CN111603472A

CN111603472A - Application of tin-mesoporphyrin and artemisinin in preparation of antitumor drugs in combined manner

Info

Publication number: CN111603472A
Application number: CN202010519985.4A
Authority: CN
Inventors: 苏畅; 曹奕鸥; 肖立俊; 张淳
Original assignee: Central Hospital Of Minhang District Shanghai
Current assignee: Central Hospital Of Minhang District Shanghai
Priority date: 2020-06-09
Filing date: 2020-06-09
Publication date: 2020-09-01

Abstract

The invention discloses an application of tin-mesoporphyrin and artemisinin in preparation of antitumor drugs in a combined manner. The tin-mesoporphyrin obviously inhibits the expression of heme oxygenase-1 and increases the apoptosis induction effect of artemisinin on tumor cells. The invention utilizes the effect of tin-mesoporphyrin on inhibiting the activity of heme oxygenase-1 of tumor cells, reduces the metabolism of heme in the tumor cells, improves the apoptosis induction effect of artemisinin on the tumor cells, and improves the anti-tumor effect of artemisinin. The invention has very obvious anti-tumor effect by combining tin-mesoporphyrin and artemisinin and has wide application prospect.

Description

Application of tin-mesoporphyrin and artemisinin in preparation of antitumor drugs in combined manner

Technical Field

The invention belongs to the field of antitumor drugs, and particularly relates to an application of tin-mesoporphyrin and artemisinin in preparation of an antitumor drug.

Background

The artemisinin and the derivatives thereof have good effects of inhibiting the proliferation of tumor cells of lung cancer, prostatic cancer, cervical cancer and the like and promoting the apoptosis of the tumor cells, have extremely low toxicity to normal tissue cells, and have good application prospect in the aspect of anticancer treatment. However, artemisinin drugs do not inhibit all types of tumor cells well, and even the sensitivity of artemisinin varies widely between different cell lines of tumors at the same site. The direct target of the antitumor effect of the artemisinin is intracellular heme, the intracellular heme can mediate the cytotoxicity of the artemisinin, the increase of a precursor substance for the synthesis of the heme can enhance the effect of the artemisinin, and the acceleration of the metabolism of the intracellular heme also obviously weakens the cytotoxic effect of the artemisinin. Heme oxygenase-1 is an antioxidant defense enzyme widely present in mammals and is an important rate-limiting enzyme for degrading heme. The expression level of heme oxygenase-1 is increased in a plurality of tumor tissues (gastric cancer, lung cancer, breast cancer, rectal cancer and the like) and is abnormally high expressed. The high expression of heme oxygenase-1 accelerates the metabolism of heme in tumor cells, and inhibits the anticancer effect of artemisinin. The tin-mesoporphyrin has a structure similar to heme, can be combined with heme oxygenase-1, but is not degraded per se, so that the combination of heme oxygenase-1 and heme can be reduced, and the tin-mesoporphyrin can inhibit the activity of heme oxygenase-1, and is clinically used for treating hyperbilirubinemia, hematoporphyria and infantile glucose-6-phosphate dehydrogenase deficiency of newborns.

Disclosure of Invention

The invention aims to solve the technical problems and provides the application of the combination of tin-mesoporphyrin and artemisinin in preparing the antitumor drugs.

The invention is realized by the following technical scheme:

an application of tin-mesoporphyrin and artemisinin in preparing antineoplastic medicines is disclosed.

Furthermore, the tin-mesoporphyrin obviously inhibits the expression of heme oxygenase-1 and increases the apoptosis induction effect of artemisinin on tumor cells.

A pharmaceutical composition comprises tin-mesoporphyrin and artemisinin.

Furthermore, the pharmaceutical composition obviously inhibits the expression of heme oxygenase-1 and increases the apoptosis induction effect of artemisinin on tumor cells.

The invention has the beneficial effects that:

the invention utilizes the effect of tin-mesoporphyrin on inhibiting the activity of heme oxygenase-1 of tumor cells, reduces the metabolism of heme in the tumor cells, improves the apoptosis induction effect of artemisinin on the tumor cells, and improves the anti-tumor effect of artemisinin.

Drawings

FIG. 1 is a graph comparing the heme content of tumor cells with that of normal tissues, which highly express heme oxygenase-1, according to the present invention;

FIG. 2 is a graph showing the results of tin-mesoporphyrin of the present invention significantly inhibiting heme oxygenase-1 expression and increasing artemisinin induced apoptosis in tumor cells;

FIG. 3 is a schematic diagram of the action of tin-mesoporphyrin according to the present invention.

Detailed Description

In order to make the technical solutions of the present invention better understood by those skilled in the art, the technical solutions of the present invention are further described below by way of specific embodiments with reference to the drawings of the specification.

Example 1

Collecting colon cancer tissue and normal colon tissue specimen above 5CM beside cancer, and performing no other treatment before operation. And (3) quickly immersing a part of the sample into liquid nitrogen, preserving the part in a refrigerator at minus 80 ℃ for the detection of the content of the heme, fixing the part by using formaldehyde, embedding the part by using conventional paraffin, and preparing a 4-micron section.

Immunohistochemical staining of HO-1: HO-1 rabbit anti-human polyclonal antibody, according to 1: 200 diluted to be used as a primary antibody; an immunohistochemical S-ABC method is adopted, and the dyeing step is strictly carried out according to the instruction of a DAB color development kit. The known positive reaction plate is used as a positive control, and rabbit anti-irrelevant serum is used as a negative control to replace primary antibody. Observing the stained tissue section with a microscope under a microscope, and carrying out quantitative evaluation according to staining intensity: the light yellow is 1 point, namely weak positive; brown yellow is 2 points, namely positive;the brown color is 3 points, i.e. strong positive. Then, organizing each case under a high power lens (400x) to randomly take 4 visual fields, and the proportion of visual field cells to the visual field cells is scored according to the following standard: positive expression rate<1% is 0min, 2% -24% is 1 min, 25% -49% is 2 min, 50% -74% is 3 min, and not less than 75% is 4 min. Product of staining intensity and percentage of positive cells>2 is divided into positive (+) for immunohistochemistry, otherwise is counted as negative (-) for immunohistochemistry.

And (3) detecting the concentration of heme and porphyrin of the tumor cells by a fluorescence method: selecting a tissue specimen by an experienced doctor, cleaning, removing fat and necrotic tissues, and finely cutting into paste under an aseptic condition; digesting the cells for 0.5 to 1.5 hours by a cell dispersing agent at 37 ℃ in a shaking table, filtering the cells by a 308 mu m filter screen and collecting the cells (1 x 10)⁵) Centrifuging at 4 ℃ at 12000 r/min for 5min to obtain cell precipitate, washing with water, and adding into 1.5 ml amber centrifuge tube: adding 500 mu L of 20mmol/L oxalic acid into centrifuge tubes, standing overnight at 4 ℃ in a dark room for 16h, adding 500 mu L of 2mmol/L oxalic acid into each centrifuge tube, taking a half sample (for detecting porphyrin concentration) to react at normal temperature to serve as an experimental control group, and heating the other half sample (for detecting total concentration of porphyrin and heme) in a water bath at 95 ℃ for 30 mim; naturally cooling, centrifuging at 12000 r/min for 5min, placing 200 μ l in black 96-well plate, and performing fluorescence position detection (excitation wavelength is 400nm, emission wavelength is 620 nm), wherein the difference between the two groups is the concentration of hemoglobin to be detected.

Spearman grade correlation analysis the correlation between the HO-1 expression and the heme content of colon cancer tumor tissues.

The experimental results show (fig. 1): colorectal cancer tumor cells highly express heme oxygenase-1, and the content of heme is obviously reduced compared with normal tissues.

Example 2

The human colon cancer cell line Lovo cells in the logarithmic growth phase are inoculated in a 96-well culture plate, and the number of the cells in each well is 2x10³Cultured for 12h and then divided intoInduced (CoPP), blocked (SnMP) and control (control). Co-culturing the inducing group and a HO-1 specific inducer with the final concentration of 50mmol/L cobalt porphyrin CoPP for 36 h; co-culturing the blocking group with 50mmol/L CoPP and 50mmol/LHO-1 specificity blocker tin-mesoporphyrin (SnMP) for 36 h; the control group was not treated specifically.

Centrifuge and remove media, resuspend with fresh media, at 5 × 10³One/well was seeded in 96-well plates. And after 24h of culture, removing the culture medium, adding 20 mu l of newly configured serum-free culture medium containing 5mg/ml MTT into each well, incubating for 4h at 37 ℃, centrifuging for 15min at 3000 r/min, removing supernatant, adding 200 mu 1 DMSO, slightly oscillating for 10min, and measuring the OD value by using an enzyme-labeling instrument under the conditions of 570nm detection wavelength and no reference wavelength.

Adjusting cell density to 10⁶And/ml, taking 100 mu l of cell suspension, washing with cold PBS for 2 times, adding Annexin V-PE and 7-AAD according to the specification of an Annexin V-PE/7-AAD apoptosis detection kit, setting three multiple holes and a PBS negative control hole, detecting the cell scattering distribution condition by a flow cytometer, and quantitatively analyzing the apoptosis.

The experimental results show (fig. 2): tin-mesoporphyrin (SnMP) is added into the tumor cell culture environment, so that the expression of heme oxygenase-1 (HO-1) can be obviously inhibited, and the apoptosis induction effect of artemisinin on tumor cells is increased; on the contrary, the addition of cobalt porphyrin (CoPP) can increase the expression of heme oxygenase-1 of tumor cells and weaken the apoptosis induction effect of artemisinin on the tumor cells.

The applicant declares that the above description is only a specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and it should be understood by those skilled in the art that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are within the scope and disclosure of the present invention.

Claims

1. An application of tin-mesoporphyrin and artemisinin in preparing antineoplastic medicines is disclosed.

2. The use of a tin-mesoporphyrin in combination with artemisinin for the preparation of an antitumor medicament according to claim 1, wherein: the tin-mesoporphyrin obviously inhibits the expression of heme oxygenase-1 and increases the apoptosis induction effect of artemisinin on tumor cells.

3. A pharmaceutical composition characterized by: including tin-mesoporphyrin and artemisinin.

4. A pharmaceutical composition according to claim 3, wherein: the pharmaceutical composition obviously inhibits the expression of heme oxygenase-1 and increases the apoptosis induction effect of artemisinin on tumor cells.