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Overcoming TNF-α and Drug Resistance of Human Renal Cell Carcinoma Cells by Treatment with Pentoxifylline in Combination with TNF-α or Drugs: The Role of TNF-α mRNA Downregulation in Tumor Cell Sensitization
0022-5347 /94/15161697$03.00/0 VoL 151,
THE JOURNAL OF UROLOGY
Copyright© 1994 by AMERICAN UROLOG!CAL ASSOCIATION, !NC.
June 1994 in U.S.A.
OVERCOMING TNF-a AND DRUG RESISTANCE OF HUMAN RENAL CELL CARCINOMA CELLS BY TREATMENT WITH PENTOXIFYLLINE IN COMBINATION WITH TNF-a OR DRUGS: THE ROLE OF TNF-a mRNA DOWNREGULATION IN TUMOR CELL SENSITIZATION YOUICHI MIZUTANI, BENJAMIN BONAVIDA, YOSHINO RI NIO AND OSAMU YOSHIDA* From the Department of Urology and First Department of Surgery, Faculty of Medicine, Kyoto University, Kyoto, Japan and the Department of Microbiology and Immunology, UCLA School of Medicine, Los Angeles, California
ABSTRACT
Previous studies have demonstrated that one of the possible mechanisms responsible for the resistance of tumor cells to tumor necrosis factor-Cl' (TNF-a) is the expression of TNF-a mRNA and/or protein. Pentoxifylline (PTX) suppressed TNF-a gene transcription and downregulates the expression of TNF-a mRNA and the secretion of TNF-a protein in macrophages and monocytes. This study investigates whether PTX downregulates the expression of TNF-a mRNA and/or protein in renal cell carcinoma (RCC) cells and whether PTX enhances the sensitivity of TNF-aresistant RCC cells to TNF-a. Further, we explored whether PTX enhances the sensitivity of RCC cells to agents other than TNF-l\' by downregulation of the expression of TNF-a mRNA and protein. The R4 human RCC cell line constitutively expressed TNF -a mRNA and protein and was resistant to TNF-a. When R4 cells were incubated with PTX, the level of TNF-a mRNA and protein was markedly reduced. Pentoxifylline and TNF-a together overcame the resistance of R4 cells to TNFa. The Rll human RCC cell line did not constitutively express TNF-a mRNA or protein, and was resistant to TNF-a. The expression of TNF-ll' mRNA in Rll cells, but not the production of TNFa protein, was induced by TNF -a. When PTX was used in combination with TNF -a, the level of TNF-a mRNA induced by TNF-a was markedly reduced. The combination of PTX and TNF-a overcame the resistance of Rll cells to TNF-a. Pentoxifylline also enhanced the sensitivity of R4 cells to interferon-a. Pentoxifylline and anti-TNF -Cl' monoclonal antibody augmented the sensitivity of R4 cells to cis-diamminedichloroplatinum (II) (CDDP). This study demonstrated that PTX, in combination with TNF-ll', IFN-a or CDDP, overcame the drug resistance to RCC cells and that downregulation of TNF-a mRNA by PTX may be related to the cytotoxicity enhanced by the combination. The implications of these findings for clinical therapy are discussed. KEY WORDS:
carcinoma, renal cell; cisplatinum; interferon-alpha; pentoxifylline; tumor necrosis factor
Pentoxifylline (PTX) [3, 7-dimethyl-l (5-oxohexyl)-xanthine] was originally introduced as a hemorrheologic agent for the treatment of peripheral vascular disease. 1 ' 2 It augments erythrocyte flexibility and decreases blood viscosity, resulting in increased microcirculatory blood flow. 2 ' 3 It also inhibits platelet aggregation, increases fibrinolytic activity and stimulates prostacyclin release. 4 · 5 It has been reported that as a methylxanthine derivative related to aminophylline and caffeine, PTX inhibits intracellular cyclic nucleotide phosphodiesterase, resulting in an increased intracellular concentration of cyclic adenosine monophosphate (cAMP), which impairs a number of cellular immune functions. 6 · 7 Pentoxifylline significantly reduces tumor necrosis factor-a (TNF-a) protein production and TNF-a mRNA transcription in murine macrophages and human monocytes through the cAMP pathway. 8 • 9 Tumor necrosis factor-a was originally identified in the serum of mice challenged with bacillus Calmette-Guerin and endotoxin. It has a wide range of biological activities, including cytotoxic and cytostatic antitumor activity. 10- 12 Recombinant human TNF-a has been developed, and its antitumor activity against certain cancers involving renal cell carcinoma (RCC) Accepted for publication December 2, 1993. * Requests for reprints: Department of Urology, Faculty of Medicine, Kyoto University, 54 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606, Japan. This work was supported by a Grant-in-Aid (No. 3454385 and No. 5671314) from the Japanese Ministry of Education, Science and Culture.
is currently being investigated in phase I and phase II clinical trials. 13- 15 Its antitumor effect may be linked to the generation of free radicals, the inhibition of protein synthesis and the induction of programmed cell death. 16· 17 Although TNF-a appears to be a promising anticancer agent, most tumor cells, including RCC, are resistant to ito 18 · 19 It has been reported that one possible mechanism responsible for the resistance of tumor cells to TNF-a is the expression of TNF-a mRNA and/or protein. 20- 23 We demonstrated that cisdiamminedichloroplatinum II (CDDP) and buthionine sulfoximine downregulate the expression of TNF-a mRNA and enhance the susceptibility of tumor cells to TNF-a. 24 - 26 In this study we investigated whether PTX downregulates the expression ofTNF-a mRNA and/or protein in RCC cells and whether PTX augments the sensitivity of TNF -a-resistant RCC cells to TNF-a. In addition, we studied whether PTX enhances the sensitivity of RCC cells to other agents as well as TNF -a by downregulation of the expression of TNF-a mRNA and/or protein. Furthermore, we have explored the possible underlying mechanism involved in the reversal of resistance. MATERIALS AND METHODS
Tumor cells. The human renal cell carcinoma (RCC) cell lines R4 and Rll were supplied by Dr. Hans Stotter, Bethesda, Maryland (Histopathology: adenocarcinoma). 25 The ovarian tumor line ADlO was supplied by Dr. Robert Ozols. 27 The cell lines were maintained in RPMI-1640 medium (M.A. Bioprod-
1697
1698
TNF-a mRNA DOWNREGULATION IN TUMOR CELL SENSITIZATION 2
A
3
4
2
C
TNF-a
3
4
TNF-«
/l-actin a-actin
50
B
D
30
40 20
i .!l .z,.!ii
30
.,
20
ll:
10
10
0 4
Lane numbers
Lane numbers
FIG. 1. Effect of PTX on level of TNF-a mRNA expression in R4 and R11 cells. R4 and R11 cells were incubated with PTX and/or TNF-a for 4 hours. Total RNA was then separated, Northern blotted and probed for TNF-a and /3-actin as described in Materials and Methods. Each lane is mRNA for TNF-a and /3-actin of R4 cells (A) and R11 cells (C) after the various treatments. A, C: lane 1: medium only; lane 2: PTX at 100 µg./ml.; lane 3: TNF-a at 10 ng./ml.; lane 4: PTX at 100 µg./ml. and TNF-a at 10 ng./ml. simultaneously. Densitometry scan of bands in A and C shows relative levels of mRNA compared with controls (B, D ). Experiments were repeated three times. TABLE
1. The effect of PTX on TNF-a secretion by R4 and Rll cells
TABLE 3.
The enhanced sensitivity of Rll cells to TNF-a by PTX Percent Cytotoxicity (mean ± S.D.)•
TNF-a secretion (pg./ml.)" PTX (µg./ml.)
Treatmentb
Cell line Control (Medium) PTX R4 Rll
177.0 ± 19.3 1.0 ± 1.4
20.9 ± 6.6 0.0 ± 5.5
TNF-a 279.4 ± 5.9 1.1 ± 4.0
• Tumor necrosis factor-a secretion was measured by ELISA as described in Materials and Methods. The results are expressed as the means ± SD of 2 different experiments. b R4 and Rll cells were treated with PTX at 100 µg./ml. and/or TNF-a at 10 ng./ml. for 4 hours, washed three times in complete medium and incubated in complete medium for 24 hours. After incubation, the level of TNF-a protein in the supernatant was measured. TABLE 2.
0
PTX+TNF-a 144.4 ± 31.8 0.8 ± 4.5
The enhanced sensitivity of R4 cells to TNF-a by PTX
TNF-a (ng./ml.)
0 1 10 100
0 1.9 ± 1.4 4.2 ± 1.0 4.5 ± 1.8
TABLE 4.
0 1.2 ± 0.6 2.5 ± 2.0 4.2 ± 2.0
1 1.9 ± 14.2 ± 18.1 ± 27.3 ±
10 1.8 1.6 2.4 2.lb
9.9 ± 20.7 ± 25.6 ± 34.4 ±
100
± 1.6 ± 1.4 ± 1.6 ± 0.4b
15.0 ± 15.9 ± 20.2 ± 34.7 ±
2.4 1.6 2.5 4.6b
Percent Cytotoxicity (mean ± SD)'
20.6 ± 30.9 ± 33.2 ± 44.3 ±
2.6 2.4 2.5b 4.0b
IFN-a (U/ml.) 0
100 1.4 2.4 2.0b 5.8b
3.2 9.3 15.1 25.6
The enhanced sensitivity of R4 cells to IFN-a by PTX
PTX (µg./ml.)
TNF-a (ng./ml.) 0
0 1 10 100
± 0.2 ± 1.5 ± 1.6 ± 0.2
• The cytotoxic effect of PTX and TNF-a on Rll cells was assessed in a 1day MTT assay. The results are expressed as the means ± SD of 3 different experiments. b The values are significantly higher than those obtained by treatment with PTX alone plus those obtained by treatment with TNF-a alone at p < 0.05.
Percent Cytotoxicity (mean± SD)' PTX (µg./ml.)
10
1 0.4 2.2 5.3 10.2
0 1 10 100
0 1.8± 2.0 2.8 ± 2.0 5.8 ± 0.6
10 1.3 ± 10.9 ± 12.0 ± 16.1 ±
100 2.0 1.2 1.6 1.4
2.8 ± 11.0 ± 16.1 ± 24.1 ±
2.6 1.3 2.6 2.lb
1000 4.3 ± 11.1 ± 17.9 ± 26.0 ±
1.0 1.2 2.4 2.0b
• The cytotoxic effect of PTX and TNF-a on R4 cells was assessed in a 1-day MTT assay. The results are expressed as the means ± SD of 3 different experiments. b The values are significantly higher than those obtained by treatment with PTX alone plus those obtained by treatment with TNF-a alone at p < 0.05.
• The cytotoxic effect of PTX and IFN-a on R4 cells was assessed in a 1-day MTT assay. The results are expressed as the means ± SD of 3 different experiments. b The values are significantly higher than those obtained by treatment with PTX alone plus those obtained by treatment with IFN-a alone at p < 0.05.
ucts) supplemented with 1% L-glutamine (Gibco, Bio-Cult, Glasgow, Scotland, United Kingdom), 1% nonessential amino acid (Gibco), 1% Fungibact solution (Irvine Scientific, Irvine, California) and 10% heat-inactivated fetal bovine serum (Gibco), hereafter referred to as complete medium. When the
tumor cell lines were used as target cells, they were treated with trypsin-ethylene-diamine-tetraacetic acid (TrypsinEDTA, ICN Biomedical Inc.), washed three times and suspended in complete medium. Reagents. Pentoxifylline (Lot. No. 91H0194) and cis-diam-
1699
TNF-a mRNA DO'NNREGULATION IN TUMOR CELL SENSITIZATION 5- The cytotoxic effect of PTX and IFN-a on RI 1 cells
TABLE
2
3
4
5
Percent Cytotoxicity (mean± SD)· PTX (µg-/mL)
IFN-a (U/mL)
0 1 10 100
MDR-1
100
0
10
0 0-6 ± L2 6A ± LS 6A ± L6
D-2 ± OA 3-0 ± LS 6-9 ± L1 lOA± U
2-8 4-2 9-5 13-7
± ± ± ±
1000 D-8 L6 LO 2-6
4-6 4-6 9-8 lU
± ± ± ±
LS L6 L6 2-2
a The cytotoxic effect of PTX and IFN-a on Rll cells was assessed in a 1-day MTT assay- The results are expressed as the means ± SD of 3 different experiments-
TABLE
6- The enhanced sensitivity of R4 cells to CDDP by PTX Percent Cytotoxicity (mean ± S-D-)·
PTX (µg-fmL)
CDDP (µg-/mL) 0
0 1 10
100
JO
0-1 L9 15-9 16-7 25-5
0 L3 ± L6 L9 ± 0-8 6-2 ± L4
± ± ± ±
0-4 2-4 2-6 2-6b
8-8 19-7 23-7 32-1
± ± ± ±
L2 2-8 3-0 3-2b
39-3 54-6 57-1 65-1
± ± ± ±
4-0 3-2 3-2b 506b
a The cytotoxic effect of PTX and CDDP on R4 cells was assessed in a 1-day MTT assay- The results are expressed as the means ± SD of 3 different experimentsb The values are significantly higher than those obtained by treatment with PTX alone plus those obtained by treatment with CDDP alone at p < 0-05-
The effect of PTX on the sensitivity of RI I cells to CDDP
TABLE 7-
Percent Cytotoxicity (mean ± SD)· PTX (µg-/mL) 0 1 10 100
CDDP (µg-fmL) 0
0-1
0 OA±OA LO± 0-6 5-5 ± LS
0-5 ± LO D-6 ± 1-2 0-2 ± 0-4 7-7±2-6
10 2-8 3-1 3-8 13-2
± ± ± ±
0-6 0-4 2-4 2-2
15-6 10-3 14-9 2U
± ± ± ±
0-2 D-8 1-6
2-8
• The cytotoxic effect of PTX and CDDP on Rll cells was assessed in a 1-day MTT assay- The results are expressed as the means ± SD of 3 different experiments. TABLE 8-
The enhanced sensitivity of R4 cells to CDDP by anti-TNFa MAb Percent Cytotoxicity (mean± SD)" CDDP (µg-/mL)
Treatment
Medium Control Ab Anti-TNF-a MAb
0
0-1
0
1-6 ± 1-6 L1 ± LS 14-6 ± OAb
LS± 0-2 L5 ± D-2
10 7-l±L6 3-3 ± 1-8 21-9 ± 2-2b
44-0 ± 2-5 41-6 ± 3-2 6L9 ± 2-9b
• The cytotoxic effect of CDDP in combination with anti-TNF-a MAb, which neutralized 30 ng- TNF-a on R4 cells, was assessed in a 1-day MTT assay- The results are expressed as the means ± SD of 3 different experimentsh Values are significantly higher than those of combination treatment with CDDP and control Ab at p < 0-05-
minedichloroplatinum II (CDDP: Lot- No. 70H3412) were purchased from Sigma Chemical Co-, St. Louis, Missouri- Tumor necrosis factor-a (5 >< 10 7 U/mg-, Lot- No- 4906) was kindly supplied by Pepro Tech Inc- Interferon-a (Lot. No- R410231) was obtained from Roche Co., Ltd-, Tokyo, Japan. The TNF cDNA and /3-actin cDNA used in making probes for Northern blot analysis were gifts from Smith-Kline-French. The MDR1 cDNA was a gift from Carsten Lincke at The Netherlands Cancer Institute- Anti-TNF-a monoclonal antibody (MAb) (IgG 1 ) and murine IgG 1 isotype control antibody were generous gifts from Dr- G. Trinchieri- 28 Antiserum directed against TNFa was raised in rabbits by means of intramuscular injection of 5 µg. TNF-a in complete Freund's adjuvant. The rabbits were boosted 3 weeks later, and 7 days thereafter were bled by venous puncture. The serum was then tested for neutralizing activityThe rabbits were housed in accordance with UCLA guidelines-
FIG- 2- Effect of PTX andanti-TNF-a MAb on expression ofMDR1 mRNA in R4 cells- R4 cells were incubated with PTX or anti-TNFa MAb for 4 hours- Total RNA was then separated, Northern blotted and probed for MDR-1 and {J-actin as described in Materials and Methods- Each lane is mRNA for MDR-1 and {J-actin of R4 cells after following treatment: lane 1: medium only; lane 2: PTX at 100 µg-/mL; lane 3: IgG 1 control antibody; lane 4: anti-TNF-a MAb that neutralized 30 ng- TNF-a; lane 5: ADlO ovarian tumor cell line as positive controL These results are representative of three experiments-
Alkaline phosphatase linked goat-anti-rabbit IgG was purchased from CaltagCytotoxicity assay. Tumor cell lysis was determined using the microculture tetrazolium dye (MTT) assay as previously described. 24-26 Briefly, 100 µL of target cell suspension (2 x 104 cells) was added to each well of 96-well flat-bottom microtiter plates (Corning Glass Works, Corning, New York), and the plates were incubated for 24 hours at 37C in a humidified 5% CO2 atmosphere- After incubation, 100 µL of drug solution or complete medium for control was placed in the wells, and the plates were incubated for 24 hours at 37C in a humidified 5% CO2 atmosphere- Thereafter, 20 µL of MTT working solution (5 mg-/mL, Sigma Chemical Co-) was added to each culture well, and the cultures were incubated for 4 hours at 37C in a humidified 5% CO2 atmosphere- The culture medium was removed from the wells and replaced with 100 µl. of isopropanol (Sigma Chemical Co-) supplemented with 0.05 N HCl. The absorbance of each well was measured with a microculture plate reader (Titertek Multiskan MCC/340, Flow Laboratories, Rickmansworth, United Kingdom) at 540 nm. The percentage cytotoxicity was calculated according to the following formula: Percentage cytotoxicity = [l - (absorbance of experimental wells/absorbance of control wells)] X 100. Northern blotting. Cytoplasmic RNA from tumor cells was prepared as described in detail elsewhere- 24-26 Briefly, 40 µg- per lane of tumor cell RNA was electrophoresed in 1-2% agarose2.2 M. HCHO gels in lX MOPS buffer (200 mM- MOPS, 50 mM- sodium acetate, 10 rnM. sodium EDTA). The RNA was transferred to Zeta Probe nylon membranes (Bio Rad Laboratories, San Francisco, California) in 20x SSC (3 NL NaCl, 0-3 IvL sodium citrate, pH 7.0)- Fifty to 100 ng. of cDNA probe was labeled with [a 32 P] dCTP (New England Nuclear, Boston, Massachusetts) by random oligo-primer extension- The nylon membranes were ultraviolet-crosslinked, then prehybridized at 45C overnight in 50% formamide (Bethesda Research Laboratories, Bethesda, Maryland), 5x Denhardts (Ficoll, polyvinylpyrrolidone, bovine serum albumin), 0.1 % sodium dodecyl sulfate (SDS, Bethesda Research Laboratories), 100 µg-/mL salmon sperm DNA (Sigma Chemical Co.), and 5X SSC- The radiolabeled probe was added at a concentration of 1 x 106 cpm./ml. of hybridization buffer (6X SSC, 0-5% SDS, 5x Denhardts, 100 µg-/ml. salmon sperm DNA), and the blot was incubated overnight at 65C. The hybridized membranes were then washed with 2X SSC and 0.1% SDS twice for 20 minutes at room temperature and with O.lx SSC and 0.1 % SDS twice for 20 minutes at 65C and exposed to Kodak XAR-5 X-ray filmTumor necrosis factor-a ELISA. Tumor necrosis factor-a protein in the supernatant of the tumor cells was quantified by ELISA. 27 The murine anti-TNF-a MAb, B154.7.l andB154.9-1
1700
TNF-a mRNA DOWNREGULATION IN TUMOR CELL SENSITIZATION
were specific for different epitopes of the TNF-a molecule. B154.7.1 derived from ascites fluid was purified by affinity chromatography using protein A. B154.9.l from ascites was partially purified by 50% ammonium sulfate precipitation. Wells of 96-well ELISA plates were coated with 50 µl. of both antibodies for at least 1 day, then stored as long as 4 weeks at 4C. To set up the assay, coated plates were washed 3 times and blocked with ELISA phosphate-buffered saline containing 1 % bovine serum albumin for 1 hour. The plates were washed twice, and 50 µl. of tumor supernatants and TNF -a standard were added to the wells. After an overnight incubation, the plates were washed 3 times, and 50 µl. ofpolyclonal anti-TNFa Ab was added to each well. After a 2-hour incubation, alkaline phosphatase-conjugated goat anti-rabbit IgG (Caltag) was added to each well and incubated for an additional 2 hours. Finally, the plates were washed and incubated with substrate (Sigma 104). Plates were read 2 hours later at 405 nm. using a Titertek Multiscan MCC/240 ELISA reader. Statistical analysis. All determinations were performed in triplicate, and the results are expressed as the means ± standard deviation (SD). Statistical significance was determined by Student's t test. A p-value of 0.05 or less was considered significant. RESULTS
The effect of PTX on the expression of TNF-a mRNA and protein in R4 and Rll cells. Untreated R4 cells constitutively expressed the mRNA for TNF-a (fig. 1, A, B), and secreted TNF-a protein (table 1). Exposing R4 cells to PTX resulted in a marked reduction of TNF-a mRNA and protein expression. In contrast, the incubation of R4 cells with TNF-a upregulated the level of mRNA and protein for TNF-a. The TNF-a mRNA expression was optimally enhanced following TNF-a treatment at 10 ng./ml. for 1 hour (data not shown). A combination of PTX and TNF-a downregulated the TNF-a enhanced expression of TNF-a mRNA and protein. Treatment of R4 cells with PTX and/or TNF-a had no effect on the /j-actin mRNA level. R11 cells did not constitutively express TNF-a mRNA or protein (fig. 1, C, D, table 1). Pentoxifylline had no effect on the expression of TNF-a mRNA or protein in R11 cells. Pretreatment of R11 cells with TNF-a resulted in the induction of mRNA for TNF-a, while the TNF-a treatment did not induce TNF -a protein. When PTX was used in combination with TNF-a, the level of TNF-a mRNA induced by TNF-a in R11 cells was markedly downregulated. In contrast, treatment of R11 cells with PTX and/or TNF-a had no effect on the /j-actin mRNA level. These results showed that PTX downregulated TNF-a mRNA in RCC cells as well as in macrophages and monocytes. The enhanced sensitivity of R4 cells and Rll cells to TNF-a by PTX. One possible mechanism responsible for the resistance of tumor cells to TNF-a is the expression of TNF-a mRNA and/or protein. 20 -23 Pentoxifylline downregulated the TNF-a expression (fig. 1, A-D, table 1). We examined whether or not PTX in combination with TNF-a could overcome the resistance of R4 and R11 cells to TNF -a by means of a one-day MTT assay. Pentoxifylline enhanced the sensitivity of R4 cells to TNF-a (table 2). The susceptibility of R11 cells to TNF-a was also augmented by PTX (table 3). The cytotoxic effect of PTX in combination with IFN-a on R4 and Rll cells. Pentoxifylline downregulated the TNF-a mRNA and protein expression. Because TNF-a is an immunoregulatory cytokine, we explored the possibility that PTX could enhance the susceptibility of R4 cells to IFN-a, the most commonly used immunotherapeutic agent against RCC, by downregulation of TNF-a expression. Pentoxifylline and IFNa, when used in combination, overcame the resistance of TNFa producing R4 cells to IFN-a (table 4), whereas PTX did not augment the susceptibility of R11 cells, which do not produce TNF-a, to IFN-a (table 5). Pentoxifylline had no effect on the cytotoxic activity of IFN-'Y against R4 cells (data not shown).
The effect of PTX on the sensitivity of R4 and Rl 1 cells to CDDP. A possible mechanism of TNF-a resistance in tumor cells is endogenous TNF -a expression, the endogenous TNF -a exerting its protective function against exogenous TNF -a by inducing manganous superoxide dismutase (MnSOD) production.20-23, 29 • 30 Tumor necrosis factor-a and CDDP exert their cytotoxic activity in part through free radical production. 16· 31 We examined whether PTX could overcome the resistance of R4 cells to CDDP as well as to TNF-a, by downregulating endogenous TNF -a expression. Pentoxifylline enhanced the sensitivity of TNF-a producing R4 cells to CDDP in a one-day MTT assay (table 6). However, treatment of the non-TNF-aproducing R11 cells with PTX at the concentrations used in this study did not augment their sensitivity to CDDP (table 7). The effect of anti-TNF-a MAb on the sensitivity of R4 cells to CDDP. We explored the possibility that anti-TNF-a MAb could enhance the susceptibility of R4 cells to CDDP. AntiTNF-a MAb enhanced the sensitivity of R4 cells to CDDP (table 8). The effect of PTX or anti-TNF-a MAb on the expression of MDR-1 mRNA in R4 cells. One of the possible mechanisms of drug resistance is overexpression of the MDR-1 gene. 32 • 33 R4 cells did not constitutively express MDR-1 mRNA. Neither PTX nor anti-TNF-a MAb had an effect on the expression of mRNA for MDR-1 in R4 cells (fig. 2). DISCUSSION
The present study demonstrated that PTX enhanced the sensitivity of R4 and R11 RCC cells to TNF-a and reversed tumor cell resistance to TNF-a. Clinically, it is interesting that the PTX-mediated effect was optimal at a concentration of 100 µg./ml., the level observed in the blood of human cancer patients treated with PTX by local arterial injection, intravenous injection, or oral administration, without severe side effects. 34• 35 R4 cells were resistant to TNF -a and expressed TNF -a mRNA and protein constitutively. Exposing R4 cells to PTX markedly reduced the expression of TNF -a mRN A and protein. In contrast, treatment with TNF -a alone enhanced the level of TNF -a mRN A and protein. A combination of PTX and TNFa downregulated the level of TNF-a mRNA and protein enhanced by TNF -a alone. It has been reported that there is a correlation between the expression of TNF-a mRNA and/or protein in tumor cells and their resistance to TNF -a. 20-23 Thus, one possible mechanism of the enhanced cytotoxic effect of PTX and TNF -a used in combination on R4 cells may be PTXinduced downregulation of TNF-a mRNA and protein. Conceivably, treatment of R4 cells with TNF-a enhances the expression of TNF-a mRNA and protein, and these cells may become even more resistant to TNF -a, but the addition of PTX may diminish TNF-a-induced resistance by downregulating TNF-a mRNA and protein. R11 cells were also resistant to TNF -a, but did not express TNF-a mRNA constitutively. However, treating R11 cells with TNF-a induced TNF-a mRNA. Treatment with PTX alone did not induce TNF-a mRNA. Incubating R11 cells with a combination of PTX and TNF-a markedly downregulated TNF-a-induced expression of TNF-a mRNA. A proposed mechanism of TNF-a resistance in tumor cells is the endogenous production of TNF-a mRNA and/or protein. 20-23 The PTX-induced downregulation of TNF-a-induced TNF-a mRNA in R11 cells may play a role in the enhanced cytotoxicity observed with the combination of PTX and TNF-a. Conceivably, like R4 cells, treatment of R11 cells with TNF-a induces mRNA for TNF-a, making R11 cells even more resistant to TNF-a. The addition of PTX may diminish the TNF-a-induced resistance of R11 cells by downregulating their TNF -a mRNA. It has been reported that the presence of mRNA for TNF-a does not correlate with translation and secretion of TNF-a protein. 36· 37 Treatment of Rl 1 cells with TNF -a induced TNF -
TNF-a IllRNA DOWNREGULATION IN TUMOR CELL SENSITIZATION a mRNA expression, but TNF-a-treated Rll cells did not secrete detectable levels of TNF-a protein. Accordingly, the resistance of Rll cells to TNF-a may be associated with TNFa-induced TNF-a mRNA expression in the absence of TNF-a protein secretion. Although downregulation of TNF-a mRNA by PTX is suggestive of enhanced sensitivity, the precise mechanism of overcoming resistance of R4 and Rll cells to TNF-a by a combination of PTX and TNF-a is not fully understood. One possibility is that the antitumor effect of TNF-a has been partly linked to DNA damage 16• 17 and PTX interferes with the repair of DNA damage by prematurely inducing mitosis. 38• 39 This may result in an enhanced cytotoxic effect of PTX and TNF-a. Pentoxifylline and the human-derived cytokine IFN-a acted synergistically in their cytotoxic activity against TNF-a-producing R4 cells, but not against non-TNF-a-producing Rll cells, although the synergy was modest. The possible mechanism responsible for the enhanced cytotoxicity seen with the combination of PTX and IFN-a may be associated with the downregulation of TNF ~a mRNA and protein by PTX. Interferon-a is relatively effective against RCC, not only through its direct cytotoxic activity, but also through its ability to augment the cytotoxic activity of natural killer cells and other lymphocytes. 40 • 41 Although the mechanism responsible for the enhanced cytotoxicity of this combination is not yet fully understood, the findings are nevertheless of clinical relevance. Several possible mechanisms of CDDP resistance of tumor cells have been reported such as alterations in the transmembrane transport of CDDP, cytosolic quenching of CDDP due to increased levels of sulfhydryl compounds, decreased CDDPbinding to DNA and enhanced DNA adduct repair capability. Alterations in the transmembrane transport of CDDP in tumor cells result in reduced intracellular accumulation of CDDP and resistance to CDDP, since there is an inverse correlation between intracellular accumulation of CDDP and resistance to it. 42- 44 Evidence for the cytosolic quenching of CDDP, either by glutathione or by sulfhydryl containing proteins, has been obtained in tumor cell lines that are resistant to CDDP in vitro. Some resistant cells have higher levels of intracellular glutathione or metallothionein. 45 • 46 Cis-diamminedichloroplatinum II inhibits DNA synthesis. This process is mediated by binding to DNA47 and the formation of intrastrand or interstrand crosslinks in DNA. 48 Cell-mediated augmentation of the DNA repair capability plays a major role in CDDP resistance in several mammalian cell lines studied. 49 Endogenous TNF-a exerts its protective function against exogenous TNF-a by inducing MnSOD expression. 29 • 30 Tumor necrosis factor-a and CDDP exert their cytotoxic activity in part through free radical generation. 16• 31 We examined the possibility that PTX could overcome the resistance of R4 cells to CDDP, as well as to TNF-a, by downregulating endogenous TNF-a expression. Combination treatment with PTX and CDDP resulted in enhanced cytotoxicity against TNF-a-producing R4 cells, but not against Rll cells, which did not express TNF-a mRNA and protein. Anti-TNF-a MAb enhanced the sensitivity of R4 cells to CDDP. These results suggest that one of the mechanisms responsible for the enhanced cytotoxicity seen with the combination of PTX and CDDP may be associated with the downregulation of TNF -a protein secretion by PTX. Although downregulation of TNF -a secretion by PTX is a potential mechanism for reversal of CDDP-resistance, the precise mechanism for overcoming resistance of R4 cells to CDDP by a combination of PTX and CDDP is not yet fully understood. The MDR-1 gene has been proposed as a possible mechanism of tumor cell resistance to anticancer chemotherapeutic drugs. 32• 33 We considered the possibility that PTX and antiTNF-a MAb might reduce the level of MDR expression in R4 cells, so sensitizing the tumor cells to CDDP, but this possibility
1701
was excluded since PTX and anti-TNF-a MAb had no effect on MDR-1 mRNA expression in R4 cells. Cis-diamminedichloroplatinum II mediates a significant inhibitory effect on DNA synthesis. Pentoxifylline prevents delays in the cell cycle transit through the G2 phase and does not allow time for the repair of DNA cross-links. 38• 39 Accordingly, CDDP-induced DNA-damaged R4 cells treated with PTX may proceed through the cell cycle more quickly (no arrest in the G2 phase) and may divide before DNA repair can be completed. Thus, the combination of PTX and CDDP results in enhanced cytotoxicity against R4 cells. Further studies are needed to support this hypothesis. Interferon-a and TNF-a are relatively effective against RCC, but the response rate is low. 40 • 41 Although the most effective treatment for RCC remains surgery, efficacy has been only demonstrated for regional resectable disease. Thus, a more effective therapy is necessary for these patients. This study has demonstrated that PTX, in combination with TNF-a, IFN-a, or CDDP, can overcome the drug-resistance of RCC cells and that the downregulation of TNF-a mRNA and/or protein may play a role in this enhanced cytotoxicity. These data suggest that therapy with PTX in combination with TNF-a, IFN-a, or CDDP may be beneficial in overcoming the resistance of RCC to conventional forms of therapy. Acknowledgments. We thank Dr. Linda Sakimura for her help in preparation of this manuscript. REFERENCES
1. Muller, R. and Lehrach, F.: Haemorheology and cerebrovascular disease. Multifunctional approach with pentoxifylline. Curr. Med. Res. Opin., 7: 253, 1981. 2. Grigoleit, H. G. and Jacobi, G.: Microrheological effects of vasoactive compound 3,7-dimethyl-aa-(5-oxo-hexyl)-xanthine (Pentoxifylline, BL 191). Vasa, 6: 274, 1977. 3. Aviado, D. M. and Porter, J. M.: Pentoxifylline. A new drug for the treatment of intermittent claudication. Mechanism of action, pharmacokinetics, clinical efficacy, and adverse effects. Pharmacotherapy, 4: 297, 1984. 4. Muller, R.: Pentoxifylline-A biomedical profile. J. Med., 10: 307, 1979. 5. Poggesi, L., Scarti, L., Boddi, M., Masotti, G. and Serneri, G. G.N.: Pentoxifylline treatment in patients with occlusive peripheral arterial disease. Circulatory changes and effects on prostaglandin synthesis. Angiol. J. Vase. Dis., 36: 628, 1985. 6. Bessler, H., Gilgal, R., Djaldetti, M. and Zahavi, I.: Effect of pentoxifylline on the phagocytic activity, cAMP levels, and superoxide anion production by monocytes and polymorphonuclear cells. J. Leukoc. Biol., 40: 747, 1986. 7. Tannenbaum, C. S., and Hamilton, T. A.: Lipopolysaccharideinduced gene expression in murine peritoneal macrophages is selectively suppressed by agents that elevate intracellular cAMP. J. Immunol., 142: 1274, 1989. 8. Han, J., Thompson, P. and Beutler, B.: Dexamethasone and pentoxifylline inhibit endotoxin-induced cachectin/tumor necrosis factor synthesis at separate points in the signaling pathway. J. Exp. Med., 172: 391, 1990. 9. Waage, A., Sorenson, M. and Stordal, B.: Differential effect of oxpentifylline on tumor necrosis factor and interleukin-6 production. Lancet, 3: 543, 1990. 10. Carswell, E. A., Old, L. J., Kassel, R. L., Green, S., Fiore, N. and Williamson, B.: An endotoxin-induced serum factor that causes necrosis in tumors. Proc. Natl. Acad. Sci. U.S.A., 72: 3666, 1975. 11. Old, L. J.: Tumor necrosis factor (TNF). Science, 230: 630, 1985. 12. Ruggiero, V., Latham, K. and Baglioni, C.: Cytostatic and cytotoxic activity of tumor necrosis factor on human cancer cells. J. Immunol., 138: 2711, 1987. 13. Shirai, T., Yamaguchi, H., Ito, H., Todd, C. W. and Wallace, R. B.: Cloning and expression in Escherichia coli of the gene for human tumor necrosis factor. Nature, 313: 803, 1985. 14. Feinberg, B., Kurzurock, R., Talpaz, M., Blick, M., Saks, S. and Gutterman, J. U.: Phase I trial of intravenously-administered recombinant tumor necrosis factor-alpha in cancer patients. J. Clin. Oncol., 6: 1328, 1988. 15. Lenk, H., Tanneberger, St., Muller, U., Ebert, J. and Shiga, T.:
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TNF-a mRNA DOWNREGULATION IN TUMOR CELL SENSITIZATION
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THE JOURNAL OF UROLOGY
Copyright© 1994 by AMERICAN UROLOG!CAL ASSOCIATION, !NC.
June 1994 in U.S.A.
OVERCOMING TNF-a AND DRUG RESISTANCE OF HUMAN RENAL CELL CARCINOMA CELLS BY TREATMENT WITH PENTOXIFYLLINE IN COMBINATION WITH TNF-a OR DRUGS: THE ROLE OF TNF-a mRNA DOWNREGULATION IN TUMOR CELL SENSITIZATION YOUICHI MIZUTANI, BENJAMIN BONAVIDA, YOSHINO RI NIO AND OSAMU YOSHIDA* From the Department of Urology and First Department of Surgery, Faculty of Medicine, Kyoto University, Kyoto, Japan and the Department of Microbiology and Immunology, UCLA School of Medicine, Los Angeles, California
ABSTRACT
Previous studies have demonstrated that one of the possible mechanisms responsible for the resistance of tumor cells to tumor necrosis factor-Cl' (TNF-a) is the expression of TNF-a mRNA and/or protein. Pentoxifylline (PTX) suppressed TNF-a gene transcription and downregulates the expression of TNF-a mRNA and the secretion of TNF-a protein in macrophages and monocytes. This study investigates whether PTX downregulates the expression of TNF-a mRNA and/or protein in renal cell carcinoma (RCC) cells and whether PTX enhances the sensitivity of TNF-aresistant RCC cells to TNF-a. Further, we explored whether PTX enhances the sensitivity of RCC cells to agents other than TNF-l\' by downregulation of the expression of TNF-a mRNA and protein. The R4 human RCC cell line constitutively expressed TNF -a mRNA and protein and was resistant to TNF-a. When R4 cells were incubated with PTX, the level of TNF-a mRNA and protein was markedly reduced. Pentoxifylline and TNF-a together overcame the resistance of R4 cells to TNFa. The Rll human RCC cell line did not constitutively express TNF-a mRNA or protein, and was resistant to TNF-a. The expression of TNF-ll' mRNA in Rll cells, but not the production of TNFa protein, was induced by TNF -a. When PTX was used in combination with TNF -a, the level of TNF-a mRNA induced by TNF-a was markedly reduced. The combination of PTX and TNF-a overcame the resistance of Rll cells to TNF-a. Pentoxifylline also enhanced the sensitivity of R4 cells to interferon-a. Pentoxifylline and anti-TNF -Cl' monoclonal antibody augmented the sensitivity of R4 cells to cis-diamminedichloroplatinum (II) (CDDP). This study demonstrated that PTX, in combination with TNF-ll', IFN-a or CDDP, overcame the drug resistance to RCC cells and that downregulation of TNF-a mRNA by PTX may be related to the cytotoxicity enhanced by the combination. The implications of these findings for clinical therapy are discussed. KEY WORDS:
carcinoma, renal cell; cisplatinum; interferon-alpha; pentoxifylline; tumor necrosis factor
Pentoxifylline (PTX) [3, 7-dimethyl-l (5-oxohexyl)-xanthine] was originally introduced as a hemorrheologic agent for the treatment of peripheral vascular disease. 1 ' 2 It augments erythrocyte flexibility and decreases blood viscosity, resulting in increased microcirculatory blood flow. 2 ' 3 It also inhibits platelet aggregation, increases fibrinolytic activity and stimulates prostacyclin release. 4 · 5 It has been reported that as a methylxanthine derivative related to aminophylline and caffeine, PTX inhibits intracellular cyclic nucleotide phosphodiesterase, resulting in an increased intracellular concentration of cyclic adenosine monophosphate (cAMP), which impairs a number of cellular immune functions. 6 · 7 Pentoxifylline significantly reduces tumor necrosis factor-a (TNF-a) protein production and TNF-a mRNA transcription in murine macrophages and human monocytes through the cAMP pathway. 8 • 9 Tumor necrosis factor-a was originally identified in the serum of mice challenged with bacillus Calmette-Guerin and endotoxin. It has a wide range of biological activities, including cytotoxic and cytostatic antitumor activity. 10- 12 Recombinant human TNF-a has been developed, and its antitumor activity against certain cancers involving renal cell carcinoma (RCC) Accepted for publication December 2, 1993. * Requests for reprints: Department of Urology, Faculty of Medicine, Kyoto University, 54 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606, Japan. This work was supported by a Grant-in-Aid (No. 3454385 and No. 5671314) from the Japanese Ministry of Education, Science and Culture.
is currently being investigated in phase I and phase II clinical trials. 13- 15 Its antitumor effect may be linked to the generation of free radicals, the inhibition of protein synthesis and the induction of programmed cell death. 16· 17 Although TNF-a appears to be a promising anticancer agent, most tumor cells, including RCC, are resistant to ito 18 · 19 It has been reported that one possible mechanism responsible for the resistance of tumor cells to TNF-a is the expression of TNF-a mRNA and/or protein. 20- 23 We demonstrated that cisdiamminedichloroplatinum II (CDDP) and buthionine sulfoximine downregulate the expression of TNF-a mRNA and enhance the susceptibility of tumor cells to TNF-a. 24 - 26 In this study we investigated whether PTX downregulates the expression ofTNF-a mRNA and/or protein in RCC cells and whether PTX augments the sensitivity of TNF -a-resistant RCC cells to TNF-a. In addition, we studied whether PTX enhances the sensitivity of RCC cells to other agents as well as TNF -a by downregulation of the expression of TNF-a mRNA and/or protein. Furthermore, we have explored the possible underlying mechanism involved in the reversal of resistance. MATERIALS AND METHODS
Tumor cells. The human renal cell carcinoma (RCC) cell lines R4 and Rll were supplied by Dr. Hans Stotter, Bethesda, Maryland (Histopathology: adenocarcinoma). 25 The ovarian tumor line ADlO was supplied by Dr. Robert Ozols. 27 The cell lines were maintained in RPMI-1640 medium (M.A. Bioprod-
1697
1698
TNF-a mRNA DOWNREGULATION IN TUMOR CELL SENSITIZATION 2
A
3
4
2
C
TNF-a
3
4
TNF-«
/l-actin a-actin
50
B
D
30
40 20
i .!l .z,.!ii
30
.,
20
ll:
10
10
0 4
Lane numbers
Lane numbers
FIG. 1. Effect of PTX on level of TNF-a mRNA expression in R4 and R11 cells. R4 and R11 cells were incubated with PTX and/or TNF-a for 4 hours. Total RNA was then separated, Northern blotted and probed for TNF-a and /3-actin as described in Materials and Methods. Each lane is mRNA for TNF-a and /3-actin of R4 cells (A) and R11 cells (C) after the various treatments. A, C: lane 1: medium only; lane 2: PTX at 100 µg./ml.; lane 3: TNF-a at 10 ng./ml.; lane 4: PTX at 100 µg./ml. and TNF-a at 10 ng./ml. simultaneously. Densitometry scan of bands in A and C shows relative levels of mRNA compared with controls (B, D ). Experiments were repeated three times. TABLE
1. The effect of PTX on TNF-a secretion by R4 and Rll cells
TABLE 3.
The enhanced sensitivity of Rll cells to TNF-a by PTX Percent Cytotoxicity (mean ± S.D.)•
TNF-a secretion (pg./ml.)" PTX (µg./ml.)
Treatmentb
Cell line Control (Medium) PTX R4 Rll
177.0 ± 19.3 1.0 ± 1.4
20.9 ± 6.6 0.0 ± 5.5
TNF-a 279.4 ± 5.9 1.1 ± 4.0
• Tumor necrosis factor-a secretion was measured by ELISA as described in Materials and Methods. The results are expressed as the means ± SD of 2 different experiments. b R4 and Rll cells were treated with PTX at 100 µg./ml. and/or TNF-a at 10 ng./ml. for 4 hours, washed three times in complete medium and incubated in complete medium for 24 hours. After incubation, the level of TNF-a protein in the supernatant was measured. TABLE 2.
0
PTX+TNF-a 144.4 ± 31.8 0.8 ± 4.5
The enhanced sensitivity of R4 cells to TNF-a by PTX
TNF-a (ng./ml.)
0 1 10 100
0 1.9 ± 1.4 4.2 ± 1.0 4.5 ± 1.8
TABLE 4.
0 1.2 ± 0.6 2.5 ± 2.0 4.2 ± 2.0
1 1.9 ± 14.2 ± 18.1 ± 27.3 ±
10 1.8 1.6 2.4 2.lb
9.9 ± 20.7 ± 25.6 ± 34.4 ±
100
± 1.6 ± 1.4 ± 1.6 ± 0.4b
15.0 ± 15.9 ± 20.2 ± 34.7 ±
2.4 1.6 2.5 4.6b
Percent Cytotoxicity (mean ± SD)'
20.6 ± 30.9 ± 33.2 ± 44.3 ±
2.6 2.4 2.5b 4.0b
IFN-a (U/ml.) 0
100 1.4 2.4 2.0b 5.8b
3.2 9.3 15.1 25.6
The enhanced sensitivity of R4 cells to IFN-a by PTX
PTX (µg./ml.)
TNF-a (ng./ml.) 0
0 1 10 100
± 0.2 ± 1.5 ± 1.6 ± 0.2
• The cytotoxic effect of PTX and TNF-a on Rll cells was assessed in a 1day MTT assay. The results are expressed as the means ± SD of 3 different experiments. b The values are significantly higher than those obtained by treatment with PTX alone plus those obtained by treatment with TNF-a alone at p < 0.05.
Percent Cytotoxicity (mean± SD)' PTX (µg./ml.)
10
1 0.4 2.2 5.3 10.2
0 1 10 100
0 1.8± 2.0 2.8 ± 2.0 5.8 ± 0.6
10 1.3 ± 10.9 ± 12.0 ± 16.1 ±
100 2.0 1.2 1.6 1.4
2.8 ± 11.0 ± 16.1 ± 24.1 ±
2.6 1.3 2.6 2.lb
1000 4.3 ± 11.1 ± 17.9 ± 26.0 ±
1.0 1.2 2.4 2.0b
• The cytotoxic effect of PTX and TNF-a on R4 cells was assessed in a 1-day MTT assay. The results are expressed as the means ± SD of 3 different experiments. b The values are significantly higher than those obtained by treatment with PTX alone plus those obtained by treatment with TNF-a alone at p < 0.05.
• The cytotoxic effect of PTX and IFN-a on R4 cells was assessed in a 1-day MTT assay. The results are expressed as the means ± SD of 3 different experiments. b The values are significantly higher than those obtained by treatment with PTX alone plus those obtained by treatment with IFN-a alone at p < 0.05.
ucts) supplemented with 1% L-glutamine (Gibco, Bio-Cult, Glasgow, Scotland, United Kingdom), 1% nonessential amino acid (Gibco), 1% Fungibact solution (Irvine Scientific, Irvine, California) and 10% heat-inactivated fetal bovine serum (Gibco), hereafter referred to as complete medium. When the
tumor cell lines were used as target cells, they were treated with trypsin-ethylene-diamine-tetraacetic acid (TrypsinEDTA, ICN Biomedical Inc.), washed three times and suspended in complete medium. Reagents. Pentoxifylline (Lot. No. 91H0194) and cis-diam-
1699
TNF-a mRNA DO'NNREGULATION IN TUMOR CELL SENSITIZATION 5- The cytotoxic effect of PTX and IFN-a on RI 1 cells
TABLE
2
3
4
5
Percent Cytotoxicity (mean± SD)· PTX (µg-/mL)
IFN-a (U/mL)
0 1 10 100
MDR-1
100
0
10
0 0-6 ± L2 6A ± LS 6A ± L6
D-2 ± OA 3-0 ± LS 6-9 ± L1 lOA± U
2-8 4-2 9-5 13-7
± ± ± ±
1000 D-8 L6 LO 2-6
4-6 4-6 9-8 lU
± ± ± ±
LS L6 L6 2-2
a The cytotoxic effect of PTX and IFN-a on Rll cells was assessed in a 1-day MTT assay- The results are expressed as the means ± SD of 3 different experiments-
TABLE
6- The enhanced sensitivity of R4 cells to CDDP by PTX Percent Cytotoxicity (mean ± S-D-)·
PTX (µg-fmL)
CDDP (µg-/mL) 0
0 1 10
100
JO
0-1 L9 15-9 16-7 25-5
0 L3 ± L6 L9 ± 0-8 6-2 ± L4
± ± ± ±
0-4 2-4 2-6 2-6b
8-8 19-7 23-7 32-1
± ± ± ±
L2 2-8 3-0 3-2b
39-3 54-6 57-1 65-1
± ± ± ±
4-0 3-2 3-2b 506b
a The cytotoxic effect of PTX and CDDP on R4 cells was assessed in a 1-day MTT assay- The results are expressed as the means ± SD of 3 different experimentsb The values are significantly higher than those obtained by treatment with PTX alone plus those obtained by treatment with CDDP alone at p < 0-05-
The effect of PTX on the sensitivity of RI I cells to CDDP
TABLE 7-
Percent Cytotoxicity (mean ± SD)· PTX (µg-/mL) 0 1 10 100
CDDP (µg-fmL) 0
0-1
0 OA±OA LO± 0-6 5-5 ± LS
0-5 ± LO D-6 ± 1-2 0-2 ± 0-4 7-7±2-6
10 2-8 3-1 3-8 13-2
± ± ± ±
0-6 0-4 2-4 2-2
15-6 10-3 14-9 2U
± ± ± ±
0-2 D-8 1-6
2-8
• The cytotoxic effect of PTX and CDDP on Rll cells was assessed in a 1-day MTT assay- The results are expressed as the means ± SD of 3 different experiments. TABLE 8-
The enhanced sensitivity of R4 cells to CDDP by anti-TNFa MAb Percent Cytotoxicity (mean± SD)" CDDP (µg-/mL)
Treatment
Medium Control Ab Anti-TNF-a MAb
0
0-1
0
1-6 ± 1-6 L1 ± LS 14-6 ± OAb
LS± 0-2 L5 ± D-2
10 7-l±L6 3-3 ± 1-8 21-9 ± 2-2b
44-0 ± 2-5 41-6 ± 3-2 6L9 ± 2-9b
• The cytotoxic effect of CDDP in combination with anti-TNF-a MAb, which neutralized 30 ng- TNF-a on R4 cells, was assessed in a 1-day MTT assay- The results are expressed as the means ± SD of 3 different experimentsh Values are significantly higher than those of combination treatment with CDDP and control Ab at p < 0-05-
minedichloroplatinum II (CDDP: Lot- No. 70H3412) were purchased from Sigma Chemical Co-, St. Louis, Missouri- Tumor necrosis factor-a (5 >< 10 7 U/mg-, Lot- No- 4906) was kindly supplied by Pepro Tech Inc- Interferon-a (Lot. No- R410231) was obtained from Roche Co., Ltd-, Tokyo, Japan. The TNF cDNA and /3-actin cDNA used in making probes for Northern blot analysis were gifts from Smith-Kline-French. The MDR1 cDNA was a gift from Carsten Lincke at The Netherlands Cancer Institute- Anti-TNF-a monoclonal antibody (MAb) (IgG 1 ) and murine IgG 1 isotype control antibody were generous gifts from Dr- G. Trinchieri- 28 Antiserum directed against TNFa was raised in rabbits by means of intramuscular injection of 5 µg. TNF-a in complete Freund's adjuvant. The rabbits were boosted 3 weeks later, and 7 days thereafter were bled by venous puncture. The serum was then tested for neutralizing activityThe rabbits were housed in accordance with UCLA guidelines-
FIG- 2- Effect of PTX andanti-TNF-a MAb on expression ofMDR1 mRNA in R4 cells- R4 cells were incubated with PTX or anti-TNFa MAb for 4 hours- Total RNA was then separated, Northern blotted and probed for MDR-1 and {J-actin as described in Materials and Methods- Each lane is mRNA for MDR-1 and {J-actin of R4 cells after following treatment: lane 1: medium only; lane 2: PTX at 100 µg-/mL; lane 3: IgG 1 control antibody; lane 4: anti-TNF-a MAb that neutralized 30 ng- TNF-a; lane 5: ADlO ovarian tumor cell line as positive controL These results are representative of three experiments-
Alkaline phosphatase linked goat-anti-rabbit IgG was purchased from CaltagCytotoxicity assay. Tumor cell lysis was determined using the microculture tetrazolium dye (MTT) assay as previously described. 24-26 Briefly, 100 µL of target cell suspension (2 x 104 cells) was added to each well of 96-well flat-bottom microtiter plates (Corning Glass Works, Corning, New York), and the plates were incubated for 24 hours at 37C in a humidified 5% CO2 atmosphere- After incubation, 100 µL of drug solution or complete medium for control was placed in the wells, and the plates were incubated for 24 hours at 37C in a humidified 5% CO2 atmosphere- Thereafter, 20 µL of MTT working solution (5 mg-/mL, Sigma Chemical Co-) was added to each culture well, and the cultures were incubated for 4 hours at 37C in a humidified 5% CO2 atmosphere- The culture medium was removed from the wells and replaced with 100 µl. of isopropanol (Sigma Chemical Co-) supplemented with 0.05 N HCl. The absorbance of each well was measured with a microculture plate reader (Titertek Multiskan MCC/340, Flow Laboratories, Rickmansworth, United Kingdom) at 540 nm. The percentage cytotoxicity was calculated according to the following formula: Percentage cytotoxicity = [l - (absorbance of experimental wells/absorbance of control wells)] X 100. Northern blotting. Cytoplasmic RNA from tumor cells was prepared as described in detail elsewhere- 24-26 Briefly, 40 µg- per lane of tumor cell RNA was electrophoresed in 1-2% agarose2.2 M. HCHO gels in lX MOPS buffer (200 mM- MOPS, 50 mM- sodium acetate, 10 rnM. sodium EDTA). The RNA was transferred to Zeta Probe nylon membranes (Bio Rad Laboratories, San Francisco, California) in 20x SSC (3 NL NaCl, 0-3 IvL sodium citrate, pH 7.0)- Fifty to 100 ng. of cDNA probe was labeled with [a 32 P] dCTP (New England Nuclear, Boston, Massachusetts) by random oligo-primer extension- The nylon membranes were ultraviolet-crosslinked, then prehybridized at 45C overnight in 50% formamide (Bethesda Research Laboratories, Bethesda, Maryland), 5x Denhardts (Ficoll, polyvinylpyrrolidone, bovine serum albumin), 0.1 % sodium dodecyl sulfate (SDS, Bethesda Research Laboratories), 100 µg-/mL salmon sperm DNA (Sigma Chemical Co.), and 5X SSC- The radiolabeled probe was added at a concentration of 1 x 106 cpm./ml. of hybridization buffer (6X SSC, 0-5% SDS, 5x Denhardts, 100 µg-/ml. salmon sperm DNA), and the blot was incubated overnight at 65C. The hybridized membranes were then washed with 2X SSC and 0.1% SDS twice for 20 minutes at room temperature and with O.lx SSC and 0.1 % SDS twice for 20 minutes at 65C and exposed to Kodak XAR-5 X-ray filmTumor necrosis factor-a ELISA. Tumor necrosis factor-a protein in the supernatant of the tumor cells was quantified by ELISA. 27 The murine anti-TNF-a MAb, B154.7.l andB154.9-1
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TNF-a mRNA DOWNREGULATION IN TUMOR CELL SENSITIZATION
were specific for different epitopes of the TNF-a molecule. B154.7.1 derived from ascites fluid was purified by affinity chromatography using protein A. B154.9.l from ascites was partially purified by 50% ammonium sulfate precipitation. Wells of 96-well ELISA plates were coated with 50 µl. of both antibodies for at least 1 day, then stored as long as 4 weeks at 4C. To set up the assay, coated plates were washed 3 times and blocked with ELISA phosphate-buffered saline containing 1 % bovine serum albumin for 1 hour. The plates were washed twice, and 50 µl. of tumor supernatants and TNF -a standard were added to the wells. After an overnight incubation, the plates were washed 3 times, and 50 µl. ofpolyclonal anti-TNFa Ab was added to each well. After a 2-hour incubation, alkaline phosphatase-conjugated goat anti-rabbit IgG (Caltag) was added to each well and incubated for an additional 2 hours. Finally, the plates were washed and incubated with substrate (Sigma 104). Plates were read 2 hours later at 405 nm. using a Titertek Multiscan MCC/240 ELISA reader. Statistical analysis. All determinations were performed in triplicate, and the results are expressed as the means ± standard deviation (SD). Statistical significance was determined by Student's t test. A p-value of 0.05 or less was considered significant. RESULTS
The effect of PTX on the expression of TNF-a mRNA and protein in R4 and Rll cells. Untreated R4 cells constitutively expressed the mRNA for TNF-a (fig. 1, A, B), and secreted TNF-a protein (table 1). Exposing R4 cells to PTX resulted in a marked reduction of TNF-a mRNA and protein expression. In contrast, the incubation of R4 cells with TNF-a upregulated the level of mRNA and protein for TNF-a. The TNF-a mRNA expression was optimally enhanced following TNF-a treatment at 10 ng./ml. for 1 hour (data not shown). A combination of PTX and TNF-a downregulated the TNF-a enhanced expression of TNF-a mRNA and protein. Treatment of R4 cells with PTX and/or TNF-a had no effect on the /j-actin mRNA level. R11 cells did not constitutively express TNF-a mRNA or protein (fig. 1, C, D, table 1). Pentoxifylline had no effect on the expression of TNF-a mRNA or protein in R11 cells. Pretreatment of R11 cells with TNF-a resulted in the induction of mRNA for TNF-a, while the TNF-a treatment did not induce TNF -a protein. When PTX was used in combination with TNF-a, the level of TNF-a mRNA induced by TNF-a in R11 cells was markedly downregulated. In contrast, treatment of R11 cells with PTX and/or TNF-a had no effect on the /j-actin mRNA level. These results showed that PTX downregulated TNF-a mRNA in RCC cells as well as in macrophages and monocytes. The enhanced sensitivity of R4 cells and Rll cells to TNF-a by PTX. One possible mechanism responsible for the resistance of tumor cells to TNF-a is the expression of TNF-a mRNA and/or protein. 20 -23 Pentoxifylline downregulated the TNF-a expression (fig. 1, A-D, table 1). We examined whether or not PTX in combination with TNF-a could overcome the resistance of R4 and R11 cells to TNF -a by means of a one-day MTT assay. Pentoxifylline enhanced the sensitivity of R4 cells to TNF-a (table 2). The susceptibility of R11 cells to TNF-a was also augmented by PTX (table 3). The cytotoxic effect of PTX in combination with IFN-a on R4 and Rll cells. Pentoxifylline downregulated the TNF-a mRNA and protein expression. Because TNF-a is an immunoregulatory cytokine, we explored the possibility that PTX could enhance the susceptibility of R4 cells to IFN-a, the most commonly used immunotherapeutic agent against RCC, by downregulation of TNF-a expression. Pentoxifylline and IFNa, when used in combination, overcame the resistance of TNFa producing R4 cells to IFN-a (table 4), whereas PTX did not augment the susceptibility of R11 cells, which do not produce TNF-a, to IFN-a (table 5). Pentoxifylline had no effect on the cytotoxic activity of IFN-'Y against R4 cells (data not shown).
The effect of PTX on the sensitivity of R4 and Rl 1 cells to CDDP. A possible mechanism of TNF-a resistance in tumor cells is endogenous TNF -a expression, the endogenous TNF -a exerting its protective function against exogenous TNF -a by inducing manganous superoxide dismutase (MnSOD) production.20-23, 29 • 30 Tumor necrosis factor-a and CDDP exert their cytotoxic activity in part through free radical production. 16· 31 We examined whether PTX could overcome the resistance of R4 cells to CDDP as well as to TNF-a, by downregulating endogenous TNF -a expression. Pentoxifylline enhanced the sensitivity of TNF-a producing R4 cells to CDDP in a one-day MTT assay (table 6). However, treatment of the non-TNF-aproducing R11 cells with PTX at the concentrations used in this study did not augment their sensitivity to CDDP (table 7). The effect of anti-TNF-a MAb on the sensitivity of R4 cells to CDDP. We explored the possibility that anti-TNF-a MAb could enhance the susceptibility of R4 cells to CDDP. AntiTNF-a MAb enhanced the sensitivity of R4 cells to CDDP (table 8). The effect of PTX or anti-TNF-a MAb on the expression of MDR-1 mRNA in R4 cells. One of the possible mechanisms of drug resistance is overexpression of the MDR-1 gene. 32 • 33 R4 cells did not constitutively express MDR-1 mRNA. Neither PTX nor anti-TNF-a MAb had an effect on the expression of mRNA for MDR-1 in R4 cells (fig. 2). DISCUSSION
The present study demonstrated that PTX enhanced the sensitivity of R4 and R11 RCC cells to TNF-a and reversed tumor cell resistance to TNF-a. Clinically, it is interesting that the PTX-mediated effect was optimal at a concentration of 100 µg./ml., the level observed in the blood of human cancer patients treated with PTX by local arterial injection, intravenous injection, or oral administration, without severe side effects. 34• 35 R4 cells were resistant to TNF -a and expressed TNF -a mRNA and protein constitutively. Exposing R4 cells to PTX markedly reduced the expression of TNF -a mRN A and protein. In contrast, treatment with TNF -a alone enhanced the level of TNF -a mRN A and protein. A combination of PTX and TNFa downregulated the level of TNF-a mRNA and protein enhanced by TNF -a alone. It has been reported that there is a correlation between the expression of TNF-a mRNA and/or protein in tumor cells and their resistance to TNF -a. 20-23 Thus, one possible mechanism of the enhanced cytotoxic effect of PTX and TNF -a used in combination on R4 cells may be PTXinduced downregulation of TNF-a mRNA and protein. Conceivably, treatment of R4 cells with TNF-a enhances the expression of TNF-a mRNA and protein, and these cells may become even more resistant to TNF -a, but the addition of PTX may diminish TNF-a-induced resistance by downregulating TNF-a mRNA and protein. R11 cells were also resistant to TNF -a, but did not express TNF-a mRNA constitutively. However, treating R11 cells with TNF-a induced TNF-a mRNA. Treatment with PTX alone did not induce TNF-a mRNA. Incubating R11 cells with a combination of PTX and TNF-a markedly downregulated TNF-a-induced expression of TNF-a mRNA. A proposed mechanism of TNF-a resistance in tumor cells is the endogenous production of TNF-a mRNA and/or protein. 20-23 The PTX-induced downregulation of TNF-a-induced TNF-a mRNA in R11 cells may play a role in the enhanced cytotoxicity observed with the combination of PTX and TNF-a. Conceivably, like R4 cells, treatment of R11 cells with TNF-a induces mRNA for TNF-a, making R11 cells even more resistant to TNF-a. The addition of PTX may diminish the TNF-a-induced resistance of R11 cells by downregulating their TNF -a mRNA. It has been reported that the presence of mRNA for TNF-a does not correlate with translation and secretion of TNF-a protein. 36· 37 Treatment of Rl 1 cells with TNF -a induced TNF -
TNF-a IllRNA DOWNREGULATION IN TUMOR CELL SENSITIZATION a mRNA expression, but TNF-a-treated Rll cells did not secrete detectable levels of TNF-a protein. Accordingly, the resistance of Rll cells to TNF-a may be associated with TNFa-induced TNF-a mRNA expression in the absence of TNF-a protein secretion. Although downregulation of TNF-a mRNA by PTX is suggestive of enhanced sensitivity, the precise mechanism of overcoming resistance of R4 and Rll cells to TNF-a by a combination of PTX and TNF-a is not fully understood. One possibility is that the antitumor effect of TNF-a has been partly linked to DNA damage 16• 17 and PTX interferes with the repair of DNA damage by prematurely inducing mitosis. 38• 39 This may result in an enhanced cytotoxic effect of PTX and TNF-a. Pentoxifylline and the human-derived cytokine IFN-a acted synergistically in their cytotoxic activity against TNF-a-producing R4 cells, but not against non-TNF-a-producing Rll cells, although the synergy was modest. The possible mechanism responsible for the enhanced cytotoxicity seen with the combination of PTX and IFN-a may be associated with the downregulation of TNF ~a mRNA and protein by PTX. Interferon-a is relatively effective against RCC, not only through its direct cytotoxic activity, but also through its ability to augment the cytotoxic activity of natural killer cells and other lymphocytes. 40 • 41 Although the mechanism responsible for the enhanced cytotoxicity of this combination is not yet fully understood, the findings are nevertheless of clinical relevance. Several possible mechanisms of CDDP resistance of tumor cells have been reported such as alterations in the transmembrane transport of CDDP, cytosolic quenching of CDDP due to increased levels of sulfhydryl compounds, decreased CDDPbinding to DNA and enhanced DNA adduct repair capability. Alterations in the transmembrane transport of CDDP in tumor cells result in reduced intracellular accumulation of CDDP and resistance to CDDP, since there is an inverse correlation between intracellular accumulation of CDDP and resistance to it. 42- 44 Evidence for the cytosolic quenching of CDDP, either by glutathione or by sulfhydryl containing proteins, has been obtained in tumor cell lines that are resistant to CDDP in vitro. Some resistant cells have higher levels of intracellular glutathione or metallothionein. 45 • 46 Cis-diamminedichloroplatinum II inhibits DNA synthesis. This process is mediated by binding to DNA47 and the formation of intrastrand or interstrand crosslinks in DNA. 48 Cell-mediated augmentation of the DNA repair capability plays a major role in CDDP resistance in several mammalian cell lines studied. 49 Endogenous TNF-a exerts its protective function against exogenous TNF-a by inducing MnSOD expression. 29 • 30 Tumor necrosis factor-a and CDDP exert their cytotoxic activity in part through free radical generation. 16• 31 We examined the possibility that PTX could overcome the resistance of R4 cells to CDDP, as well as to TNF-a, by downregulating endogenous TNF-a expression. Combination treatment with PTX and CDDP resulted in enhanced cytotoxicity against TNF-a-producing R4 cells, but not against Rll cells, which did not express TNF-a mRNA and protein. Anti-TNF-a MAb enhanced the sensitivity of R4 cells to CDDP. These results suggest that one of the mechanisms responsible for the enhanced cytotoxicity seen with the combination of PTX and CDDP may be associated with the downregulation of TNF -a protein secretion by PTX. Although downregulation of TNF -a secretion by PTX is a potential mechanism for reversal of CDDP-resistance, the precise mechanism for overcoming resistance of R4 cells to CDDP by a combination of PTX and CDDP is not yet fully understood. The MDR-1 gene has been proposed as a possible mechanism of tumor cell resistance to anticancer chemotherapeutic drugs. 32• 33 We considered the possibility that PTX and antiTNF-a MAb might reduce the level of MDR expression in R4 cells, so sensitizing the tumor cells to CDDP, but this possibility
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was excluded since PTX and anti-TNF-a MAb had no effect on MDR-1 mRNA expression in R4 cells. Cis-diamminedichloroplatinum II mediates a significant inhibitory effect on DNA synthesis. Pentoxifylline prevents delays in the cell cycle transit through the G2 phase and does not allow time for the repair of DNA cross-links. 38• 39 Accordingly, CDDP-induced DNA-damaged R4 cells treated with PTX may proceed through the cell cycle more quickly (no arrest in the G2 phase) and may divide before DNA repair can be completed. Thus, the combination of PTX and CDDP results in enhanced cytotoxicity against R4 cells. Further studies are needed to support this hypothesis. Interferon-a and TNF-a are relatively effective against RCC, but the response rate is low. 40 • 41 Although the most effective treatment for RCC remains surgery, efficacy has been only demonstrated for regional resectable disease. Thus, a more effective therapy is necessary for these patients. This study has demonstrated that PTX, in combination with TNF-a, IFN-a, or CDDP, can overcome the drug-resistance of RCC cells and that the downregulation of TNF-a mRNA and/or protein may play a role in this enhanced cytotoxicity. These data suggest that therapy with PTX in combination with TNF-a, IFN-a, or CDDP may be beneficial in overcoming the resistance of RCC to conventional forms of therapy. Acknowledgments. We thank Dr. Linda Sakimura for her help in preparation of this manuscript. REFERENCES
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