General Pharmacology 33 (1999) 179–186

In vitro and in vivo antitumor activity of YoshixolTR against murineL1210 leukemic cells

Satoshi Tanakaa, Shozo Koyamaa,*, Hisao Haniua, Yoshihiro Yamaguchia,1,Jiro Motoyoshiyab

aShinshu University School of Medicine, Department of Physiology, Division 2, Asahi, Matsumoto, Nagano 390, JapanbDepartment of Chemistry, Shinshu University Faculty of Textile Science and Technology, Ueda, Nagano 386, Japan

Received 13 August 1998; accepted 30 October 1998

Abstract

In this report, antiproliferative effects of YoshixolTR in vitro and in vivo were investigated in murine L1210 cells. A proliferationof L1210 cells in vitro was inhibited by YoshixolTR in a dose- and time-dependent manner. This inhibition showed an arrest atthe G0/G1 stage of the cell cycle, followed by a flow cytometric measurement. YoshixolTR induced apoptosis-like cell death identi-fied by histological observations (scanning electron and transmission electron microscopy), DNA fragmentation, and a smallerincrease in lactate dehydrogenase (LDH). In the in vivo experiments, YoshixolTR (5 ml/kg of body weight, on days 1, 3, and 5)was injected intraperitoneally in mice inoculated with L1210 cells. No marked prolongation of survival occurred between the con-trol group and treated group. However, a survival curve in the treated group showed a shift toward a possible longer survivaltime. Additionally, on the basis of apoptosis-like cell death due to YoshixolTR as indicated above, a possibility of immunotherapyas a tumor vaccine has been examined. A vaccination of rabbit anti-serum, which consisted of components from the L1210 cellskilled by YoshixolTR, produced a dramatic improvement of viability in the leukemic mice. In conclusion, YoshixolTR has an anti-leukemic potency with a new biological mechanism and an inductive potency of super-antigens as immunotherapeutic agentsagainst malignant tumors. 1999 Elsevier Science Inc. All rights reserved.

Keywords: Leukemia; L1210 cell; YoshixolTR; Apoptosis; Antitumor agent; Quantum thermodynamic cell death; Vaccination

Acute leukemia, like other cancers, is a progressive unexpected accidents related to atomic energy supply.Therefore, an advancement of therapeutic challengesclonal disorder driven by mutation. Leukemic cells pro-

liferate primarily in the bone marrow and lymphoid tis- has required more scientific approaches, even in view ofmedical cost performances.sues, where they interrupt normal hematopoiesis and

immunity. Although the cause of most acute leukemia Murine L1210 leukemia, which is an acute lymphoidleukemia cell line (Law et al., 1949), has been intro-is not known, some factors have been reported. Regi-

mens for induction of remission and consolidation ther- duced into use for investigations for screenings of manyanticancer agents (De Graaf et al., 1993; Kasamatsu etapy and bone-marrow transplantation have improved

the outlook for patients with acute leukemia (Burnett al., 1995). For example, treatment of only 5-Fluoro-uracil (5-FU), which inhibits DNA synthesis, causes aand Eden, 1997). However, it has been shown that

5-year survival rates have not improved sufficiently for slight increase in survival of mice inoculated with L1210leukemia over 15 years of surveillance of the study cells (Kline et al., 1966a, 1966b). An increased life span(Hernandez et al., 1995). In addition, various industrial of mice with L1210 cells was achieved with cis-Diam-activities have been linked to a risk of incidence of leu- minedichloroplatinum (Cisplatin), of which an effect iskemic diseases, e.g., destruction of the ozone layer and the platination of DNA (Moran and Straus, 1981).

However, a beneficial antitumor effect of Cisplatin hasbeen limited by a severe nephrotoxicity in clinical use.

* Corresponding author. Tel.: 181-236-37-2598; fax: 181-263-36- An optimal combination therapy of more than two3644 drugs consistently showed greater antileukemic activityE-mail address: skoyama@gipac.shinshu-u.ac.jp (S. Koyama)

(Dionet and Verrelle, 1984; Kline et al., 1966a, 1966b).1 Present address: Kyorin University School of Medicine, Depart-ment of Emergency Medicine, Mitaka 181, Tokyo, Japan. Combination chemotherapy increases a tumoricidal ef-

0306-3623/99/$ – see front matter 1999 Elsevier Science Inc. All rights reserved.PII : S0306-3623(99)00006-3

180 S. Tanaka et al. / General Pharmacology 33 (1999) 179–186

fect and reduces the harmful side effects of each thera- plemented with 10% FBS (Dai-Nippon PharmaceuticalCo., Tokyo, Japan) at 378C in an incubator with 5% car-peutic agent (Frank et al., 1968).

From a different point of view, it has been reported bon dioxide. L1210 cells in exponential phase of growthwere harvested by centrifugation and counted by a he-that an increased antitumor immunity on the host has

an effect on L1210-bearing mice (Kataoka and Oh- mocytometer using trypan blue exclusion to check via-bility. In in vitro tests of viability, concentration of Yos-hashi, 1985b) and on the tumor-specific cellular and hu-

moral immunity induced by anti-idiotype antibodies hixolTR were in a range between 1 mM and 1 mM. Onlysingle-cell suspensions of more than 90% viability as(Raychaudhuri et al., 1987). Additionally, it has been

shown that immunogenic cells, obtained following drug determined by trypan blue exclusion were used in the invivo experiments.treatment of murine leukemias, could be used for gen-

erating successful immunochemotherapy regimens1.2. Morphological observations in in vitro experimentsagainst the parental tumor (Nicolin et al., 1981).

On a view of cell death, apoptosis as a natural cell Twenty-four hours after treatment with YoshixolTR,death has been distinguished from necrosis on histologi- each sample for scanning electron microscopy (SEM)cal investigation (Kerr et al., 1972). Since this advoca- observation was prepared. YoshixolTR at 10 mM wastion of a new medical definition, aspects of apoptosis used. L1210 leukemia cells were mounted on the sur-have been characterized biochemically as an occurrence face of an aluminum cup on a flat copper plate. After 5of DNA ladder or fragmentation. DNA is segmented be- min, the plate was dipped several times in phosphatetween nucleosomes. Correspondingly, it has been sug- buffer solution (pH 7.4) to remove unattached biologi-gested that a process of DNA ladder or fragmentation cal materials. The adhered materials on the plate weremight be initiated by extracellular signal molecules, such prefixed in cold 2% glutaraldehyde in phosphate bufferas hormones, cytokines, and viral infections (Wyllie et solution for 1 h and postfixed in 1% OsO4 solution inal., 1984; Majno and Joris, 1995). Sensitivity of cancer phosphate buffer solution for 1 h. The plate with samplecells to induction of apoptosis has been recognized to be was subsequently dehydrated with graded ethanol solu-one of important events during future chemotherapy. tions, with isoamyl acetate three times, and dried with

Our earlier work has demonstrated characteristic cy- carbon dioxide by the critical point method. The driedtotoxic effects of YoshixolTR (as a parent compound; 4, surfaces were mounted and sputter-coated with gold4-dimethyl-6-methylene-2-cyclohexen-1-one), which was palladium and carbon. The samples were sputter-identified as the new major chemical candidate of the coated with 50 A of gold palladium and observed at 15neutral wood oil from Chamaecyparis obtusa (Kiso- keV in a JSM-6000F scanning electron microscopeHinoki). YoshixolTR is not only of interest scientifically, (JEOL, Tokyo, Japan). For an observation of internalas in the existence of possibly quantum thermodynamic aspects by a transmission electron microscope, thesecell death (QTCD: apoptosis-like cell death) of HeLa L1210 cells were prepared 24 h after the treatment withcells, but also has a potent antibiotic and/or antitumor YoshixolTR (10 mM). The cells were prefixed with 2.5%agent (Koyama et al., 1997a, 1997b). The purpose of glutaraldehyde in 0.1 M phosphate buffer (PB) at a pHthis study was to reveal cellular events on cultured of 7.4 for 1 h and postfixed in 1% osnium tetroxide inL1210 leukemia cell lines after treatment with Yoshi- PBS for 1 h. Thereafter, L1210 leukemia cells were de-xolTR and antileukemic effects of YoshixolTR in vivo. We hydrated in ethanol and embedded in Epon 812. Their

sections were cut with a diamond knife and Reichert-demonstrated here that YoshixolTR suppresses L1210ultramicrotome (REICHERT-OVm3, Vienna, Aus-cell proliferation and induces apoptosis-like cell deathtria) and routinely stained with uranyl acetate and leadon L1210 cells in vitro and arrest in the G0/G1 phase ofcitrate, and then grids were air-dried and viewed underthe cell cycle. The slope of the mortality curve in intra-a JEOL-JEM1200EX electron microscope (JEOL,peritoneal inoculation of L1210 cells in vivo was shiftedTokyo, Japan) with an accelerating voltage of 80 kV.toward a prolonged survival in the group treated with

YoshixolTR intraperitoneally. Additionally, a vaccina-1.3. DNA gel electrophoresistion of rabbit anti-serum against killed L1210 cells by

YoshixolTR dramatically improved the survival slope on Cells grown in suspension to a density 3 3 105 cell/mlthe experimental leukemic mice. were incubated without or with YoshixolTR (0.1 mM).

After 3 h of incubation, cells were collected and centri-fuged (1,200 rpm for 5 min). For DNA extraction, cells

1. Materials and methods were lysed in Tris buffer (10 mM Tris, 10 mM EDTAcontaining 0.5% Triton X-100) for 20 min at 48C. Lysis1.1. Cell lines and culturecells were treated with RNase A (Boehringer-Mann-heim, Mannheim, Germany) for 1 h at 378C, with pro-Tumor cells of murine L1210 leukemia were obtained

from Cancer Research Institute (Tokyo, Japan) and teinase K (Boehringer-Mannheim) for 1 h at 378C. DNAwas analyzed by agarose gel electrophoresis. DNA waswere cultured in MEM (Gibco, Gaithersburg, MD) sup-

S. Tanaka et al. / General Pharmacology 33 (1999) 179–186 181

visualized by staining with ethidium bromide. DNA phosphate, 0.9 mM cholesterol of lipophilized powderMass Ladder (Life Technologies, Gaithersburg, MD) [Sigma]) on days 1, 3, and 5. Only 0.1 ml of liposomalwas used as a marker. solution (Sigma) was administrated intraperitoneally in

the control group.1.4. Flow cytometric analysis

1.7. VaccinationFor an analysis of flow cytometry, L1210 leukemiacells were plated on sterile 35-mm tissue culture dishes When the number of cells becomes approximatelyand incubated at 378C in 5% carbon dioxide for 24 and 1 3 106, 10 mM of YoshixolTR was added into the culture48 h. The treated group received 10 mM of YoshixolTR. medium to induce cell death. After confirming cellOn the hours of the experiment, L1210 leukemia cells death of more than 90% (after 12 h), the culture me-were collected by centrifugation, resuspended in phos- dium was centrifuged at 3,000 rpm for 5 min. The super-phate buffer saline (PBS), and fixed in 70% ethanol at natant was removed. After adding physiological saline48C for 30 min. Then, cells were stained with 300 ml pro- of 0.9 cc into the sedimented component and stirring, apidium iodide (Molecular Probes, Eugene, OR) and centrifuging separation was performed at 1,000 rpm fortreated with 2 ml RNase A (Sigma, St. Louis, MO) be- 5 min. This procedure was repeated twice. Then, afterfore cells were incubated at 378C for 30 min. Cellular adding physiological saline of 0.9 cc in the rinsed resid-DNA content was determined using a FACS Calibur ual of sediment and stirring, the mixed solution was(Becton Dickinson, San Jose, CA) flow cytometer filtered by a minipore filter of cellulose acetate withequipped with an algon-ion laser turned to 488 nm. 0.45-mm pores. This filtered solution of 1 cc was in-

jected intravenously into a Japanese white rabbit. One1.5. Lactate dehydrogenase assaymonth after the injection, the rabbit was sacrificed by

In addition, we have measured the lactate dehydro- bleeding under Nembutal anesthesia. The serum wasgenase (LDH) activity in the medium from the cells as obtained after removal of cell components by centrifu-an index of cellular injury. L1210 cells were exposed to gation. CDF1 mice were treated intraperitoneally with10 mM YoshixolTR for 1 h and replaced with fresh MEM 0.2 ml of rabbit serum one week before the intraperito-containing with 10% FBS. After 24 and 48 h, an avail- neal transplantation of L1210 cells. As a control chal-able enzymatic test kit for LDH (Kyokuto Pharmaceu- lenge, the same amount of rabbit normal serum ob-tical Co. Ltd, Tokyo, Japan) was used. Cells with or with- tained from another sacrificed rabbit was treated. Theout treatments were centrifuged (1,000 rpm for 5 min), preparation of cells was the same as the above-and the supernatants were collected and transferred to described in vivo experiment. Survival rate of animalsthe plates. Nitrozolium blue mixture with Tris-buffer was after the transplantation of cells were investigated toadded to each plate. After 30 min of incubation at 378C, test the immunochemotherapeutic effect of YoshixolTR

0.1 ml of 1 M HCl solution was added to stop the reac- in vivo.tion. The absorbance at 560 nm (DU640, Beckman In-struments, Fullerton, CA) was measured. Results were 1.8. Providing of YoshixolTR

expressed as a percentage of LDH activity.The synthesis process and quality of YoshixolTR has

1.6. In vivo experiments been previously reported (Koyama et al., 1997a). TheYoshixolTR used here was provided partly from SSS Co.Six-week-old male CDF1 mice were purchased from(Matsumoto, Japan), which is the technology and li-Charles River (Tokyo, Japan). The care and use of thecensing transfer company. The test material used hereanimals were in accordance with institutional guide-as the parent compound of YoshixolTR is 4,4-dimethyl-lines. Each animal group consisted of 10 mice. The ani-6-methylene-2-cyclohexen-1-one. The NMR analysis ofmals were fed with a commercial pellet diet and waterthis synthesized YoshixolTR used here is IR(neat): 1670ad libitum. For in vivo injection, cells were incubated(C5O), 1620 (C5C) cm-1.1H NMR (60 MHZ, CCl4),for 18 h at 378C in the CO2 incubator. Cells were washedd1.15 (s, 6H, C(CH3)2), 2.57 (bs, 2H, CH2), 5.20, 5.93twice with PBS prior to transplantation in mice, and cell(2m, 2H, CH25), 5.87(d, J510, 1H, 2-H), 6.63(d,viability was tested by trypan blue exclusion assay.J510Hz, 1H, 3-H).L1210 leukemia was induced in mice by intraperito-

neal administration of 1 3 105 cells in 0.1 ml of PBS(0 hours). Those mice were 100% lethal for the period

2. Resultsof days 9–10 (Dionet and Verrelle, 1984). We observedsurvival hourly, daily change in weight, and physiologi- 2.1. Effect on cell viabilitycal behavior, such as the appetite and movements of

Fig. 1 shows trypan blue counts when L1210 cellsmice. Mice were treated with intraperitoneal injectionwere exposed to 0.1 mM of YoshixolTR for 1 h. Incuba-of 5 ml/kg of 0.22 M YoshixolTR (saline solution with 6.3

mM L-a-phosphatidylcholine [egg yolk], 1.8 mM dicetyl tion of L1210 leukemia cells with YoshixolTR resulted in

182 S. Tanaka et al. / General Pharmacology 33 (1999) 179–186

apoptosis-like aspect after the treatment with Yoshix-olTR. These aspects consisted of a reduced cell size, nu-clear condensation and breakdown, nuclear membraneinvaginations, and formation of vacuoles (Fig. 3b).Most of the chromatin aggregated in large compactgranular masses that abutted on the nuclear membrane.

2.3. Effect on DNA gel electrophoresis

As shown in Fig. 4 (lane 3), after 24 h of incubation,electrophoresis of total cellular DNA from L1210 cellstreated with YoshixolTR exhibited a DNA fragmenta-tion with a ladder-like smear pattern.

2.4. Effect on cell cycle

In addition, we performed cell cycle analysis to de-termine whether the changes in cell cycle progressionare associated with the antiproliferative activity of Yo-shixolTR in L1210 cells. Fig. 5 shows the results of DNA

Fig. 1. Effects of YoshixolTR on L1210 cell viability. content and cell cycle progression in L1210 cells. Re-duced DNA content (Sub-G1 peak) and marked de-crease of G0/G1 proportion (approximately 31% at 24 h,

dose-dependent inhibition of cell proliferation in paral-12% at 48 h) of L1210 cells treated with YoshixolTR

lel with increases in the percentage of cell death.were observed in a dose- and time-dependent manner.

2.2. Morphological observations2.5. Activity of lactate dehydrogenase

We then examined the morphological changes in-The release of LDH, a marker of increased mem-duced by YoshixolTR for 24 h by electron microscopy.

brane permeability and lethal cell injury, was measured.Scanning electron micrographs showed a variable cell-As shown in Fig. 6, LDH levels in the medium aftersize shrinkage, bearing resemblance to a kind of nuts12 h exposure to YoshixolTR did not increase signifi-and/or shells, after the treatment with YoshixolTR. Pigcantly. Even when most cells died over time, the lowernose–like pores and a cavity-like crevasse were found inlevels of LDH remained.cells treated with YoshixolTR. The surfaces of L1210

cells after the treatment lose a dendric process, as 2.6. Effect of YoshixolTR on mice inoculatedshown in Fig. 2b, compared with Fig. 2a as control with L1210 cellsL1210 cells. Cell–cell contact disappeared. Transmis-

Fig. 7 shows the survival curves of 10 mice in eachsion electron microscopy (TEM, Fig. 3) showed severalgroup. The difference of mean survival times was notcharacteristic changes. An aspect of the cells after treat-significant. However, the slope of mortality curve in in-ment with YoshixolTR showed a reduced cell size and atraperitoneal transplantation of L1210 cells in vivo wasloss of cell–cell contact. There was also a clear absenceshifted toward prolonged survival times in the groupof many granulated materials in the cytoplasm. Also,

we were able to demonstrate that those cells exhibit an treated intraperitoneally with YoshixolTR. Forty percent

Fig. 2. Morphological changes in L1210 cells observed by SEM after 24 h with 10 mM of YoshixolTR. (a) Control and (b) treatment. Bars, 1 mm.

S. Tanaka et al. / General Pharmacology 33 (1999) 179–186 183

Fig. 3. Morphological changes in L1210 cells observed by TEM after 24 h with 10 mM of YoshixolTR. (a) Control and (b) treatment. Bars, 1 mm.

of mice showed a survive longer than that of control cell particles of L1210 in vitro induced by YoshixolTR,dramatically enhanced survival. All animals in the con-group. Treatment with YoshixolTR did not show any al-

terations in movement and weight loss, compared with trol group died on day 9 after the transplantation ofL1210 cells; however, 70% of the animals survived untila single 5-FU treatment. 5-FU caused a depression of

movements, loss of appetite, and weights loss (data not day 12. All of the vaccinated animals died on day 15.shown), which investigations were consistent with onthe previous report (Houghton et al., 1979).

3. DiscussionAdditionally, as shown in Fig. 8, an intraperitonealchallenge of rabbit serum, which has been treated with In the present study, we demonstrated that Yoshi-

xolTR has an inhibitory potency of proliferation and/orcytotoxic effect on leukemia L1210 cells in vitro. In ad-

Fig. 4. DNA fragmentation assay after treatment with YoshixolTR.From the left to right lanes represent marker, control, and treatedpreparations, respectively. Fig. 5. Cell cycle determined by flow cytometry detection.

184 S. Tanaka et al. / General Pharmacology 33 (1999) 179–186

Fig. 8. Vaccinated mice survival curve after intraperitoneal trans-plantation of L1210 cells.Fig. 6. LDH levels after treatment with YoshixolTR.

al., 1993; Shi et al., 1994). Here, we investigated thosedition, YoshixolTR induces cell arrest of L1210 cells inoverall identifications in this study, as well as the previ-the G0/G1 phase of the cell cycle and causes an apopto-ous investigation on the HeLa cells (Koyama et al.,sis-like cell death.1997b) and on murine B16 melanoma cells (Koyama etIt has been known that, on a cellular level, an ultra-al., 1999).structural aspect of zeiosis or apoptosis is characterized

As a general mechanism of natural cell death in-by plasma membrane blebbing, loss of cell–cell contact,duced by YoshixolTR, we would like again to indicateclumping and rounding of cells, cytoplasmic vacuoliza-the existence of quantum thermodynamic cell deathtion, cytoplasmic condensation, and redistribution of(QTCD) as a physiological concept, even in the obser-cytoplasmic organelles (Kerr et al., 1972; Wyllie et al.,vation of lymphoid cells originated from the mesoderm.1980; Searle et al., 1982; Cotter et al., 1990; Majno andBecause of the events demonstrated in this study, weJoris, 1995). Unlike necrosis, apoptosis is a physiologi-have disclosed our valid concept of QTCD in the previ-cal mode of death during which the dying cell plays anous papers (Koyama et al., 1997a, 1997b, 1999), whichactive part in its own demise (Cotter et al., 1990). As thehave been investigated on several kinds of prokaryoteshallmark of apoptosis, additionally, DNA fragmenta-and eucaryotic cell. Although the medical term of “apo-tion has been used as one of the biochemical indicatorsptosis,” advocated by morphological findings, is beauti-(Bissonnette et al., 1992; Cohen et al., 1992; Obeid etful and/or wonderful, we would like repeatedly to pres-ent QTCD to open a new avenue of understanding inthe functional and/or physiological event of the deter-minant death in animals as well as cells, and for devel-oping therapeutic drugs along a physiological conceptof the natural event existing between life and death.

Recently, a scientific approach to develop a new che-motherapy against malignant tumors—including thoseassociated with leukemia—as well as biological inter-ests have been focused on defining a mechanism ofapoptosis and/or natural cell death (Fisher, 1994; Kerr,1994). Also, it has been implicated in a signal transduc-tion pathway related to cell growth and differentiation.Although recent studies have been focused on the cellcycle, namely, G1 checkpoint proteins such as p53,which leads cells to either growth arrest in G1 or apo-ptosis, it has been shown that the p53 tumor suppressorgene is a critical regulator of tumorigenesis, as demon-strated by the loss of functional p53 in more than halfFig. 7. Mice survival curve after intraperitoneal treatment with Yo-

shixolTR. of human cancer (Lowe et al., 1993; Lowe, 1995). Con-

S. Tanaka et al. / General Pharmacology 33 (1999) 179–186 185

versely, expression of bcl-2, which was first identified at ditionally, the preliminary results of a vaccination dem-onstrated here could be strengthened to understand athe site of translocations common to follicular lympho-more exact mechanism of an acquired drug resistancemas (Tsujimoto et al., 1985), could prevent this gene-and to develop a new therapeutic challenge for severalrelated cell death of myeloid and lymphoid cells (Vaux,kinds of immunization for infectious diseases as well as1993). However, these genes related to cell death couldmalignant tumors.not explain an integrated physiological pathway of

overall events of apoptosis (Vaux et al., 1992; Lowe,1995) and/or natural cell death. Our reports here as well

Acknowledgmentsas the recent paper from our group (Koyama et al.,1999) disclosed that a discovery of the simple chemical The authors thank Mr. K. Kametani and Ms. K. Su-substance originating from natural products such as zuki for their technical support of electron microscopicYoshixolTR might open a new avenue to understanding observations and Ms. N. Taguchi for her secretarial anda physiological function of protein expression encoded laboratory work. Finally, we deeply thank our familiesby mRNA as well as DNA and its significance, as an for their daily support and the executives of SSS Co.overall and analogously equivalent–dynamic physiologi- (Matsumoto, Nagano) and members of A & M Co. (Age-cal system existing between life and death of cells. In a matsu, Kiso, Nagano) for their spirituous encouragement.recent paper (Koyama et al., 1999), we demonstratedphysiologically that YoshixolTR caused cell death from a

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