Jpn. J. Cancer Res. (Gann), 78, 193-201; February, 1987
`
`The Inhibition of Neoplastic Cell Proliferation with Human Natural Tumor Necrosis
`Factor
`
`Masahiro NoBUHARA, * 1 Toshinori KANAMORt, * 1 Yoshikazu AsHIDA, • 1 Hiromi OotNO, • 1
`Yoshifumi HoRISAWA, *1 Kazuyuki NAKAYAMA,*' Tetsuya AsAMI, • 1 Munehiro lKETANI, *'
`Kouichi NooA, *' Syunsaku AN00H* 2 and Masashi KuR1MoT0* 2
`.,Research Laboratories for Cell Science, Machida Pharmaceutical Co., Ltd., 1-1, Kamiya 1-chome, Kita-ku,
`Tokyo J15 and * 1Fujisaki Institute, Hayashibara Biological Laboratories, Inc., 675-1, Fujisaki, Okayama 702
`
`Purified human natural tumor necrosis factor (n-TNF) was prepared by stimulating human
`leukemic B cell line (BALL- I ) with Sendai virus. The colony formations of all of 18 human
`cancer-derived abnormal cell lines were suppressed by 10'- 106 U/ml of n-TNF, while n-TNF was
`nontoxic to all human normal fibroblast cells. This i11 vitro inhibition of cell growth was reversible.
`In breast adenocarcinoma MCF7 cells treated with n-TNF a specific decrease of DNA synthesis
`was observed, and DNA histograms showed a block at G, in the cell cycle. In vivo studies revealed
`that n-TNF suppressed the tumor growth of murine Meth A sarcoma, human renal adeno(cid:173)
`carcinoma (ACHN), malignant melanoma (SK-MEL- 28) and glioblastoma (U- 373 MG).
`Isobologram analysis showed that n-TNF synergistically inhibited cell growth in combination with
`human natural interferon (IFN)-a. 111 vivo synergism of n-TNF and IFN-a was also found in the
`U-373MG tumor model implanted into nude mice.
`
`Key words: Tumor necrosis factor -
`
`Interferon - Synergism
`
`Human cells produce many kinds of cyto(cid:173)
`kines in response to many types of stimuli
`such as endotoxin, virus, bacteria and so on.
`Tumor necrosis factor (TNF) was found as a
`kind of cytokine which showed in vitro L cell
`in vivo hemorrhagic
`killing activity and
`necrotic activity on murine Meth A sarcoma
`implanted into mice. 'l It was originally thought
`that TNF was secreted from monocyte/
`• J> However,
`macrophage cell population. 2
`> recently found that a hemato(cid:173)
`Rubin et a/. 4
`poietic-derived Luk II cell line secreted TNF
`tumor promoter,
`on stimulation with a
`mezerein. T hey purified two types of human
`natural TNFs with molecular weights of
`70,000 daltons and 19,000-25,000 daltons by
`SDS-polyacrylamide gel electrophoresis (SDS(cid:173)
`PAGE) under reducing conditions. The amino
`acid sequence and cDNA sequence of both
`rabbit TNF from macrophage-like cells 1
`> and
`human TNF from monocyte-like cells6l have
`recently been determined.
`We have found that a human leukemic B
`> secreted human nat(cid:173)
`cell (BALL-1 cell) line1
`ural TNF (n-TNF) into the culture fluid to(cid:173)
`gether with human natural interferon ( IFN)(cid:173)
`a on stimulation with Sendai virus. The
`
`78(2)
`
`1987
`
`crude n-TNF in the culture fluid was purified
`to homogeneity on SOS-PAGE in order to
`determine the profiles of the in vitro anti(cid:173)
`proliferative and in vivo antitumor activities.
`
`MATERIALS AND METHODS
`Cell Lines MCF7'> was supplied by Dr. M. Namba
`(Kawasaki Med. Sch.), MKN-1'1 and MKN-28' 1
`were from Dr. T. Motoyama (Niigata Univ. Sch.
`Med.), HEC-1C1°1 was from Dr. T . Kuwata (Chiba
`Univ.), KP! 111 was from Dr. K. Tanaka (Kyushu
`Univ.), SEKI-F 12> was from Dr. M. Sekiguchi
`(Inst. Med. Sci., Univ. Tokyo), Sk-MEL-28"> was
`from Dr. F. Takaku (Tokyo Univ.), U-373MG">
`was from Otsuka Pharm. Co., Ltd ., and HF8209 1»
`and HF821 I were from Dr. J. lmanishi (Kyoto
`Pref. Univ. Med.). M08 was established by the
`authors. A549,'6> A-498,"> BT-20,"> SK-LU-J, 11>
`HT-144,"> SK-CO-I,''> HeLa, 1•> ACHN, 20> U-138
`MG 1'> and KB 21> were supplied by the American
`Type Culture Collection. BALL- I cells, which was
`used for n-TNF production, were established from
`a patient with acute lymphoblastic leukemia by
`> in 1976. BALL-I cells were nega(cid:173)
`Miyoshi et al. 1
`tive for EBNA, virtually 100% surface lg-positive
`and human thymus-related antigen-negative.
`Culture Media, Animals and Reagents MEM,
`methionine-free MEM, Ham's F -12 and RPMI
`
`193
`
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`

`1640 were purchased from Nissui Seiyaku Co.,
`(D(cid:173)
`Ltd., Japan. Dulbecco's modified MEM
`MEM) was purchased from Life Technologies,
`Inc., USA. L-15 medium and non-essential amino
`acid mixture were purchased from Flow Labo(cid:173)
`ratories, Inc., USA. All sera were obtained from
`Irvine Scientific, USA, through Nippon Bio-Supply
`Center, Japan. The 15.5 mm</> well dishes, 35 mm</>
`Petri dishes and 96-microwell plates were pur(cid:173)
`chased from A/S Nunc, Denmark and 60 mm</>
`Petri dishes from Corning Glass Works, USA.
`Mitomycin C (MMC), fluorouracil (5-FU) and
`doxorubicin hydrochloride (DXR) were purchased
`from Kyowa Hakka Kogyo Co., Ltd., Japan, and
`nimustine hydrochloride (ACNU) was purchased
`from Sankyo Co., Ltd., Japan. [3H]Thymidine,
`3H]uridine, [31S]methionine and Bolton-Hunter
`[
`reagent for protein iodination (uil) were supplied
`by Amersham International plc. UK. RNase and
`iodide were purchased from Sigma
`propidium
`Chemical Co., USA, and pronase P was obtained
`from Kaken Pharmaceutical Co., Japan. CBF1
`mice and BALB/c nu/nu mice were purchased
`from Shizuoka Laboratory Center, Japan and
`Nippon Clea, Japan, respectively.
`The purified human natural IFN-a was prepared
`from the culture fluid of BALL- I cells stimulated
`with Sendai virus by the authors as described by
`Imanishi et al.m and Tanimoto.n> Sendai virus was
`also prepared by the authors. 2•>
`Sepharose gel coupled with murine anti-IFN-a
`monoclonal antibody (NK-2 Sepharose gel ) was
`purchased from Celltech Ltd., UK. An ultra(cid:173)
`filtration membrane (AIP-3013, MW 6000-cut)
`was purchased from Asahi Chemical Industry Co.,
`Ltd., Japan. Phenyl-Sepharose gel and Sephadex
`G-200 gel were obtained from Pharmacia, Sweden.
`Murine anti-n-TNF monoclonal antibody (3D6
`antibody) was prepared by the authors (unpub(cid:173)
`lished).
`n-TNF Preparations BALL-I cells suspended in
`RPMI 1640 (5 X 106 cells/ml) were stimulated
`overnight with about 100 HAU/ml of Sendai virus
`at 35°. The cells were discarded by centrifugation
`and the supernatant was concentrated by using an
`ultrafiltration membrane (AIP-3013). The concen(cid:173)
`trated solution containing n-TNF was applied to a
`phenyl-Sepharose column. n-TNF was then eluted
`in O.OlM sodium
`with 60% ethylene glycol
`phosphate-buffered saline (PBS), followed by dial(cid:173)
`ysis against PBS. The partially purified n-TNF
`solution was passed through an NK-2 Sepharose
`gel column to remove IFN-a. n-TNF was purified
`to homogeneity by affinity chromatography on a
`3D6 antibody coupled-Sepharose gel column by
`eluting with 0.035M ethylamine, followed by gel
`filtration on a Sephadex G-200 column with X 2
`PBS (maned fraction No. H-7-1). The purified
`
`M. NOBUHARA, ET AL.
`n-TNF showed a single homogeneous band with a
`molecular weight of 17,000 to 18,000 daltons on
`SOS-PAGE by the method of Laemli.m Protein
`sequencing and cDNA sequencing of n-TNF26
`> in(cid:173)
`dicated it to be almost identical with human TNF
`secreted from a premyelocytic leukemic cell (HL-
`60) line. 6> All in vitro and in vivo experiments in this
`study were carried out by using purified n-TNF
`preparations with a specific activity of about 109 U/
`mg protein.
`Titration of n-TNF was carried out by measur(cid:173)
`ing the cytopathic effeci (CPE) against murine Ln,
`in MEM sup(cid:173)
`cells, which were subcultured
`plemented with 10% bovine serum in a 96-micro(cid:173)
`well plate ( 1 x 10• cells/well). Actinomycin D ( 1.6
`µg/ml final concentration) and n-TNF were
`simultaneously added to all microwells and the
`plate was incubated overnight at 37°. The final
`concentration of n-TNF giving half CPE was arbi(cid:173)
`trarily defined as SO Laboratory units/ml. A Japan
`Standard Reference for TNF was recently supplied
`by Dr. S. Yamazaki, NIH, Japan, and 1 JRU was
`equivalent to 350 Laboratory units.
`In vitro Cell Proliferation Inhibition Table I shows
`the compositions of media employed for in vitro
`colony formation. One hundred to three thousand
`cells of each cell line were inoculated in 35 mm</> or
`60 mm</> Petri dishes and cultured at 37° under~%
`CO,/95% air for 7- 13 days either in the presence
`or absence of n-TNF, followed by Giemsa stain(cid:173)
`ing.m The number of colonies consisting of more
`than 20 cells was counted in order to calculate the
`n-TNF concentration which decreased colony for(cid:173)
`mation by 50% (IC!O) ,
`In order to clarify whether or not the anti(cid:173)
`proliferative activity of n-TNF was reversible,
`MCF7 cells were cultured by the mass culture
`method in the presence of 104-10' U/ml of n-TNF
`for 2 days, then culture was continued in fresh
`medium without n-TNF for another 7 days.
`Isobologram analysis of the combination of
`n-TNF with human IFN-a or chemotherapeutics
`such as MMC, 5-FU, DXR and ACNU was
`performed as described by Steel and Peckham. 21> In
`the case of HEC-1 C cells, which are totally resis(cid:173)
`tant to human IFN-a, the combination effect of
`n-TNF (0 or 350 U/ml) and IFN-a (0, 101, or 104
`IU/ml) was analyzed in triplicate at each concen(cid:173)
`tration by the method of Dannecker et a/,2'l
`The effects of n-TNF on DNA, RNA and pro(cid:173)
`tein syntheses were also investigated by using MCF
`7 cells cultured in the presence of 106 U/ml of
`n-TNF for 24 hr according to the method of Fuse
`and Kuwata, 30> The DNA histogram ofMCF7 cells
`treated with 106 U/ml of n-TNF was analyzed for
`36 hr in a fluorescence-activated ceU sorter (FACS
`IV, Becton Dickinson Immunocytometry Systems,
`USA) by the method of Crissman et al. 31> n-TNF
`
`194
`
`Jpn. J. Cancer Res. (Gann)
`
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`

`protein was iodinated by the method of Littman et
`a/.">
`In vivo Antitumor Animal Experiments The in vivo
`experiments were carried out with murine Meth A
`sarcoma intradermally (id) implanted into CBF,
`(U-373MG),
`and human glioblastoma
`mice,
`human renal adenocarcinoma (ACHN) and human
`malignant melanoma (SK-MEL-28) which were
`subcutaneously (sc) implanted into BALB/c nu/
`nu mice. The n-TNF was intravenously (iv) or
`intratumorally (itu) administered either alone or in
`combination with human natural IFN-a.
`
`HUMAN NATURAL TUMOR NECROSIS FACTOR
`the in vitro inhibition spectra against the 18
`abnormal cell lines did not show any spe(cid:173)
`cificity regarding the origin of the tumors
`from which the cell lines had been established.
`MCF7 cells, which were most sensitive to
`n-TNF in vitro, were employed in order to
`clarify how n-TNF would affect the intra(cid:173)
`cellular macromolecule synthesis. Figure 1 in(cid:173)
`dicates that over a 24 hr period, 106 U/ml of
`n-TNF specifically suppressed DNA synthesis
`and brought about very little decrease of
`RNA and protein syntheses. As shown in Fig.
`2, the DNA histogram analysis revealed that
`control (untreated) MCF7 cells constantly
`divided during 36 hr under these experimental
`conditions. On the other hand, a major por(cid:173)
`tion of S-, G2- and M-phase cells had dis(cid:173)
`appeared almost completely after a 24 hr
`
`RESULTS
`In vitro Experiments As shown in Table I, the
`colony formation of all of 18 human cancer(cid:173)
`derived abnormal cell lines was inhibited by
`n-TNF but that of three normal fibroblasts
`was not suppressed. The results also show that
`
`Table I.
`
`Cell line
`
`In vitro Antiproliferative Activity of n-TNF against Twenty-one Human Cell Lines
`re,.
`Culture
`media''
`(U/ml)
`
`Origin
`
`A. Extremely sensitive group
`Breast adenocarcinoma
`MCF7
`Renal carcinoma
`A-498
`Lung carcinoma
`A549
`B. Highly sensitive group
`Glioblastoma
`U-373MG
`SEKI-F
`Malignant melanoma
`Breast carcinoma
`BT-20
`Uterine cervical carcinoma
`HeLa
`Glioblastoma
`U-138MG
`HT-144
`Malignant melanoma
`Lung adenocarcinoma
`SK-LU-I
`C. Sensitive group
`SK-MEL-28
`MKN-1
`HEC-lC
`ACHN
`KPI
`MKN-28
`KB
`SK-CO-I
`D. Resistant group
`I.O X 106<
`E
`Normal diploid fibroblast
`HF8209
`I.OX 106<
`E
`Normal diploid fibroblast
`HF82! 1
`'I.O X 106<
`E
`Normal diploid fibroblast
`M08
`The colony count was carried out by using a colony counter (Colony Analyzer CA-7, Orient Instruments Ltd.,
`Japan).
`a) A, 10% fetal bovine serum/D-MEM; B, 10% fetal bovine serum/MEM; C, 10% fetal bovine serum/
`RPMI 1640; D, 10% fetal bovine serum/Ham's Fl2; E, 20% fetal bovine serum/L-15; F, 20% fetal
`bovine serum/MEM; G, 20% fetal bovine serum/MEM/non-essential amino acids/2mM sodium pyruvate.
`All media were supplemented with IOmM HEPES.
`
`2.8 X 101
`3.0 X 101
`4.3 X 101
`
`1.8 X 102
`2.5 X 10'
`2.7 X 10'
`3.0Xl02
`4.5 X 102
`4.7 X 102
`5.4 X 102
`
`1.0 X 10'
`1.1 X 10'
`2.4 X 10'
`4.0 X IO'
`4.5 X 10'
`8.3 X 10'
`4.0 X 104
`2.3 X JOl
`
`A
`D
`A
`
`G
`F
`D
`A
`E
`E
`B
`
`D
`C
`A
`A
`D
`A
`A
`B
`
`Malignant melanoma
`Stomach adenocarcinoma
`Uterine adenocarcinoma
`Renal adenocarcinoma
`Renal pelvic carcinoma
`Stomach adenocarcinoma
`Oral epidermoid carcinoma
`Colon adenocarcinoma
`
`78(2) 1987
`
`195
`
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`

`M. NOBUHARA, ET AL.
`
`Inhibition of DNA, RNA and protein
`Fig. 1.
`syntheses of MCF7 ceJls. • , DNA; o, RNA; 6,
`protein. CeJls were inoculated in 15.5 mm¢ well
`dishes at a concentration of 4 X 10' cells/ml/dish in
`D-MEM supplemented with 5% bovine serum, and
`cultured for 3 days at 37°. The culture was con(cid:173)
`tinued for another 0, 3, 8, 16 and 24 hr in the
`presence of 106 U/ml of n-TNF. At the indicated
`time,
`the medium was changed
`to
`fresh
`methionine-free MEM containing 106 U/ml of
`n-TNF, and the cells were pulse-labeled for 1 hr
`with (3H)thymidine (0.55 µCi/weJI), [3H]uridine
`(5.5 µCi/well) or [3'S)methionine (5.5 µCi/well)
`as described in "Materials and Methods." The
`trichloroacetic acid-insoluble
`isotopic count in
`fractions was measured in a liquid scintillation
`counter (Liquid Scintillation System LSC-700,
`Aloka). The figures indicate the relative values as
`compared with control groups at each time.
`
`8
`16
`Incubation Time (hr)
`
`24
`
`1000
`
`12hr
`
`24hr
`
`500
`
`0
`
`!:!J.
`Q)
`(.)
`....
`0 ... Q)
`.0 1000
`E
`::::l z
`
`500
`
`DNA Content
`
`Fig. 2. DNA histogram of MCF7 ceJls treated with n-TNF. (a) Control (b) 106 U/ml. CeJls
`were inoculated in 60 mm¢ Petri dishes at a concentration of 2.5 X 10' ceJls/10 ml/dish in
`D-MEM supplemented with 5% calf serum, and cultured for 2 days at 37°. The cells were treated
`with n-TNF for another 12, 24 or 36 hr. At the indicated time, ceJls were detached from the dishes
`with 0.25% pronase P, treated with 1 % sodium azide and fixed with 70% ethanol. They were
`stocked at - 20° until analysis. Flow cytometric analysis was conducted after treating the cells
`with 50 µg/ml of propidium iodide and 1 mg/ml of RNase as described in "Materials and
`Methods."
`
`treatment with 106 U/ml of n-TNF. These
`results strongly suggest that n-TNF mainly
`blocked the G1-phase of the cell cycle.
`Figure 3 shows that n-TNF also inhibited
`the cell growth of MCF7 cells in the mass
`culture method, and immediate reprolifera(cid:173)
`tion of MCF7 cells was observed on changing
`to fresh culture medium without n-TNF at a
`
`growth rate similar to that of the control cells.
`Isobologram analysis indicated clear syn(cid:173)
`ergistic effects ofn-TNF and human IFN-a or
`ACNU in the inhibition of colony formation,
`as shown in Fig. 4. It was also observed that 5-
`FU and MMC inhibited synergistically, and
`DXR inhibited additively the cell prolifera(cid:173)
`tion of ACHN cells in the presence of n-TNF
`
`196
`
`Jpn. J. Cancer Res. (Gann)
`
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`

`HUMAN NATURAL TUMOR NECROSIS FACTOR
`
`1X105 -
`
`FICIFN
`1.0
`
`' ' ',,
`
`0.5
`
`{a)
`
`' ' ' ' ' ' ' ' ' ',
`
`FICIFN
`
`' ' ',
`
`{b)
`
`' ' ' ' ' ' ' ' '
`
`' ' '
`
`o-l--.....:=:=::==~
`
`1X104
`
`r ~
`
`8
`6
`4
`Incubation Period (days)
`
`FICIFN
`
`FIG ACNU
`
`,b
`
`Fig. 3. Reversible in_hibition by n-TNF of the cell
`proliferation of MCF7 cells. O, Control; (cid:143) , 10• U/
`ml; 6, 105 U/ml. Cells were inoculated into a 96-
`. microwell plate at a concentration of0.2 X 10• cells/
`0.2 ml/well in D-MEM supplemented with 5%
`bovine serum. One day later, n-TNF was added to
`give a final concentration of 10•. or 105 U/ml, and
`the culture was continued for a further 2 days
`(indicated by hatched bars). After the 2-day treat(cid:173)
`ment with n-TNF, cells were cultured for another 7
`days in fresh medium without n-TNF.
`
`(data not shown). Interestingly, HEC-lC
`cells, which are totally resistant to IFN-a
`> also
`because they lack the IFN receptor, 10
`showed synergistic inhibition of colony for(cid:173)
`mation by the combination of n-TNF and
`IFN-a.
`Receptor analysis shows that all the cell
`lines including a normal fibroblast cell line
`expressed the n-TNF receptor on the cell sur(cid:173)
`face (Table II). Even normal fibroblast cells,
`which are resistant to n-TNF, expressed the
`receptor on the cell surface with similar Kd
`value and receptor number to those of sensi(cid:173)
`tive abnormal cell lines. We could not observe
`any correlation between n-TNF sensitivity
`and affinity/number of the receptor.
`In vivo Experiments The tumor mass increase
`of Meth A sarcoma was significantly inhibited
`by both iv and itu administration of n-TNF at
`doses of 2 X 105-2 X 106 U/mouse, as shown in
`Table III. In addition, a high frequency of
`complete tumor regression was observed in
`both itu and iv administration groups. The
`
`78(2) 1987
`
`0.5
`FICTNF
`
`0
`
`0.5
`FICTNF
`
`1.0
`
`Isobologram studies on the combination
`Fig. 4.
`of n-TNF and human IFN-a (a, b, c) or ACNU
`(d). (a) Renal adenocarcinoma ACHN, (b) malig(cid:173)
`nant melanoma SK-MEL-28, (c) glioblastoma U-
`373MG, (d) renal adenocarcinoma ACHN. Either
`200 cells/2 ml for SK-MEL-28 cells or 500 cells/2
`ml for ACHN and U-373MG cells was inoculated
`into 35 mm¢ dishes in the media listed in• Table I
`and cultured for 7-9 days in the presence of n-TNF
`(0-1 X 10(cid:141) U/ml) and human IFN-a (0-5 x 10' IU/
`ml) or ACNU (0-40 µg/ml). After Giemsa stain(cid:173)
`ing, the number of colonies was counted in order
`to obtain the fractional inhibitory concentration
`(FIC) for n-TNF (FICmp) and human IFN-a
`(FIC1PN) or ACNU (FIC;.CN1J) by the method de(cid:173)
`scribed in "Materials and Methods."
`
`antitumor activity by the itu route, however,
`seemed to be 3 to 10 times stronger than that
`by the iv route.
`Table IV shows that intratumoral adminis(cid:173)
`tration of n-TNF also effectively suppressed
`the mass increase of human tumors implanted
`into BALB/c nu/nu mice. In particular,
`ACHN tumor was a very sensitive model; 5
`out of 6 mice were completely cured by daily
`itu administration of 2 X 106 U/mouse. The
`combined effect of n-TNF and human IFN-a
`on U-373MG glioblastoma was also studied in
`vivo by daily itu administration (Table IV).
`
`197
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`

`M. NOBUHARA, ET AL.
`
`Cell line
`
`Kd
`(M X IO")
`
`IC,a''
`(U/ml)
`
`Medium
`used"
`
`Table II. n-TNF Receptor Analysis in the Absence or the Presence of Human IFN-a
`No. of
`receptor/cell
`( X 10- ')
`A
`2.8 X 101
`7.7
`7.8
`MCF7
`B
`3.0X 101
`3.9
`1.7
`A-498
`B
`1.8 X 102
`5.6
`3.5
`U-373MG
`A
`2.4X 103
`9.8
`8.4
`HEC-IC
`C
`2.3 X 10'
`2.3
`2.7
`SK-CO-I
`LOX 106<
`D
`7.2
`8.1
`HF8209
`Cells were inoculated into 35 mm¢ dishes (6-18 X IO' cells/dish) and incubated overnight at 37°. "'1-n-TNF
`(ca. 3.8 mCi/mg protein) in 0. I% HSA/D-MEM was added to each dish (2 X 10'- l X IO' cpm/dish), f~llowed
`by incubation at 37° for 2 hr either in the presence or absence of 1.4 µg/dish of cold n-T~F protem. The
`specific binding of n-TNF on the cell surface was counted in the I% SDS cell lysate by usmg an autowell
`gamma system ARC-300 (Aloka, Tokyo).
`a) Each IC50 value is cited from Table I.
`b) A, 5% bovine serum/D-MEM; B, 10% fetal bovine serum/MEM/non-essential amino acids/2mM
`sodium pyruvate; C, 10% fetal bovine serum/MEM; D, 10% bovine serum/D-MEM.
`
`Table III.
`
`Group
`
`Route
`
`Complete
`regression
`0/6
`0/6
`1/6
`3/6
`0/6
`1/6
`4/6
`
`In vivo Antitumor Effect of n-TNF on Meth A Sarcoma
`Tumor weight''
`Dose
`(mg)
`(U/mouse)
`1520 ± 256
`861 ± 108*
`529 ± 132 * *
`176±84**
`1520± 130
`644±159*
`52±44*
`
`2X 10'
`6X 10'
`2 X 106
`
`iv
`iv
`iv
`iv
`itu
`itu
`itu
`
`Control
`n-TNF
`n-TNF
`n-TNF
`Control
`2 X 10'
`n-TNF
`6 X 10'
`n-TNF
`'P< 0.05, "P< 0.01.
`a) Mean± SE.
`Ascitic Meth A sarcoma cells (2 X JO' cells/0.1 ml), which had been serially passaged in BALB/c mice, were
`id implanted into CBF, mice. Seven to nine days later, when the tumor became palpable, n-TNF was
`administered iv or itu once every three days for 15 days. On day 15 the excised tumor was weighed and
`complete regression was macroscopically checked.
`
`The results show that the combined effect of
`n-TNF and human IFN-a in vivo was definite(cid:173)
`ly synergistic, in agreement with the results of
`the in vitro isobologram studies;
`
`DISCUSSION
`
`We have shown here that human leukemic
`B cells (BALL-1 cell line) produced n-TNF
`on viral stimulation with Sendai virus. Al(cid:173)
`though n-TNF effectively inhibited the cell
`proliferation of this human cancer-derived
`cell line in a reversible manner, n-TNF did not
`affect the colony formation of normal fibro(cid:173)
`biast cell lines. It is suggested that this inhibi(cid:173)
`tion is mainly due to the specific reduction of
`DNA synthesis at the G,-phase of the cell
`cycle (Figs. 1 and 2).
`
`198
`
`This specific in vitro antiproliferative activ(cid:173)
`ity against abnormal cells coincides well with
`other reports.''·!4> With the aim of explaining
`the mechanism of specific inhibition of abnor(cid:173)
`mal cell growth, several studies have been
`conducted from the viewpoint of the affinity/
`5> Our results dem(cid:173)
`number ofTNF receptors. 3
`l-l
`onstrate that the sensitivity of each cell line is
`not directly dependent upon the affinity/
`number of the receptors. Baglioni et a/. 35> re(cid:173)
`cently found a positive correlation between
`the degree of sensitivity to TNF and the
`number of the receptors, but other groupsll,!4l
`could not observe any correlation between
`them. It is possible that Baglioni et al. might
`have obtained positive results just by chance,
`because only three cell lines were used in their
`
`Jpn. J. Cancer Res. (Gann)
`
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`

`Table IV.
`
`Group
`
`6XJ0'
`2X 10'
`
`6X 10'
`2 X IO'
`
`2 X 10'
`
`2 X 10'
`6 X 10' IU
`2 X 10'
`+6 x 10• IU
`
`126 ± 11
`84 ± 13•
`49 ± 3 .. -
`
`74± 12
`6 ± 3**
`2 ± 2 ..
`
`721±113
`226±40**
`868 ± 175
`714 ± 134
`625 ± 164
`
`166 + 77• •
`
`HUMAN NATURAL TUMOR NECROSIS FACTOR
`In vivo Antitumor Effect of n-TNF on Human-derived Tumors
`Tumor weight•l
`Dose
`(mg)
`(U/mouse)
`SK-MEL-28 (malignant melanoma)
`Control
`n-TNF
`n-TNF
`ACHN (renal adenocarcinoma)
`Control
`n-TNF
`n-TNF
`U-373MG (glioblastoma)
`Exp. I Control
`n-TNF
`Exp. 2 Control
`n-TNF
`IFN-a
`n-TNF
`+IFN-a
`' P<0.05, .. P<0.01.
`a) Mean ± SE.
`One tumor fragment (2- 3 mm in size), which had been serially passaged in BALB/c nu/nu mice, was sc
`implanted into BALB/c nu/nu mice. Ten days to three weeks later, when the tumor became palpable, the daily
`itu administration of n-TNF and IFN-a was started for 14 days (SK-MEL-28 and U-373MG) or 28 days
`(ACHN) . On day 14 (SK-MEL-28 and U-373MG) or on day 28 (ACHN), the excised tumor was weighed
`and complete regression was macroscopically checked.
`
`Complete
`regression
`
`0/6
`0/6
`0/6
`
`0/6
`3/6
`5/6
`
`0/6
`0/6
`0/5
`0/5
`0/5
`
`0/5
`
`study. It seems likely that an unknown in(cid:173)
`tracellular mechanism(s) involving the recep(cid:173)
`tor must exist to explain these experimental
`results.
`In in vivo studies, n-TNF showed a strong
`antitumor activity against both murine and
`human solid tumors, which often completely
`regressed in the cases of murine Meth A and
`human ACHN tumors (Tables III and IV).
`and
`clear hemorrhagic
`a
`Furthermore,
`necrotic appearance of Meth A tumor was
`commonly observed as early as on day 1 of
`ad(cid:173)
`intratumoral
`and
`intravenous
`both
`ministrations of n-TNF. A similar necrotic
`appearance was also observed in the murine
`Lewis lung carcinoma model (unpublished
`data), while n-TNF induced no apparent ne(cid:173)
`crosis in any human tumor model, even a
`highly sensitive one such as ACHN. These
`observations suggest that the antitumor mech(cid:173)
`anism(s) of n-TNF are somewhat different
`the human tumor model (nu/nu
`between
`mice) and the murine tumor model (CBF)
`mice). It was observed that severe destruction
`of the vascular system causes hemorrhagic
`necrosis in the case of murine solid tumor as
`
`78(2) 1987
`
`judged from the histopathological findings on
`Meth A tumor (data not shown), but this
`destruction may not occur in human tumor
`models. Therefore, it is presumed that T cell(cid:173)
`related function or some deficient gene(s) in
`BALB/c nu/nu mice might be involved in the
`hemorrhagic necrosis.
`The studies of combined n-TNF and human
`IFN-a provided direct evidence that they syn(cid:173)
`ergistically inhibit in vitro cell proliferation
`and in vivo tumor growth. An in vitro syn(cid:173)
`ergistic cytotoxic effect of TNF and IFN-r
`reported by Aggarwal
`has recently been
`et al. 1'> They stated that IFN-r stimulated the
`number of TNF receptors on the cell surface
`two- to three-fold, with a similar Kd value to
`those of IFN-r-untreated cells, and speculated
`that this increment of the number possibly
`augments TNF activity. Williamson et a/. 31
`reported that the cell-killing activity of par(cid:173)
`tially purified human natural TNF, which was
`secreted from a hematopoietic cell line stim(cid:173)
`ulated with tumor promoter, was augmented
`by combining it with IFN-a and -r. It is
`reasonable, therefore, to think that the syn(cid:173)
`ergism observed in our in vitro studies can also
`199
`
`>
`
`NOVARTIS EXHIBIT 2089
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`

`M. NOBUHARA, ET AL.
`be explained as resulting from the same mech(cid:173)
`anism as proposed by Aggarwal! et al. 16
`>
`In the model using nu/nu mice it is thought
`that human IFN-a inhibits the tumor growth
`directly but not through any host-mediated
`system because of the species-dependent
`specificity of IFN-a. Accordingly, participa(cid:173)
`tion of a host-mediated mechanism in the nu/
`nu mice system seems unlikely to be able to
`explain our in vivo synergy.
`It would seem to be worth setting up a
`clinical trial to examine whether the anti(cid:173)
`tumor activity of n-TNF can be augmented
`by combined therapy with human IFN-a
`through direct and probably also indirect
`mechanisms.
`
`ACKNOWLEDGMENTS
`We thank Miss M. Katayama and Mr. K. Baba
`for their help in the in vitro studies and Mr. S.
`Murakami and Mr. Y. Harada for their help in the
`in vivo experiments. We also thank Miss Y.
`Yamaguchi and Miss R. Nakano for typing the
`manuscript.
`(Received Nov. 11, 1986/Accepted Dec. 27, 1986)
`
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