JPH01187094A - Tumorous cell growth inhibitory factor - Google Patents

Abstract

PURPOSE:To contrive improvement in growth inhibitory action of a tumorous cell growth inhibitory factor useful for treating tumor, etc., on human pulmonary cancerous cells, etc., by specifying the molecular weight and stable pH region. CONSTITUTION:Human-derived pulmonary cancerous cells (e.g., PC 12) having the ability to produce a tumorous cell growth inhibitory factor are inoculated into an RPMI1640 culture medium containing 0.1-15wt.% calf blood serum and then cultivated at 37 + or -5 deg.C in the vapor phase of an atmosphere containing 7wt.% O2, 5wt.% CO2 and 88wt.% N2 under ordinary pressure for 1-15 days to provide a culture, which is then centrifuged to afford a supernatant liquid. The resultant supernatant liquid is subsequently concentrated by ultrafiltration, etc., fractionated and purified by affinity chromatography, etc., to collect the aimed tumorous cell growth inhibitory factor, having 52000 + or -5000 molecular weight and stable at pH7.0-9.0.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a tumor cell proliferation inhibitor useful as a medicine.

[Prior Art] Tumors progress due to abnormal proliferation of cells. We believe that if there is a substance that acts on tumor cells and suppresses their proliferation, it would be effective in tumor treatment. Among substances that suppress proliferation, synthetic chemical drugs generally have low specificity and have strong side effects. In contrast, cell growth inhibitory factors derived from tissue culture cells may have high specificity and are considered to be excellent therapeutic agents with few side effects. In particular, it would be useful if a factor that suppresses the growth of solid tumors, which are considered to be intractable, could be found.

[Problems to be Solved by the Invention] The present invention provides tissue culture cell-derived Ili useful for tumor treatment.
The purpose of the present invention is to provide an lXgs cell growth inhibitor.

[Means for Solving the Problems] The present invention is a tumor cell proliferation inhibitor that has a molecular weight of 52,000±5.000 and is stable at p) l'7.0 to 9.0.

The tumor cell growth suppressor of the present invention has the property of exhibiting strong growth suppressive activity against human tumor cells, particularly human lung cancer cells.

The tumor cell growth inhibitory factor of the present invention can be produced by culturing cells that produce the factor and collecting and separating it from the culture supernatant.

The producing cells may be of any kind as long as they have the ability to produce the tumor cell growth inhibitory factor of the present invention.

Preferably, human-derived lung cancer cells PC-12 (Ni1nj
o H, et al, Br, J, Cancer
, 39°15-23. (1979)). The production medium is not particularly limited, but PC-12
For cells, RPM11640 is preferred.

Serum is fetal bovine serum or calf serum at 0. 1-15%
Use with. Furthermore, a serum-free medium may be used if necessary.

PC-12 cells are usually grown at 37°C + 0.5°C at normal pressure. The culture is carried out at 5°C, and the culturing time is about 1 to 15 days, preferably about 3 to 7 days. The gas phase used for culture is PC-
Although any material that allows the growth of 12 cells may be used, a composition of 7% oxygen, 5% carbon dioxide, and 88% nitrogen is particularly preferred. When culturing, in addition to flat culture, beads may be used and aeration and rotation may be used.

When culturing PC-12 cells, the cells may be planted and cultured in a serum-free medium from the beginning. However, when cells are cultured in a medium containing mammalian serum (e.g., fetal bovine serum, calf serum, human serum, horse serum) and reach subconfluence, cells can be collected using known methods such as decantation and centrifugation. It is desirable to culture in a serum-free medium.

After culturing PC-12 cells, a culture supernatant can be obtained using known means such as centrifugation.

Culture supernatants can be concentrated by ultrafiltration. The concentrate is
Fractional purification can be performed using known methods such as gel filtration, affinity chromatography, and ion exchange chromatography.

The tumor cell growth inhibitory factor of the present invention obtained by the above method has a molecular weight of 5.2000±5,000 and a pH of 7.0 to 7.0.
It is stable at 9.0 and exhibits strong growth inhibition, especially against human lung cancer cells.

Tumor cell proliferation inhibitory activity can be measured by known methods. For example, there is a method in which cells are stained with crystal violet, the dye is extracted, and proliferation is determined using the concentration as an index.

When the tumor cell proliferation inhibitor of the present invention is used as a pharmaceutical, it may be used as a powder as it is, or in combination with other pharmacologically acceptable carriers, excipients, and diluents in a pharmaceutical composition (e.g., injection, It can be safely administered parenterally or orally to warm-blooded animals (eg, humans) in the form of tablets, capsules, liquids, ointments).

Injection preparations are carried out according to conventional methods using, for example, physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as tablets and capsules can also be prepared according to conventional methods.

[Examples] The present invention will be explained below with reference to Examples, but the present invention should not be limited thereto.

Example I Preparation and purification of serum-free culture supernatant of PC-12 cells: PC-12 cells were incubated in RPM11 containing 10% newborn calf serum.
640, and when the cultured cells reached subconfluence, the medium was removed, replaced with serum-free RPM11640, and cultured for 24 to 96 hours. After culturing, the serum-free culture supernatant was collected and concentrated approximately 450 times using an ultrafilter YM-5 manufactured by Amicon. This concentrated liquid was
ttrogel AcA 54 (2,5cm0X 1
Gel filtration was performed using a filter (10 cm). The active fractions were collected and passed through DEA E-cellutose (Seikagaku Corporation) (2.8cmJ2rX20cm) equilibrated with 20mM phosphate buffer y-pH7.0.

After thorough washing with 100 mM phosphate buffer pH 7.0, the activity was eluted with varying concentrations of 0 to 0.5 M NaCl. The activity is about 0. It was eluted at a concentration of IMNaC1. The obtained sample had a C3o (dilution rate) of about 607 per mg of protein.

Example 2 Properties of crudely purified tumor cell growth inhibitory factor: The molecular weight of the tumor cell growth inhibitory factor obtained in Example 1 was determined by gel filtration method and LKB Ultrogel AcA 54 (
Measurement was made using a 2.5 cm x 110 cm). The standard proteins used were albumin (66K), ovalbumin (45K), and soybean trypsin inhibitor (21K).
K). A calibration curve was determined at the position of each protein eluted at 15.6 ml/hr. The elution position of the tumor cell growth inhibitory factor was detected based on its activity, and the molecular weight was determined from the previously prepared standard curve. The results are shown in Figure 1. Molecular weight is 52,000±5
,000.

1M acetic acid (pH 2,5), 100mM acetate buffer (pH 4,0), 100mM phosphate buffer (pH
7,0) and 100mM Tris-HCl buffer (pH
9,0) for 16 hours to check the stability of the activity. 1M
Acetic acid (pH 2,5), 100mM acetate buffer (p
H4,0), it was completely inactivated. On the other hand, 100mM phosphate buffer, 100mM Tris-HCl buffer (pH
9,0), no decrease in activity was observed.

Tumor cell growth inhibitory factor (protein concentration 82μg/ml
), DNase (1 mg/ml), RNase (1
tag/ml), Trypsin (2,5+sg/m
1) was applied at 37°C for 15 hours. DNase, R
No inactivation was observed with Nase, but with Trypsi.
It was inactivated by n.

Example 3 Suppression of growth of human lung cancer cells by tumor cell growth suppressor: The tumor cell growth suppressor obtained in Example 1 was added to PC-1
2 cells and allowed to grow. 5×10 2 cells per well and 50 μg of RPM11640 containing 10% newborn calf serum were added to a 96-well microplate. moreover,
50 μg of a sample containing tumor cell growth inhibitory factor was added. 37 in a gas phase of 7%02.5%C02, 88%N2
Culture was carried out at 60°C. After culturing, cells were stained with crystal violet, and 100 μg of 70% ethanol/ethylene glycol/citric acid mixture was added per well.
was added to extract the pigment. Measure the rescue at 590nm,
Comparison was made with a group without growth inhibitory factors.

The results are shown in Figure 2. The influence of dilution of the sample was expressed as the growth inhibition rate when the non-additive area was set as 0%.

Example 4 Effect of tumor cell growth suppressor on various cell growth: Culture supernatant concentrate obtained in Example 1 (protein concentration 0)
．． 45 mg/ml) was added to HMV-1 [Human Melanoma, Goi Sakamoto et al.; Cancer Clinic 17.278-283 (1971
)], MKN-1 (human gastric cancer, Haruto Hojo; Journal of the Niigata Medical Society, 737 (1977)), and PC-
12 cells and allowed to proliferate. The method was carried out in accordance with Example 3, except that only the type of cells to be tested was changed. The results are shown in Table 1.

Inhibition was expressed as the IC5° value of the dilution rate.

Table 1 [Effects of the Invention] The tumor cell growth suppressor of the present invention suppresses the growth of various cancer cells. It has a particularly strong growth-inhibiting effect on human lung cancer cells. Therefore, it is expected to be effective in treating tumors, especially solid tumors. In addition, it is useful as a therapeutic agent for diseases caused by abnormal cell proliferation, such as chronic rheumatoid arthritis, arteriosclerosis, and warts.

[Brief explanation of the drawing]

FIG. 1 shows the results of measuring the molecular weight of the tumor cell proliferation inhibitor of the present invention by gel filtration. FIG. 2 shows the growth-suppressing activity of the tumor cell growth-suppressing factor of the present invention. Patent applicant Toshi Co., Ltd. Figure 1 Protein amount (μg/ml) Figure Z

Claims (1)

[Claims] (1) Molecular weight is 52,000±5,000 and pH
A stable tumor cell growth inhibitor between 7.0 and 9.0.