Tumor Necrosis Factor and

Chemotherapeutic Agents

Potentiation of Cytotoxicity With Interferon Gamma Douglas E. Dawson, MD; Markus Gapany, MD; Robert C. Burgess, MD; Peter This study examined combinations of the recombinant human cytokines, tumor necrosis factor \g=a\and interferon gamma, with doxorubicin and dactinomycin as well as other drugs on six squamous cell carcinoma cell lines of head and neck origin using the 3(4,5-dimethylthiazol-2-yl)2,5\x=req-\ diphenyl-tetrazolium bromide proliferation assay. Interferon gamma significantly enhanced the cytotoxicity of tumor necrosis factor a with dactinomycin on all six cell lines investigated, while in four of six cell lines the cytotoxicity of tumor necrosis factor \g=a\with doxorubicin was significantly augmented by interferon gamma. Additional experiments showed no effect with either cytokine in combination with cisplatin, fluorouracil, methotrexate, or etoposide. These data demonstrate that human recombinant cytokines in concert with certain drugs improve in vitro cytotoxicity and may have a potential for improving in vivo therapy. (Arch Otolaryngol Head Neck Surg. 1992;118:1168-1171) \s=b\

growing information the literature suggesting Therethe efficacy of recombinant cytokines cytotoxic and human cell is

carcinoma squamous evaluated cytokines have been tumor necrosis factor (TNF- ) and interferon gamma (IFN-7). Zenner and Zimmerman1 found inconsis¬ tent effects of TNF- and IFN-7 on cultured SCC. Other studies failed to show any significant inhibition of SCC cell lines by TNF- alone after continuous incubation for as long as 72 hours.3,4 Simultaneous incubation of both TNFand IFN-7, however, was shown to result in significant synergistic cytotoxic effect on a variety of tumor cells, in¬ cluding SCC of head and neck origin.1·5"8 One explanation for the enhanced cytotoxicity of TNF- is the increased ex¬ pression of TNF- receptors by IFN-7 exposure.910 A dose-dependent cytotoxic synergism of TNF- with dacti¬ nomycin and doxorubicin previously was shown on cer¬ tain human head and neck SCC cell lines in vitro.4 This study evaluated the effect of IFN-7 in concert with TNFand selected chemotherapeutic agents on human head and neck SCC cell lines.

cytostatic agents against (SCC).14 The most frequently

MATERIALS AND METHODS

Reagents Recombinant human TNF- was obtained through the courtesy of Knoll Pharmaceuticals, Whippany, NJ. Recombinant human IFN--y was given by Charles Riggs, MD, University of Iowa, Iowa

Accepted for publication July 10, 1992. From the Otologic Medical Services of Iowa City (Iowa) (Dr Dawson), and the Department of Otolaryngology Head and Neck Surgery (Drs Gapany, Burgess, Boesen, and Headley), University of Iowa Hospitals and Clinics, Iowa City. Reprint requests to 540 E Jefferson St, Suite 401, Iowa City, IA 52245 (Dr Dawson).

Boesen, MD; Don B. Headley, MD

City. The chemotherapeutic agents were obtained through the Department of Pharmacy, University of Iowa Hospitals, via the following suppliers: dactinomycin (Actinomycin-D), Merck, Sharp & Dohme, West Point, Pa; doxorubicin (Adriamycin), Farmitalia Carlo Erba, Spa, Milan, Italy; methotrexate, Lederle Parenterals Ine, Carolina, Puerto Rico; fluorouracil (5-FU), Solopak Laboratories, Franklin Park, NY; and cisplatin (Cis-

diaminedichloroplatinum), Bristol-Meyers Oncology Products, Syracuse, NY. Cell Line Human SCC cell lines derived from the hypopharynx (FaDu), larynx (HEp-2), and oral cavity (KB-17) were obtained from the American Type Culture Collection, Rockville, Md. Cell lines from the supraglottic larynx (UM-SCC-5) and oral cavity (UM-SCC-8 and UM-SCC-25) were obtained from Thomas Carey, PhD, Uni¬ versity of Michigan, Ann Arbor. Cells were maintained in 25- or 75-cm2 tissue culture flasks (Falcon Plastics, Cockeysville, Md) with RPMI 1640 medium containing 15% fetal calf serum, antibi¬ otics, and supplements (University of Iowa Cancer Center) in a 5% carbon dioxide, 97% humidity, and 37°C environment.

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Cytotoxicity Assay Squamous cells in the exponential phase of growth were har¬ vested from culture flasks using 0.1% trypsin and 0.4% edetic acid (University of Iowa Cancer Center). The cells were washed in medium and counted via hemocytometer, and their viability was determined by 0.5% trypan blue vital dye exclusion. Only harvested aliquots showing viability of greater than 90% were

used in these studies. The cells were then transferred to 96-well microtiter plates (Falcon Plastics) at 100000 cells per well in 0.1 mL of medium. Preliminary studies demonstrated that this num¬ ber of cells was not confluent at the end of the assay and provided an

optimal 3(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium

bromide (MTT) (Sigma Chemical Co, St Louis, Mo) absorbance value. The cells were allowed to adhere by incubating for 24 hours in 5% C02 at 37°C. A 2-hour pulse of IFN- was then given to the cells by replacing the medium with that containing fresh IFN-7 at 100 U/mL and incubating at 37°C. The medium was replaced at 2 hours and various concentrations of cytotoxic agents, with or without TNF- , were added; final volume was 0.2 mL. The plates were incubated for 24 hours at 37°C in 5% C02, and cell viability in each well was estimated via the colorimetrie MTT assay according to the method of Mosmann.11 The yellow tetrazolium salt MTT is converted into a purple formazan product by living cells in direct proportion to cell number and metabolic activity.11 Briefly, 5 µ of a 10-mg/mL solution of MTT in phosphatebuffered saline (0.01 mol/L phosphate, 0.85% sodium chloride, pH 7.4) was added to each well and incubated for 4 hours at 37°C. The content of each well was then solubilized by addition of 0.175 mL of acid isopropanol (0.4N hydrochloric acid in 2-propanol) at room temperature, and absorbance of each well was measured at 570 ran by a multiple scanning spectrophotometer (Bio-Tek Instruments, Winooski, Vt). The percent viability of each well was determined from the absorbance readings at 570 nm and calcu¬ lated from the following: Percent viability=(drug±cytokine/ control cells alone) X100, where drug±cytokine indicates mean absorbance as determined by the MTT assay with chemo¬ therapeutic agent alone, with cytokine alone, or with the combi-

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nation of cytokine plus chemotherapeutic agent; and control cells alone, mean absorbance as determined by the MTT assay without chemotherapeutic agents or cytokines.

The data are means from eight replicate wells and are con¬ firmed by replicate experiments. Microscopic inspection of the wells revealed that decreased absorbance values from control wells correlated with decreased cell number and not diminished metabolic rate. Previous studies have shown that the MTT assay correlates with tritiated thymidine assays of proliferation, viabil¬ ity as determined by propidium iodide staining utilizing flow cytometry, and chromium 51 assays of cytotoxicity; as well, it has been easily adapted to SCC cell lines.4·12

RESULTS Preliminary experiments examined the various ways to expose IFN-7 to the cultures before incubation of the cell lines with TNF- and/or chemotherapeutic agents. Preincubation with IFN-7 or TNF- for as long as 24 hours did not alter cell viability or microscopic appearance in the six cell lines initially screened for their susceptibility to IFN-7 and TNF- (data not shown). Continuous incubation with IFN-7 for up to 24 hours at concentrations ranging from 6.25 to 400 U/mL failed to show any significant cytotoxic effect in any of the tested SCC cell lines. Table 1 displays cell viabilities from 91.6% to 99.9% of controls following 24-hour incubation with 100 U/mL of IFN-7. Continuous 24-hour incubation with TNF- at concentrations ranging from 3.125 to 200 ng/mL showed no effect on the cell lines' viability as determined by the MTT assay (Table 1). The cell lines were then pre¬ incubated or pulsed with IFN-7 for 2, 4, 8, and 12 hours, respectively, before 24-hour incubation with TNF- . Little cytotoxic effect was observed with any of the tested SCC cell lines when the cytokines alone were exposed simulta¬ neously or with preincubation pulsing of the IFN-7. Two cell lines showed minimal cell death when incubated with both TNF- and IFN-7, but not at clinically significant lev¬ els (Table 1). All subsequent experiments were performed utilizing a 2-hour pulse of IFN-7 before exposure to TNFand/or the chemotherapeutic agent. Table 1 gives the cell viabilities of SCC cell lines following a 2-hour pulse with IFN-7 (concentration, 100 U/mL) followed by 24-hour in¬ cubation with TNF- (concentration, 75 ng/mL); viability ranged from 87.0% to 101.4% of controls. The cell viabilities of SCC cell lines following incubation with dactinomycin or doxorubicin are given in Table 1. Drug concentrations of 0.25 mg/L for dactinomycin and 2.5 mg/L for doxorubicin were used throughout these ex¬ periments because these levels are physiologically attain¬ able and achieved the desired cytotoxicity. These drug concentrations were selected to allow cell viabilities (43.3% to 71 %) of the SCC cell lines that would demonstrate the possible effects of the investigated cytokines. Despite the initial data that were used to set drug dosages, one cell line, KB-17, proved to be especially sensitive to dactinomycin (cell viability of 13.8% with dactinomycin plus TNF- ) and doxorubicin (cell viability of 25% with doxorubicin alone) in later experiments (Table 1). The full efficacy of combi¬ such low nation cytokine therapy is potentially masked cell viability. As this laboratory has previously shown, continuous 24-hour incubation of dactinomycin with TNF- (concen¬ tration, 75 ng/mL) showed a significant enhancement of cytotoxicity on five of six cell lines investigated (FADu, HEp-2, KB-17, UM-SCC-8, UM-SCC-25) (Table l).4 The cy¬ totoxic effect of SCC cell lines exposed to dactinomycin and

by

over the cytotoxic ef¬ alone (Table 2). One cell line, UM-SCC-5, however, failed to show any significantly in¬ creased cell death with dactinomycin and TNF- as com¬ pared with drug alone. When cell lines were preincubated with IFN-7 for 2 hours and then treated with dactinomy¬ cin for 24 hours (IFN-7 was not removed from the wells after initial 2-hour pulse; see "Materials and Methods" section), significant enhancement of the drug cytotoxicity was observed on five of six cell lines studied (FaDu, KB17, UM-SCC-5, UM-SCC-8, UM-SCC-25) (Table 1). The cy¬ totoxic effects observed with these cell lines increased from 8.7% to 37.3% with dactinomycin and IFN-7 as compared with drug treatment alone (Table 2). Pretreatment with IFN-7 for 2 hours, followed by simultaneous 24-hour incubation with IFN-7, TNF- , and dactinomycin, showed significant augmentation of cell death in all the investi¬ gated cell lines as compared with exposure of the drug with TNF- alone (Table 1). The mean percent augmenta¬ tion of cytotoxic effect increased 16.0% to 73.2% with dactinomycin, TNF- , and IFN-7, as compared with drug alone (Table 2). The statistical analysis of mean cytotoxicities of the drug and TNF- compared with the drug, TNF- , and IFN-7 showed the effect of IFN-7 to be highly significant on all cell lines (P