 mg:kg cDDP on days 7, 8, and 9 after P388 innoculation led to 54% reduction in the tumor cell numberlml of ascites on day 9 (Tables I1 and 111). Thymidine incorporation into DNA after a single dose of cDDP was elevated 37% (day 71, but after the second dose was reduced 64% (day €9, and by the third dose Table CCytotoxiclty and Antlneoplastic Activity of cDDP and R r Derlvatlves againrt P388 Lymphocytic Leukemia Growth In BDF, Male Mlce
Antitumor Screen Compound (n = 5) cDDP Pt(mal)(NH,), PtCl,(dach) Pt(mal)(dach) 5-Fiuorouracil VP-16 (Etoposide) Control
Optimum Avg. Days dose' Survived mglkglday 1.5 10 0.75 17.5 12.5
-
22.8 2 23.4 ? 29.8 -t 22.6 2 21.7 2
-
3.4' 3.8' 7.6' 4.3' 3.5'
10.4 2 1.1
Cytotoxicity Yo T/C
Value' 219 225 287 217 209
-
MImLb 2.4 11.0 10.5 13.6 3.7 2.5
-
a ?/oT/C= [average days survived by treated animalslaverage days survived by control animals] x 100; %TIC > 125 are considered significant according to NCI protocol (ref 16). Values < 4 M/mL are considered significant according to NCI protocol (ref 16). p = 5 0.001 r test.
3H-OACH-Pt-MAL (17.5mg/kg/dose)
3H-DACH-Pt-CI, (0.75rng/kg/dosd
200 1000
160
! t
j120
\
W 0
a
0 \
3 40
m
W 0
E
o
-
400
E"a
(3
0
600
a
80
U
5a / , ,
*----ADACH-Pt-MAL
I
6
200
0
I
2
3
# DAILY DOSES
(1
4 2 0
vivo binding of 3H-labeled platinum compounds to P388 macromolecules after one, two, and three daily ip doses of [3H]Pt(mal)(dach)or [3H]PtC12(dach).Key: (0-0) binding to DNA; (0-0) binding to RNA; (A-A) binding to protein. Standard deviations for all values were <8 pmol/mg for each data point given (n = 6). Binding of each drug to DNA was significantlydifferent (p 5 0.001)than RNA and protein on each day. Figure &In
2
I
3
# OF DAILY DOSES Figure 1-In vivo P388 leukemic cell uptake and DNA binding of (17.5 rng/kg/ [3H]PtC12(dach)(0.75mg/kg/day) and [3H]Pt(mal)(dach) day) after one, two, and three daily ip doses. Standard deviations for all values were <6 pmol for the cell uptake and 0.5 pmol for DNA binding of drugs (n = 6). A t test demonstrated a p 5 0.001 for the cell uptake of drugs on each day. Only on day 3 was the pmoles of drug bound to DNA significantly different (p 5 0.001) between the two drugs.
2
D 40
'ADACH-Pt-MAL
0
i2 30 zj 20
*.f"---
........ .........9 DACH-Pt -C12
I 2 3 X OF DAILY DOSES Figure 2-Percentage bound to DNA of intracellular [3H]PtCl,(dach) (0.75rngikglday) and [3H]Pt(mal)(dach)(17.5mg/kg/day) P388 after one, two, and three daily ip doses. The percent drug bound to DNA between drugs was significantly different (p s 0.001) on each day. Standard deviation for all values was 4%(n = 6). (day 9) was inhibited 82% (Tables I1 and 111).Incorporation of uridine into RNA was increased, but incorporation of leucine into protein was unchanged after three daily doses of cDDP (data not shown). The effect of cDDP treatment on a number of enzyme activities, required for nucleic acid synthesis, was then determined. Many of these enzyme activities were not significantly altered by cDDP treatment for three days at the therapeutic dose, including mRNA, rRNA, and tRNA polymerase, ribonucleotide reductase, PRPP amidotransferase, OMP decarboxylase, and thymidylate synthetase activities. Some enzyme activities were slightly elevated after cDDP treatment [e.g., dihydrofolate reductase (44%) and IMP dehydrogenase (25%), with an overall increase of formate incorporation into purines (50%;p 5 0.001). Several other enzyme activities did appear to be significantly inhibited by cDPP (Tables I1 and 111).Thymidine, thymidine monophosphate, and thymidine diphosphate kinase activities showed progressively greater inhibition with sequential doses of
cDDP over the 3-day period. Carbamoyl synthetase and aspartate transcarbamoylase activities were not inhibited by a single dose. However, carbamoyl phosphate synthetase activity was inhibited significantly after two doses and aspartate transcarbamoylase activity after three doses. Since DNA synthesis was decreased preferential by the drug and some of the enzymes required to synthesize nucleotide precursors for DNA synthesis were also inhibited, the location of the metabolic block was studied further by examining deoxynucleoside triphosphate (dNTP) levels after exposure to cDDP. Deoxynucleoside triphosphate pool levels were elevated in a dose-dependent manner by cDDP treatment (Tables I1and 111).One, two, and three daily doses of cDDP caused elevations in all four dNTP pools. The effect of repeated cDDP doses on DNA polymerase activity was studied in several subcellular fractions. A single dose of cDDP increased DNA polymerase activity in the cell homogenate. The second dose caused a 38% decrease in activity which was essentially the same decrease seen at the end of day 3 (Table IV). The cDPP in the 600-g pellet was significantly inhibited after all three doses. However, the inhibition could have been due to either inhibition of the enzyme or damage to the DNA template. Since DNA polymerase activity in the cell extract is a composite of repair and replicative activities,24 DNA polymerase activity was also measured in the nuclear supernatant and in a cytoplasmic DNA polymerase a preparation. Both of these assays were strongly dependent on exogenous DNA, so damage to the DNA template was not a factor. The nuclear supernatant DNA polymerase activity was elevated 334% by a single dose of cDDP, but then declined to only a 90% stimulation following the third dose (Table IV). Cytoplasmic DNA polymerase a showed little inhibition following the first and second doses, but was inhibited 24% after the third dose (Table IV). The effects on DNA of cDDP and the other platinum drugs in this study were examined on alkaline sucrose gradients (Figure 4). A single dose of cDDP caused a shift of the DNA molecule t o a lower molecular weight compared with the control DNA, suggesting strand scission (Figure 4A). Two and three daily doses of cDDP led to a shift of the DNA to higher molecular weight DNA, but with no prominent peaks, suggesting cross-linking of the DNA. Three daily doses of PtCl,(dach) and Pt(mal)(dach) also shifted the DNA to higher molecular weight fractions (Figure 4B). Journal of Pharmaceutical Sciences / 877 Vol. 79, No. 10, October 7990
Table ll-In Vlvo Effect0 of cDDP (1.5 mg/kg/day Ip) on Enzyme Activities and d(NTP) Pool Levels ofP388 Lymphocytlc Leukemic Cells after One, Two, or Three Daya of Dosing
Treatment
Control (n = 5) Day 1 2 3
a
p
5
Thymidine
Thymidine
Carbamoyl Phosphate
Aspartate Transcarbamoylase, mole of N carbamoyl aspartateihlmg protein
density unit/h/mg protein
Monophosphate Kinase, optical density unitlhlmg protein
Diphosphate Kinase, optical density unit/h/mg protein
136332 2 14990
0.062 f 0.009
0.039 f 0.006
0.150 f 0.014
0.316 f 0.02
3.989 f 0.0485
186775 2 3891 49079 2 5674' 25107 5 982'
0.047 f 0.008' 0.044 f 0.006' 0.037 f 0.006'
0.031 f 0.010 0.024 f 0.005' 0.015 f 0.003'
0.138 f 0.012 0.125 f 0.015 0.110 f 0.007'
0.349 f 0.022 0.226 f 0.019' 0.250 f 0.017'
3.916 f 0.367 4.029 f 0.425 2.526 f 0.231"
Thymidine
DNA Synthesis, dpm/h/mg DNA
0.001. p
5
Optical
'ynthetase* mole of
citrulline/h/mg protein
0.05 ( t test).
Table Ill-Deoxyrlbonucleoslde Trlphosphate Levels'
Treatment Control (n Day 1
dATP
= 5)
7.56 f 0.73 16.08 f O M b 19.60 2 1.98' 24.41 2 O.8lb
2 3 a Expressed as
pmol/lO6cells. p
5
dCTP
dlTP 12.34 2 13.88 -t 29.65 2 51.62 2
0.001. p
5
1.29
dGTP
0.48 f 0.44 9.06 f 0.78 16.51 f 2.31' 30.43 f 0.91
0.83
'
0.61 1.84'
5.73 2 7.23 8.03 2 12.47 2
0.56
* 0.59: 0.61 3.51
Cell Number (10*/mL) 2.71 f 0.34 2.61 f 0.37 2.12 f 0.36= 1.25 f 0.36b
0.05 ( f test).
Discussion Cisplatin (cDDP) and its derivatives proved to be effective antineoplastic agents against the growth of P388 lymphocytic leukemia cells in vivo in mice as reported previously.4~6The TIC% values for the four compounds compare favorably with those for standard commercially available antineoplastic agents. Although previous studies have implicated DNA as the target of platinum anticancer agents, this is the first study to quantitate the effect of cDDP on the intracellular enzyme activities and d(NTP) pool levels required of cDDP on the intracellular enzyme activities and d(NTP) pool levels required for DNA synthesis. Our data are certainly consistent with the role of DNA as the major target of the cDDP derivatives employed in this study. There was little or no inhibition of RNA polymerases and RNA synthesis was actually elevated following cDDP treatment. Elevation of RNA synthesis following cDDP treatment has been reported previously.'~~The inhibition of the activity of enzymes involved in de novo synthesis of purines (e.g., PRPP amidotransferase and IMP dehydrogenase), orpyrimidines (e.g., OMP decarboxylase and thymidylate synthetase), or of both (e.g., dihydrofolate reductase), was not sufficient to account for the magnitude of inhibition of DNA synthesis or cell kill observed with cDDP. Thymidine, thymidine monophosphate, diphosphate kinase, CAP, and ATC enzyme activities did appear to be significantly inhibited by cDPP treatment. However, the fact that d(NTP) pool levels were elevated after drug treatment indicated that sufficient precursors were
available to maintain DNA synthesis for cell survival and growth, and that DNA synthesis was inhibited at the step involving the incorporation of deoxyribonucleotidebases into the DNA. The observed inhibition of DNA polymerase a by cDDP would appear to be a n effect of cDDP on the enzyme rather than the DNA template since the assay used was largely dependent on exogenous DNA. The addition of calf thymus DNA in the assay resulted in a threefold increase in enzyme activity, but little change in the extent of inhibition of the polymerase a activity in cDDP-treated cells.44 Additional studies have demonstrated that cDDP treatment in vivo inhibits a P388 lymphocytic leukemia cell cytoplasmic DNA polymerase a preparation by 24% and a purified DNA polymerase a by 348.45 Harder et al.11 have previously noted that DNA polymerase a was inhibited by cDDP, although they reported significant inhibition only at nonphysiological cDDP levels. Our data show that inhibition of DNA polymerase a probably makes a small, but significant contribution to the inhibition of DNA synthesis at therapeutic levels of cDDP. The elevation of the nuclear supernatant DNA polymerase activity most likely reflects increased DNA repair activity in response to the formation of platinum-DNA adducts. Harder et al.11 have previously reported that cDDP elevates DNA polymerase p activity. Although platinum-induced DNA cross-linking has been widely studied in cell culture, it has seldom been examined in vivo. The DNA strand scission observed after the first day of
Table IV-In Vlvo Effects of cDDP (1.5 mglkglday Ip) on the DNA Polymerase Actlvlty from Vsrlour Cell Fractlons of P388 Lymphocytlc Leukemic Cells ~
dpm dlTP incorporated/h/mg/protein
Cell Fraction (n
=
6)
Cell homogenate
600-9 Pellet
Nuclear supernatant Cytoplasmic a preparation a p _c
0.001. p < 0.05 (t test).
878 / Journal of Pharmaceutical Sciences Vol. 79, No. 10, Ocfober 1990
Control 7181 f 363 8161 f 655 4438 2 391 4166 2 2414
1 Day
9177
f 1011'
5060 f 321' 19257 f 4102' 42719 f 6635
Treatment 2 Day
3 Day
4452 f 502' 6689 f 546' 13935 f 843' 39588 f 1268
4883 f 359" 2367 f 95" 8432 f 668' 31657 f 2068'
333r
-
22
A
261t18
/I iI
-
-
10
II
A
-
I
14
interesting to note that the extent of interaction with DNA was very similar a t comparable therapeutic doses. The maximum binding of the [SHlPtC12(dach)and [3HlPt(mal)(dach) to P388 DNA after three doses was calculated to be approximately one platinum atod100 base pairs for in vitro studies, with a binding ratio of 0.01 (1:100, to 1platinum a t o d l . 5 x lo6 base pairs.45>61The values for drug binding to DNA for this in vivo P388 leukemia study lie between these two extremes.
9
References and Notes
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Bollom
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1. Rosenber , B.; Van Camp, L.; Trosko, J. E.; Mausour, V. H. Nature 1869,222,385. 2. Rosenberg, B.; Van Camp, L. Cancer Res. 1970,30,1799. 3. Leh, F. K. V.; Wolf, W. J . Phurm. Sci. 1976,65,323. 4. Kociba, R. T.; Sleight, S. P.; Rosenberg, B. Cancer Chemother. Rep. 1970,54,325. 5. Wolpert-DeFillippes, M. K. Cancer Treat. Rep. 1979,63, 1453. 6. Hill, J. M.; Loeb, E.; MacLellan, A.; Hill, N. 0.;Kahn, A.; King, J. J. Can. Chemother. Rep. 1975,59,647. 7. Harder, H. C.; Rosenberg, B. Znt. J . Cancer 1970,6,207. 8. Howle, J. A.; Gale, G. R. Biochem. Phummcol. 1970,19,2757. 9. Pascoe, J. M.; Roberts, J. J. Biochem. Pharmacol. 1974,23, 1345. 10. Kelman, A. D.; Peresie, H. J. Cancer Treat. Rep. 1979,63, 1445. 11. Harder, H. S.;Smith, R. G.; LeRoy, A. F. Cancer Res. 1976,36, 3821. 12. Dhara, S.C. Znd. J . Chem. 1970,8,193. 13. Ito, H.; Fuamita, J.; Saito, K. Bull Chem. Soc. Japan 1967,40, 2584. 14. S eer, R. J.; Ridway, H.; Hall, L. M.; Newman, A. D.; Howe, E.; Stewart, D. P.; Edwards, G. R.; Hall, J. M. J . Clin. Hematol. Oncol. 1975,5,335. 15. Oswald, C. B.; Wyrick, S. D.; Chane , S. G.; Shrewsbu R. 0.; Hall, I. H. Res. Comm. Chem. P a t h . h m n a c o l . 1989,6?141-58. 16. Wyrick, S . D.; Chaney, S. G. J . Labeled Compds. Radiopharmceut. 1988,25,349-357. 17. Maudlin, S. K.; Richard, F. A,; Plescia, M.; Wyrick, S. D.; Sancar, A.; Chaney, S. G. Anal. Biochem. 1988,157,129-143. 18. Geran, R. I.; Greenberg, N. H: MacDonald, M. M.; Schumacher, A. M.; Abbott, B. J. Cancer Ckemother. Rep. 1972,3,9-24. 19. Chae, C. B.; Williams, A,; Krasny, H.; Irvin, J. L.; Piantadosi, C. Cancer Res. 1970,30,2652-2660. 20. Wilson, R. G.; Bodner, R. H.; MacWhorter, G. E. Biochem. Biophys. Acta 1975,378,260-268. 21. Ashwell, G. Meth. Enzymol. 1957,3,87-90. 22. Booth, B. A.; Sartorelli, A. C. J.Biol. Chem. 1961,236,203-206. 23. Lowry, O.H.; Rosebrough, J.; Farr, A. L.; Randall, R. J. J . Bwl. Chem. 1951,193,265-275. 24. Eichler, D. C.; Fisher, P. A.; Korn, D. J . B i d . Chem. 1977,252, 40114014. 25. Sawada, H.; Tatsumi, K.; Sasada, M.; Shirakawa, S.; Nakamura, T.; Wakisaka, G. Cancer Res. 1974,34,3341-3346. 26. Hall, I. H.; Carlson, G. L.; Abernathy, G. S.; Piantadosi, C. J . Med. Chem. 1974,17,1253-1257. 27. Anderson, K. M.; Mendelson, I. S.; Guzik, G. Bwchem. Biophys. Acta 1975,383,56-66. 28. Moore, E. C.; Hurlbert, R. B. J . B i d . Chem. 1966,241,48024809. 29. Malev. F.;Ochoa, S. J . Biol. Chem. 1958,233,153f3-1543. 30. Kalman, S. M.; D a e l d , P. H.; Brzozwski, T. J . B i d . Chem. 1966, 241,1871-1877. 31. Archibald, R. M. J . Biol. Chem. 1944,156,121-142. 32. Koritz, S. B.; Cohen, P. P. J . Biol. Chem. 1954,209,145-150. 33. Appel, S.H. J . Biol. Chem. 1968,243,3024-3029. 34. Kampf, A.; Barfltnecht, R. L.; Shaffer, P. J.; Osaki, S.; Metes, M. P. J . Med. Chem. 1976,19,903-908. 35. Ho, Y . K.; Hakala, T.; Zakrezewski, S. F. Cancer Res. 1972,32, 1023-1028. - - _- - - --. 36. Woynarowski, J. M.; Konopa, J. Molec. Pharmucol. 1981, 19, 97-102. 37. S assova, M. K.; Russev, G. C.; Bolovinsku, E. V. Biochem. P i armacol. 1976,25,923-924. 38. Becker, H. J.; Lohr, G. W. Klin. Wochenschr. 1979,57, 11091115. 39. Bagnara, A. S.; Finch, L. R. Anal. Biochem. 1972,45,24. 40. Hunting, D.; Henderson, J. F. Can J . Biochem. 59,723-727.
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Figure H A ) In vivo effects of cDDP on DNA after one, two, and three daily ip doses at 1.5 mg/kg/day.Key: ( C B ) control P388 DNA; (0-0) one day of treatment with cDDP at 1.5 mg/kg/day;(A-A) two days of three days of treatment treatment with cDDP at 1.5 mg/kg/day;(0-0) with cDDP at 1.5 mg/kg/day. Standard deviations were
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Journal of Pharmaceutical Sciences I 879 Vol. 79, No. 10, October 1990
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Acknowledgments This work was supported by a grant from NIH - CA34082.