JPH0912596A - A novel proteinaceous factor that enhances tumor cytotoxicity of anticancer drugs - Google Patents

Abstract

PURPOSE: To obtain the subject new proteinic factor, derived from a culture supernatant of an epithelial tumorous cell, having a specific molecular weight and enhancing actions on tumor cytotoxic actions of an anticancer agent and capable of suppressing adverse effects of the anticancer agent due to its ability to reduce the effective amount of the anticancer agent. CONSTITUTION: This new proteinic factor is derived from a culture supernatant of an epithelial tumorous cell and has about 50000-55000 molecular weight measured by a get filtration method and further enhancing actions on tumor cytotoxic actions of an anticancer agent such as a tumor necrosis factor-α (TNF-α), bleomycin and cisplatin in a manner dependent on the concentration. The proteinic factor is obtained by culturing an epithelial tumorous cell (e.g. KB cell) in a culture medium containing a bovine fetal serum (FCS) and the culture medium is then removed after 2 days. A serum-free culture medium is added to carry out the culture for 2 days. The culture medium is centrifuged to collect a culture supernatant, which is then concentrated with an ultrafiltration membrane and fractionated according to an anion exchange column chromatography by using cytotoxic activities of the tumorous cell in the presence of the TNF-α as an index.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel proteinaceous factor derived from a culture supernatant of epithelial tumor cells, preferably KB cells, and having an action of enhancing the tumor cell damaging action of anticancer agents.

[0002]

2. Description of the Related Art Many anticancer agents used for cancer chemotherapy have been developed so far and have achieved great results in cancer treatment. However, anticancer agents inhibit nucleic acid synthesis and intracellular component synthesis essential for cell growth based on the fact that tumor cells turn over faster and grow faster than normal cells. Hematopoietic organs, mucous membranes,
It is toxic to the liver, kidneys, hair roots, etc., and causes severe pain such as anemia, vomiting, liver damage, renal damage, and hair loss to the administered patients. Moreover, since anticancer agents generally have a narrow range between the effective dose and the maximum tolerated dose, the dose should be carefully monitored.

Therefore, in order to reduce the toxicity of the anticancer drug to normal cells, a combination with a pressor has hitherto been used for the purpose of exerting an anticancer effect even with a small dose (Aki Yui et al .:
Cancer and chemotherapy, 11, 741, 1984) and combination with calcium modifiers (Tsuruo Takashi: Cancer and chemotherapy, 11, 750, 1984), administration with sustained release preparations, etc. have been tried.

On the other hand, some anticancer agents, such as bleomycin, show almost no effect of killing tumor cells by themselves and must be used in combination with other anticancer agents.

In addition, TNF (Tumor Necrosis Factor) -α
Is a protein having a function of killing tumor cells found in the serum of BCG-sensitized and endotoxin-treated mice by Carswell et al. (Carswell, EA et al., Pro
c. Natl. Acad. Sci. USA, 72: 3666-3670, 1975), which has been expected as an antitumor agent since its discovery. However, when administered to humans, there are few markedly effective cases as obtained in animal experiments, and there are considerable tumors that do not kill TNF-α at all (Naoki Watanabe et al. Medicine ", Vol. 48, 694-702, 1993).

Therefore, attempts have been made to use TNF-α in combination with other cytokines, and so far, IFN (in
terferon) -γ, IFN-α, IFN-β, IL (interleukin) -1, I
L-2, IL-4, etc. were found to enhance the tumor cell-damaging effect of TNF-α ("Tumor Necrosis Factors", B. Beu
tler ed., Raven Press, New York, 1992). However, it was clinically effective only in combination with IL-2 (Yang, SC et al., Cancer Research, 51, 3669-36).
76, 1991).

[0007]

As described above, in order to reduce the toxicity of anticancer agents, there is a demand for means that can exert a therapeutic effect even with a small dose. Further, for an anti-cancer agent which has almost no tumor cell-damaging action by itself, a means for enhancing the action is required.

[0008]

Means for Solving the Problems The present inventor believes that the above problems can be solved by using a factor that enhances the tumor cell-damaging action of an anti-cancer agent, and as a result of earnestly searching for such a factor, the epithelium It was found that a novel proteinaceous factor derived from a culture supernatant of a cancerous tumor cell and having a molecular weight of about 50,000 to about 55,000 measured by gel filtration has an action of enhancing the tumor cell-damaging action of an anticancer agent, and the present invention completed.

The present protein factor (hereinafter referred to as the present factor), when used together with an anticancer agent, remarkably enhances the cytotoxic effect of the anticancer agent on various human-derived tumor cells.
That is, by using an anticancer agent having a strong anticancer effect in combination with this factor, the effective concentration can be reduced without decreasing the effect of the anticancer agent. It is expected to reduce the side effects of anticancer drugs. In addition, by combining an anticancer agent such as bleomycin and TNF-α, which has almost no tumor cell-damaging effect alone, with this factor, tumor cell killing effect is induced. Therefore, these anticancer agents should be applied to cancer treatment. Will be possible.

The present factor alone has almost no cytotoxic effect on normal human cells, and when the concentration of the present factor is increased, it exhibits a tumor cytotoxic effect in a concentration-dependent manner. Therefore, it is expected that this factor alone can be applied as an anticancer agent.

The preparation of this factor is performed roughly as follows.

The present factor can be obtained by extraction and purification from the culture supernatant of epithelial tumor cells such as KB cells, HT-3 cells and HeLa cells, but KB cells are particularly preferably used. KB cells are an epithelial human tumor cell line
Deposit number ATCC CCL to an Type Culture Collection (ATCC)
It has been deposited as 17 and is freely available to anyone. Also, a mutant strain of KB cell can be preferably used in the present invention.

First, epithelial tumor cells are cultured according to a conventional method. The medium is not particularly limited as long as it is a medium usually used for culturing animal cells, and for example, Eagle's minium e
ssential medium, Dulbecco's modified Eagle's mediu
m (DME), RPMI1640, etc. may be used. It is preferable that the medium does not contain fetal calf serum (FCS) in order to purify this factor, but if the presence of FCS is essential for the growth of the cell line used, the cells should be cultured in a medium containing FCS. It is preferable to transfer the cells to a serum-free medium after sufficiently increasing the density and to purify this factor from the serum-free medium supernatant. Then, the culture supernatant is collected,
The supernatant is subjected to gel filtration to have a molecular weight cut-off of about 50,000 to about 60,0.
The 00 fraction is collected (it is preferable to concentrate the supernatant by removing low-molecular substances using an ultrafiltration membrane prior to gel filtration). Furthermore, when this fraction is applied to anion exchange chromatography and eluted with a buffer containing salt while increasing the salt concentration, this factor is eluted with a buffer containing about 0.05M to about 0.1M salt.

The present invention will be described below based on examples, but the present invention is not limited thereto.

[0015]

【Example】

Example 1 (Production of this factor) Extraction / purification of this factor was performed using the cytotoxic activity against SK-BR3 cells (ATCC HTB30) derived from human breast tumor cells in the presence of TNF-α as an index. The method for measuring the cytotoxic activity was as follows.

3 × 10 ⁴ of SK-BR3 cells suspended in 100 μl of DME containing 10% (v / v) FCS was spread on a 96-well plate, and CO was added at 37 ° C.
₂ Cultured overnight in an incubator. Thereafter, 50 μl of a solution containing the sample was added, and then human TNF-α (manufactured by RD System) was added so that the final concentration was 80 ng / ml, and the medium was further added to make the final volume 200 μl per well. In this state
After culturing in a CO ₂ incubator for _{2 to} 4 days, the number of viable cells was measured. The number of viable cells was measured by the crystal violet staining method, and OD ₅₉₅ was used as an index of the number of viable cells. The correlation between the number of viable cells and OD ₅₉₅ was examined in advance, and the number of viable cells was measured within a linear range.

(1) Culture of KB cells About 2 × 10 ⁸ KB cells were contained in 1,000 ml of 10% (v / v) FCS.
The cells were suspended in DME and divided into 20 150 cm ² culture dishes and cultured. After 2 days, the medium was removed, and 50 ml of serum-free medium ASF301 (manufactured by Ajinomoto Co., Inc.) was added to each dish to continue the culture. After 2 days, the medium was collected and the cells were removed by centrifugation to obtain a culture supernatant.

(2) Extraction / purification 1,000 ml of the culture supernatant obtained in (1) is passed through an ultrafiltration membrane (Filtron, manufactured by Fuji Filter Co.) having a molecular weight cutoff of 10,000 to obtain about 1/100 To a volume of about 10 ml.

Sephacryl S-300 HR is used for gel filtration
(Manufactured by Pharmacia) using a column (φ60 × 300m)
m)) and equilibrated with phosphate buffer (PBS) of pH 7.33. About 5 ml of the above concentrated culture supernatant was applied to this, and OD ₂₈₀
Elution was performed with PBS while monitoring at 1, and 15 ml of each fraction was collected. The results are shown in FIG.

Among the fractions obtained above, the even numbered fractions were examined for cytotoxic activity. A TNF-α addition group and a TNF-α non-addition group were prepared, and the culture period after adding the sample was 4 days. The results are shown in Fig. 2 (in the figure, the solid line is TNF).
-alpha-free group, wavy line indicates TNF-α-added group). From this figure, it was revealed that the maximum peak of cytotoxic effect on SK-BR3 cells in the presence of TNF-α was the fractions of fraction numbers 7 to 11. Therefore, these fractions were pooled and 50 volumes of 10 mM Tris-HCl buffer (pH 7.5) was added.
It was dialyzed twice with and subjected to the following anion exchange column chromatography.

For anion exchange column chromatography, Econo-PacQ column (Bio-Rad, φ10 × 50 mm)
The above-mentioned pooled fractions were applied to the one equilibrated with 10 mM Tris-HCl buffer (pH 7.5). The elution from the column was monitored first by OD ₂₈₀ , 5 ml of NaCl-free 10 mM Tris-HCl buffer (pH 7.5), then 0.0
5 ml each of 10 mM Tris-HCl buffer containing 5 M and 0.1 M respectively, and finally 10 mM Tris-HCl buffer containing 0.5 M NaCl (pH 7.
It was eluted in 5). Fractions of 2.5 ml were collected. The results are shown in FIG. The OD ₂₈₀ value was measured by diluting 10 times. The eluate was pooled in two fractions, and the cytotoxic activity was examined for each (culture for 4 days). The results are shown in FIG. 4 (in the figure, the solid line shows the TNF-α non-added group, and the wavy line shows the TNF-α added group). As shown in FIG. 4, fraction number 4 (combined the 7th and 8th fractions in FIG. 3), that is, 0.05M to 0.1M NaCl
A peak of cytotoxic activity was observed in the fraction eluted with
It was shown that this factor contained this factor.

The various properties of this factor are shown below. The fraction number 4 in FIG. 4 (hereinafter referred to as "protein solution containing this factor") was used as a sample. The tumor cytotoxic activity was measured by the above-mentioned method for measuring cytotoxic activity on SK-BR3 cells in the presence of TNF-α (hereinafter referred to as “tumor cytotoxic activity”). In addition, cell growth inhibition and cell killing were judged from changes in the number of cells over time and the results of observation of cell morphology with a microscope.

(Molecular weight) As a molecular weight marker, albumin (molecular weight 67,000), ovalbumin (43,000),
The molecular weight of this factor was calculated by gel filtration method using chymotrypsinogen A (25,000) and ribonuclease A (13,700). As the gel, a column (φ60 × 300 mm) packed with Sephacryl S-300 HR and equilibrated with PBS of pH 7.33 was used, and a protein solution containing this factor and the above-mentioned molecular weight marker were mixed and applied. When this was eluted with PBS and the elution position of albumin was set to fraction number 7, the maximum value of the tumor cytotoxic activity was the fraction number.
It was eluted at 9 to 10 (Fig. 5). From the graph showing the relationship between molecular weight and elution position in Fig. 5, the molecular weight of this factor is about 50,0.
Calculated from 00 to about 55,000.

(Inactivation by digestion with trypsin) To the protein solution containing this factor, trypsin was added so that the final concentration was 50 μg / ml, and the mixture was incubated at 37 ° C. for 2 hours, and then trypsin inhibitor was added at a final concentration of 200 μg / ml. Tumor cell cytotoxic activity was measured after inactivation by adding as above.

[0025] A protein solution containing this factor, to which trypsin and a trypsin inhibitor (final concentration of 200 µg / ml) were simultaneously added, was incubated at 37 ° C for 2 hours, and then the tumor cytotoxic activity was measured. -The tumor cytotoxic activity against α was set to 100%, and the tumor cytotoxic activity was measured only by adding TNF.
The enhancing activity of α on tumor cell damaging activity was set to 0%. The results are shown below.

Untreated with trypsin 74% Treated with trypsin 31% Treated with trypsin and trypsin inhibitor 100%

As shown by these results, the addition of trypsin almost completely abolished the tumor cell-damaging effect on TNF-α, and the addition of trypsin inhibitor in addition to trypsin suppressed the inactivation of this factor. To be done. From this result, this factor is considered to be proteinaceous.

(Thermal stability) A protein solution containing this factor was
After incubating at ℃, 43 ℃, 50 ℃, 55 ℃, 63 ℃, 70 ℃ and 80 ℃ for 1 hour, the tumor cytotoxic activity was measured on the 3rd and 4th day after the addition of this factor. The enhancing effect on tumor cytotoxic activity against 100% of TNF-α was defined as the value obtained by subtracting the tumor cytotoxic activity due to TNF-α alone from the tumor cytotoxic activity due to TNF-α and this factor that was not heat-treated. .

This factor showed no inactivation at 4 to 63 ° C., about 80% at 70 ° C. and about 30% at 80 ° C.
The effect of enhancing the tumor cytotoxic activity of F-α remained. From this result, the temperature at which 50% of the tumor cell-damaging activity-enhancing activity was inactivated was determined by interpolation to be about 75 ° C.

(Comparison with other known cytokines against this factor) Regarding the three points of molecular weight, identification by ELISA and tumor cell damaging action, IL-1α, IL-1β, IL-2, IL-4, IL- 6, I
FN-α, IFN-β, IFN-γ, TNF-α, TNF-β and Fas-ligan
Comparing d with this factor showed that these cytokines differed from this factor by at least two points.

Molecular Weight Table 1 shows the comparison between the present factor and these cytokines in terms of molecular weight. In this table, the molecular weight of each cytokine is Pestka, S. and Langer, JA in Ann.
Rev. Biochem. (1987) 56 : 727-777, Cytokines-A Prac
tical Approach- Ed. Balkwill, FR (1991) IRL Pre
ss and Nagata, S. and Suda, T. Immunol. Today (199
5) 16 : 39-43.

[0032]

[Table 1]

From Table 1, IL-1α, IL-1β, IL-2, IL-4,
IL-6, IFN-α and Fas-ligand are the molecular weight of this factor (about 5
It is shown that it is different from 0,000 to about 55,000).

Identification by ELISA IFN-β, IFN-γ and TNF-α form a dimer or trimer, so that their molecular weights are close to those of this factor.
It may be 40,000 to 50,000. Therefore, in order to confirm that this factor is not one of these cytokines, IFN-β, IFN-γ and
When TNF-α was measured by ELISA, none of IFN-β, IFN-γ and TNF-α was detected.

Tumor Cytotoxicity The cytotoxicity of this factor and each cytokine on SK-BR3 cells was examined for cell growth inhibition or lethality when used alone or in combination with TNF-α or cisplatin. It was Table 2 shows the results.

[0036]

[Table 2]

As shown in Table 2, the cytotoxic effect on SK-BR3 cells was observed on SK-BR3 cells both when used alone and when used in combination with TNF-α or cisplatin. This factor is the only one that has a strong growth-suppressing or lethal effect and is factor concentration-dependent.

From the above comparison, the present factor is IL-1α, IL-1
β, IL-2, IL-4, IL-6, IFN-α, IFN-β, IFN-γ, TNF-
It was found to be different from all of α, TNF-β and Fas-ligand. Therefore, this factor is considered to be a novel factor having an action of enhancing the tumor cell-damaging activity of the anticancer drug.

(Test Example) The enhancing effect of this factor on the tumor cell-damaging effect of anti-cancer agents was examined for TNF-α, cisplatin and bleomycin.

The protein solution containing the present factor used in the following test examples was prepared by the method described in Example 1 unless otherwise specified, and in some tests, the culture supernatant was concentrated about 200 times. Prepared from the ones. That is, the latter has twice the concentration of this factor as compared with that prepared in Example 1 (hereinafter referred to as a 2-fold concentration solution).

The method for measuring the tumor cytotoxic activity was carried out according to the method for measuring the cytotoxic activity in the extraction and purification of the present factor in Example 1, and the number of viable cells was measured after addition of the sample.
I went on the 4th day.

Test Example 1 (Dependence of Tumor Cytotoxicity Enhancing Activity on Anticancer Agent Concentration of this Factor) To a medium containing a constant concentration of the anticancer agent, a protein solution containing this factor was added at various dilution ratios to obtain The effect of enhancing the tumor cell-damaging activity on the anticancer drug was examined. FIG. 6 shows the results. In this figure, the horizontal axis represents the dilution factor of the protein solution containing this factor, and the vertical axis represents the cell survival rate.

A. The final dilution ratio in the well of the protein solution containing TNF-α main factor was 3456
And added TNF-α at a final concentration of 80 ng / ml. In the figure, + TNF indicates addition of TNF-α, and −TNF indicates addition of TNF-α. TNF-α alone showed only a slight cytostatic activity, but showed almost no cell-killing effect. When this factor was added to this, the cytotoxic activity was enhanced depending on the concentration of this factor.

B. A protein solution containing cisplatin main factor was added at a final concentration of 540 to 20 in a well using a double concentration solution, and cisplatin was added at a final concentration of 0.4 μg / ml. In the figure, + cisplatin indicates addition of cisplatin, and −cisplatin indicates no addition. Cisplatin alone (0.4 μg / ml)
, But cell proliferation was suppressed, but there was almost no lethal effect. When this factor was added to this, the cytotoxic activity was enhanced in a concentration-dependent manner.

C. The final dilution ratio in the well of the protein solution containing bleomycin main factor was 3456.
Bleomycin to a final concentration of 25
μg / ml was added. In the figure, + bleomycin shows bleomycin addition and −bleomycin shows no addition. Bleomycin alone did not show any cytotoxic activity, but when this factor was added to it, it showed a growth inhibitory effect in a concentration-dependent manner.

Test Example 2 (Dependence of Tumor Cytotoxic Activity Enhancement on Anticancer Agent Concentration) Antitumor agents were added at various concentrations to a medium to which the present factor was added at a constant concentration, so that the antitumor activity of the present factor was enhanced by the antitumor agent. The dependence of anticancer drug concentration was investigated. FIG. 7 shows the results. In this figure, the horizontal axis represents the concentration of the anticancer drug, and the vertical axis represents the cell survival rate.

A. TNF-α This factor was added so that the final dilution ratio in the well was 54, and TNF-α was added in the range of 0 to 160 ng / ml. In the figure, + this factor indicates that this factor was added, and-this factor indicates that PBS was added (final dilution ratio 54). At this concentration, this factor alone has a weak cytostatic effect,
In addition, TNF-α also did not show cell-killing effect in this concentration range, but by adding this factor to TNF-α, it became to show cell-killing effect, and the enhancement of the effect was due to the increase of TNF-α concentration. It was dependent.

B. A protein solution containing cisplatin main factor was added using a 2-fold concentration solution so that the final dilution ratio in the well was 60, and the cisplatin concentration was added in the range of 0 to 12.5 μg / ml. In the figure, + this factor indicates that this factor was added, and-this factor indicates that PBS (final dilution ratio 60) was added. Cisplatin alone showed a cell-killing effect at 1.58 μg / ml or higher.
At 4 μg / ml, it was mainly a cytostatic effect. However, it was shown that the curve of the graph was shifted to the left by adding this factor. That is, it was revealed that the presence of this factor exhibits equivalent cytotoxic activity at a concentration of cisplatin of about 1/4 that of the case without the addition of this factor.

C. Bleomycin main factor was added so that the final dilution ratio in the well was 54, and the bleomycin concentration was added in the range of 0 to 250 μg / ml. In the figure, + this factor is this factor, -this factor is PBS
(Final dilution ratio 54) is added.
At this concentration, the factor alone exhibited a weak growth inhibitory effect alone, and bleomycin alone showed only a weak growth inhibitory effect in this concentration range, but by adding this factor to bleomycin, It showed cytotoxicity in a bleomycin concentration-dependent manner.

Test Example 3 (Tumor cell-selective cytotoxic activity of this factor) It was confirmed that this factor, in cooperation with an anti-cancer agent, selectively exhibits cytotoxic activity on tumor cells by using TNF-α as an anti-cancer agent. Examined.

For SK-BR3 cells, U937 (human lymphoid tumor cells), KB (human epithelial tumor cells), A549 (human lung tumor cells) and TM12 (human normal fibroblasts), 1 to 3 ×
10 ⁴ was spread on a 96-well plate and cultured overnight in a CO ₂ incubator. The next day, a protein solution containing this factor was added so that the final dilution ratio was 20. In addition, the final concentration of TNF-α
80ng / ml (SK-BR3), 5ng / ml (U937), 200ng / ml (A549
, KB) and 10 ng / ml (TM12) respectively, and cultured in a CO ₂ incubator. After 3 days (KB), 4 days (SK-BR3, U937 and A549) or 6 days (TM12), live cells were added. The number was measured.

As controls, a system containing only a protein solution containing this factor, a system containing only TNF-α, and TNF-α
The number of viable cells in the system in which neither α nor the protein solution containing this factor was added was measured. 100% cell survival rate is the number of viable cells in the system in which neither TNF-α nor protein solution containing this factor is added.
The results are shown in Fig. 8. Numbers 1 to 1 attached to the graph in this figure
4 is a system in which neither TNF-α nor a protein solution containing this factor is added, 2 is a system in which only a protein solution containing this factor is added,
3 shows the results of the system in which only TNF-α was added, and 4 represents the results of the system in which both TNF-α and the protein solution containing this factor were added.

As shown in FIG. 8, this factor shows that tumor cells (SK-BR3,
U937, KB, A549), it has a strong cytotoxic effect in cooperation with TNF-α. On the other hand, it showed no cytotoxic activity against normal human cells (TM12), either alone or in the presence of TNF-α. Furthermore, TNF-
It also did not inhibit the proliferation of normal human fibroblasts by α.

From the above results, it was shown that this factor, in cooperation with TNF-α, shows a cytotoxic activity selectively on tumor cells.

[0055]

EFFECTS OF THE INVENTION This factor can be used in combination with an anticancer drug having a strong tumor cell-damaging activity to reduce the effective amount of the anticancer drug. Therefore, it is expected to suppress various side activities caused by the administration of the anticancer drug. It In addition, even if it is an anticancer agent that does not have a tumor cell-killing effect by itself, in combination with this factor, because it causes a tumor cell-killing effect, an anticancer agent that is currently considered to be ineffective alone, It is expected to expand the indication in cancer treatment. Furthermore, as the concentration of this factor increases, it exhibits a tumor cell-damaging effect even in a concentration-dependent manner. Therefore, it is expected that this factor alone can be applied as an anticancer agent.

[Brief description of the drawings]

FIG. 1 Sephac was obtained by removing a fraction of a molecular weight of about 10,000 or less from a KB cell culture supernatant using an ultrafiltration membrane.
Apply to a ryl S-300 HR column to elute the protein by OD.
_It was detected by ₂₈₀ .

FIG. 2 shows the SK-BR3 cytotoxic activity of each fraction in FIG. 1 in the case of adding TNF-α and the case of not adding TNF-α.
It is shown as the value of OD ₅₉₅ .

FIG. 3 is obtained by applying Fraction No. 7 in FIG. 1 to an Econo-PacQ column and detecting protein elution by OD ₂₈₀ .

FIG. 4 shows the SK-BR3 cytotoxic activity of each fraction of FIG. 4 pooled into two fractions as OD ₅₉₅ values for TNF-α addition and TNF-α non-addition. It is a thing.

FIG. 5 shows the molecular weight of this factor measured from the elution position by gel filtration.

FIG. 6 is a graph showing the dependence of the effect of enhancing the tumor cell-damaging activity on the anticancer agent on the concentration of this factor.

FIG. 7 is a graph showing the dependence of the enhancement of tumor cell-damaging activity by this factor on the concentration of anticancer agent.

FIG. 8 shows the cytotoxic activity of this factor on various human tumor cells and human normal cells.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI Technical display location C12R 1:91)

Claims (3)

[Claims] 1. A protein factor derived from a culture supernatant of epithelial tumor cells, having a molecular weight of about 50,000 to about 55,000 as measured by gel filtration, and having an action of enhancing the tumor cell-damaging action of an anticancer agent. 2. The epithelial tumor cell is a KB cell.
Protein factors. 3. The proteinaceous factor according to claim 1 or 2, wherein the anticancer agent is selected from the group consisting of TNF-α, bleomycin and cisplatin.