Suppression of intracellular resistance factors by adriamycin augments heat-induced apoptosis via interleukin-1β-converting enzyme activation in pancreatic carcinoma cells

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<ul><li><p>SUPPRESSION OF INTRACELLULAR RESISTANCE FACTORS BY ADRIAMYCINAUGMENTS HEAT-INDUCED APOPTOSIS VIA INTERLEUKIN-1b-CONVERTINGENZYME ACTIVATION IN PANCREATIC CARCINOMA CELLSDaisuke KOBAYASHI1, Naoki WATANABE1*, Hiroyoshi SASAKI2, Tetsuro OKAMOTO2, Naoki TSUJI2, Tsutomu SATO2,Naofumi YAMAUCHI2 and Yoshiro NIITSU21Department of Laboratory Diagnosis, Sapporo Medical University, School of Medicine, Sapporo, Japan2Fourth Department of Internal Medicine, Sapporo Medical University, School of Medicine, Sapporo, Japan</p><p>Combination of heat and various anticancer drugs canexert a synergistic antitumor effect in vitro and in vivo, thoughthe mechanism is not clear. We have previously shown thatendogenous tumor necrosis factor (enTNF) acts as an intra-cellular resistance factor to inhibit the cytotoxic effect of heatby scavenging oxygen-free radicals via the induction of manga-nous superoxide dismutase (MnSOD). Consequently, we ex-amined whether the suppression of these resistance factorsby combining anticancer drugs and heat causes an augmenta-tion of heat-induced cytotoxicity. The human pancreaticcarcinoma cell line, PANC-1, constitutively expresses appre-ciable amounts of enTNF and also demonstrates heat resis-tance. After treatment of these cells for 15 hr with adriamy-cin (ADM), the expression of enTNF was decreased by 43%,and MnSOD activity was suppressed by 55%. The cytotoxiceffects of the treatment of PANC-1 cells with ADM followedby heat were greater than the sum of those observed with theagents administrated individually. Heat-induced apoptosiswas also augmented by pretreatment with ADM. Further-more, the interleukin-1b-converting enzyme inhibitor, Ac-YVAD-CMK, reversed the augmented cytotoxicity. Our re-sults indicate that suppression of intracellular resistancefactors such as enTNF and MnSOD plays an important role inapoptosis seen after heat and ADM combined therapy. Int. J.Cancer 76:552555, 1998.r 1998 Wiley-Liss, Inc.</p><p>Hyperthemia is known to posseses a potent antitumor activity(Maeda et al., 1991). However, its antitumor effect is somewhatlimited (Roizin and Pizzo, 1988; Mivechi and Ross, 1990). Anumber of studies have attempted to augment the antitumor effectsby combining hyperthermia with anticancer drugs such as cisplatinand adriamycin (ADM) in vivo (Kakehi et al., 1990; Kusumoto etal., 1995) and in vitro (Herman et al., 1982). However, little isknown about the mechanism of the augmentation of heat-inducedcytotoxicity by anticancer drugs. Elucidation of the factors regulat-ing heat-induced cytotoxicity may improve the efficacy of hyper-thermia in anticancer therapy.</p><p>We have reported previously that intracellular oxygen freeradical (OFR) production is involved in the mechanism of heat-induced cytotoxicity (Yamauchi et al., 1992). We also observedthat enTNF exerts an intracellular protective effect against not onlyexogenous TNF (Okamoto et al., 1992), but also heat (Tsuji et al.,1992; Watanabe et al., 1997) by scavenging OFR via the inductionof manganous superoxide dismutase (MnSOD) (Himeno et al.,1992).</p><p>In the present study, we first examined whether ADM, which hasbeen widely used to treat solid tumors, downregulates intracellularresistance factors such as enTNF and MnSOD in heat-resistantPANC-1 (human pancreatic carcinoma) cells (Watanabe et al.,1997). We then went on to define the optimal sequence of heattreatment in combination with anticancer drugs. It has beenreported that apoptosis plays a major role in hyperthermic cytotox-icity (Yonezawa et al., 1996). We demonstrate here that sequentialtreatment with heat and ADM augments heat-induced apoptosis. Inaddition, the effect of the combination of heat and ADM on thefactors regulating the signal transduction of apoptosis such asinterleukin-1b-converting enzyme (ICE)-like protease, p53, andp21/WAF-1 were also examined.</p><p>MATERIAL AND METHODSCell culture</p><p>PANC-1 and MIAPaCa-2 (human pancreatic carcinoma) cellswere purchased from the ATCC (Rockville, MD) and cultured inDulbeccos modified Eagles minimal essential medium (DMEM;GIBCO, Grand Island, NY) supplemented with 10% fetal bovineserum (FBS; Flow, North Ryde, Australia) at 37C in a humidified5% CO2 incubator.</p><p>In vitro assay for cytotoxic effectsCytotoxic activity was assessed by a dye-uptake method as</p><p>described previously (Tsuji et al., 1992).Assay for enTNF expression</p><p>The expression of enTNF was measured by a indirect methodusing a fluorescein isothiocyanate (FITC)-labeled second antibody,according to a modification of the method described earlier(Pitzurra et al., 1990). Briefly, cells (1 3 104/100 l) were added tothe wells of a 96-well microculture plate (Costar, Cambridge, MA)and incubated at 37C in 5% CO2 for 18 hr. Cells were treated byADM (0.5 M) and washed twice with PBS and fixed withparaformaldehyde solution (4% v/v in PBS) for 20 min at roomtemperature. Next, cells were treated with Triton X-100 (0.2% v/vin PBS) to permeabilize the cell membrane. For blocking, 1% goatserum in PBS was added to each tube for 30 min at 37C. As a firstantibody, appropriately diluted rabbit anti-recombinant humanTNF polyclonal antibody (Asahi, Tokyo, Japan) was added to eachtube. Following 1 hr incubation at room temperature, the cells werewashed with 1% BSA (Seikagaku, Tokyo, Japan)-PBS 3 times, andthen treated with appropriately diluted FITC-labeled goat anti-rabbit IgG (E.Y. Laboratories, San Mateo, CA) as a secondantibody. Following an additional 30 min incubation at roomtemperature, cells were washed 3 times with 1% BSA-PBS.Intensity of cell fluorescence was measured in a Titertek Fluoros-kan II (Skatron, Lier, Norway; excitation 485 nm, emission 538nm). The relative fluorescence intensity (F. I.) was determinedcompared to the fluorescence intensity of non-treated PANC-1cells.</p><p>Determination of MnSOD activityMnSOD activity was assayed by the nitroblue tetrazolium</p><p>method (Oberley and Spitz, 1985). Protein concentrations weredetermined by the Bio-Rad (Hercules, CA) DC protein assay.MnSOD activity was calculated as units per mg of protein, andexpressed relative MnSOD activity was determined compared tothe activity of non-treated cells.</p><p>Grant sponsor: Ministry of Education, Science, and Culture of Japan.</p><p>*Correspondence to: Department of Laboratory Diagnosis, SapporoMedical University, School of Medicine, South-1, West-16, Chuo-ku,Sapporo 060, Japan. Fax: (81)11-622-7502. E-mail:</p><p>Received 6 October 1997; Revised 19 December 1997</p><p>Int. J. Cancer: 76, 552555 (1998)r 1998 Wiley-Liss, Inc.</p><p>Publication of the International Union Against CancerPublication de lUnion Internationale Contre le Cancer</p></li><li><p>Agarose gel electrophoresis of DNALow m.w. DNA was extracted and agarose gel electrophoresis</p><p>was performed. Briefly, 1 3 106 cells were added to each well of a100 mm culture dish (Costar) and heated or treated with ADM.Following test incubations, cells were washed twice with PBS andlysed with 100 l of lysis buffer (10mM Tris-HCl (pH7.4), 10 mMEDTA, 0.5% Triton X-100). The lysate was centrifuged at 12,000 gfor 20 min, and the supernatant was collected in an Eppendorf tube.Next, the supernatant was incubated with 2 l of proteinase K(Sigma, St. Louis, MO) at 37C for 1 hr, and incubation proceededfurther at 37C for 1 hr after 2 l of RNase A (20 mg/ml) had beenadded. After overnight precipitation with 0.5 M NaCl and 50%isopropanol at 220C, the pellets were centrifuged at 16,000 g for15 min at 4C and resuspended in 40 l H2O. Each DNA sample(10 g) was electrophoresed at 50 V for 1 hr on a 2% agarose gelimpregnated with ethidium bromide, and DNA fragments werevisualized under UV light.</p><p>RESULTSHeat sensitivity of PANC-1 and MIAPaCa-2 cells</p><p>The heat sensitivity of 2 lines derived from human pancreaticcarcinoma cells was assessed by incubating these cells at 43C for1, 2, and 3 hr, and then at 37C for a total incubation time of 48 hr.PANC-1 cells constitutively expressing appreciable amounts ofenTNF (Watanabe et al., 1996) were more resistant to heattreatment than MIAPaCa-2 cells (data not shown).Effect of ADM treatment on enTNF expression and MnSODactivity in PANC-1 cells</p><p>We have reported that enTNF acts as an intracellular resistancefactor to heat treatment by inducing MnSOD (Tsuji et al., 1992;Watanabe et al., 1997). Therefore, we examined the kinetics ofenTNF expression by an indirect antibody method to examinewhether ADM (0.5 M) inhibits enTNF synthesis. Compared withuntreated PANC-1 cells, fluorescence intensity was decreased byapproximately 43% after 15 hr of ADM treatment (Fig. 1). MnSODactivity was diminished by 55% after 15 hr of ADM treatment(Fig. 2).Augmented cytotoxicity of PANC-1 cells by the combinationof heat with ADM</p><p>We then examined the effect of sequential treatment with ADMand heat. An ADM incubation period shown to inhibit enTNFexpression and MnSOD activity was used. Cells were sequentiallyincubated at 43C for 1 hr and then treated with ADM for 15 hr orreceived no further treatment. The cytotoxic effects of the treatmentwith ADM followed by heat were greater than the sum of thoseobserved with the agents administrated individually (Fig. 3).Induction of apoptosis by sequential treatment with heatand ADM in PANC-1 cells</p><p>DNA ladder fragmentation was examined in PANC-1 cellstreated sequentially with ADM and heat, since apoptosis is knownto play a critical role in heat-induced cytotoxicity (Yonezawa et al.,1996). There was significantly increased DNA fragmentation inPANC-1 cells pretreated with heat followed by ADM compared tothe sum of those observed with the agents administrated individu-ally (Fig. 4). These observations indicate that the augmentedcytotoxic effect of heat and ADM represents an augmentation ofheat-induced apoptosis via inhibition of intracellular resistancefactors.</p><p>Effect of ICE inhibition on augmented cytotoxicityin PANC-1 cells</p><p>We examined the effect of a specific tetrapeptide interleukin-1b-converting enzyme (ICE) inhibitor, Ac-YVAD-CMK (Bachem,Torrance, CA), on the sequential treatment of PANC-1 cells withADM and heat. Administration of Ac-YVAD-CMK 5 hr before the</p><p>sequential treatment inhibited cytotoxicity in a dose-dependentmanner. Inhibition below the level of cytotoxicity of heat wasobserved, particularly with 100 M which has no inhibitorcytotoxic effects by itself (Fig. 5).</p><p>DISCUSSION</p><p>We have previously noted that OFR induction is an importantevent in heat-induced cytotoxicity (Yamauchi et al., 1992). More-over, enTNF (Okamoto et al., 1992) acts as a resistance factor tohyperthermic cytotoxicity by inducing the OFR scavenging activityof MnSOD (Tsuji et al., 1992; Watanabe et al., 1997). In the presentstudy, we hypothesized that ADM could augment the cytotoxiceffect of hyperthermia in pancreatic carcinoma cells by theinhibition of intracellular resistance factors.</p><p>We have shown that enTNF expression and MnSOD activitywere inhibited by a 15 hr ADM treatment of the heat-resistantPANC-1 cells. It has been reported that ADM downregulates theexpression of TNF mRNA, resulting in augmentated TNF cytotox-icity (Safrit and Bonavida, 1992). These investigators also foundthat the MnSOD mRNA levels in TNF resistant R4 human rectalcarcinoma cells were not decreased by ADM treatment. Theyconcluded that MnSOD plays no role in the reversal of TNFresistance by ADM. These findings differ from our observation thatintracellular MnSOD activity of PANC-1 cells was decreased by55% by a 15 hr ADM treatment. The discrepancy may be explainedby the duration of the periods of examination, since Safrit andBonavida (1992) examined the expression of MnSOD mRNA afteronly 1 hr of ADM treatment.</p><p>We next examined whether the inhibition of intracellular resis-tance factors by ADM treatment leads to an increased heat-inducedcytotoxicity. The cytotoxic effect by the sequential treatment with</p><p>FIGURE 1 Effect of ADM treatment on enTNF expression inPANC-1 cells. Cells were treated with ADM (0.5 M) for 0, 5, 10, 15,and 20 hr, and enTNF expression was measured by an indirect antibodymethod as described in Material and Methods. EnTNF expression isrepresented relative to untreated PANC-1 cells. The data represent themean 6 SD of 3 independent experiments. *p , 0.01, **p , 0.005 byStudents t-test.</p><p>553ADM AUGMENTS HEAT INDUCED APOTOSIS</p></li><li><p>ADM for 15 hr followed by heat for 1 hr were greater than the sumof those observed with the agents administrated individually.</p><p>It has been hypothesized that heat resistance induced by enTNFis due exclusively to the induction of MnSOD synthesis. However,we have previously seen a remarkable increase in heat shock</p><p>protein (HSP) (Watanabe et al., 1994), a well known resistancefactor against several cellular stresses including heat. An understand-ing of the mechanism of augmentation of heat cytotoxicity byADM will ultimately require the examination of HSP 72 levelswith ADM treatment.</p><p>Apoptosis plays an important role in heat-induced cytotoxicity inhuman cells (Yonezawa et al., 1996). We have demonstrated herean augmented DNA fragmentation only when ADM-mediatedinhibition of intracellular resistance factors was followed by heat</p><p>FIGURE 2 Effect of ADM treatment on intracellular MnSODactivity. Cells were treated with ADM (0.5 M) for 0, 5, 10, 15, and 20hr and MnSOD activity was assessed by a nitroblue tetrazolium assay.MnSOD activity is expressed as the ratio of treated to untreated cells.The data represent the mean 6 SD of 3 independent experiments. *p ,0.005 by Students t-test.</p><p>FIGURE 3 Sequential treatment of PANC-1 cells with heat andADM. Heat (43C) at 1 hr or ADM (0.5 M) at 15 hr were used forpretreatment. This was followed by washing and incubation plus theother agent. Cytotoxicity was assessed by a dye-uptake assay. The datarepresent the mean 6 SD of 3 independent experiments. A, H, and Mrepresent treatment with ADM, heat, and medium, respectively.</p><p>FIGURE 4 Induction of DNA fragmentation by sequential treatmentwith heat and ADM in PANC-1 cells. Cells were treated with heat(43C) at 1 hr or ADM (0.5 M) at 15 hr. This was followed bywashing, and incubation plus the other agent. Oligonucleosomalfragments were demonstrated by 2% agarose gel electrophoresis asdescribed in Material and Methods. A, H, and M represent ADM, heat,and medium, respectively.</p><p>FIGURE 5 Effect of a tetrapeptide ICE-inhibitor (Ac-YVAD-CMK)on heat and ADM treatment of PANC-1 cells. Various concentrations ofAc-YVAD-CMK were added 5 hr before sequential treatment withADM (0.5 M) and heat (43C). Cytotoxicity was assessed by thedye-uptake assay. The data represent the mean 6 SD of 3 independentexperiments. *p , 0.025, **p , 0.01, ***p , 0.005 by Studentst-test.</p><p>554 KOBAYASHI ET AL.</p></li><li><p>treatment. The augmentation of heat cytotoxicity by ADM is thusreflected by enhanced apoptotic cell death. There is only one singlestudy that has examined factors regulating apoptosis in heat-treatedcells (Ohnishi et al., 1996). These investigators observed thatp53-dependent p21/WAF-1 accumulation was induced by heat inglioblastoma cell lines carrying the wild ty...</p></li></ul>