CN117653632A - Application of tetrandrine as mitochondrion SIRT5 targeted inhibitor in preparation of antitumor drugs - Google Patents

Abstract

The invention belongs to the technical field of disease treatment medicines, and particularly relates to application of a traditional Chinese medicine component tetrandrine serving as a mitochondrion SIRT5 targeted inhibitor in preparation of an anti-tumor medicine. Experiments prove that the tetrandrine is a natural compound small molecule of direct targeting SIRT5, and can induce the autophagy of cells after the tetrandrine is combined with the SIRT5 in the cells, and can be used as an autophagy blocking agent after the autophagy occurs, so that the way of autophagy degradation is cut off, and the autophagy blocking of cancer cells is induced, thereby inhibiting the progress of tumors. Therefore, the tumors regulated by SIRT5 can be treated by adopting tetrandrine as an inhibiting drug, and particularly has good inhibiting effect on melanoma, gastric cancer, cervical cancer and the like.

Description

Application of tetrandrine as mitochondrion SIRT5 targeted inhibitor in preparation of antitumor drugs

Technical Field

The invention belongs to the technical field of disease treatment medicines, and particularly relates to application of a traditional Chinese medicine component tetrandrine serving as a mitochondrion SIRT5 targeted inhibitor in preparation of an anti-tumor medicine.

Background

Sirtuins belong to the widely expressed and evolutionarily conserved NAD (+) dependent deacylase/single ADP ribosyltransferase family, regulating a variety of cellular and body functions including metabolism, energy production, detoxification and oxidative stress. In mammals, there are seven Sirtuins members, sirt1-7, that are involved in tumor development as tumor suppressors or oncogenic factors. SIRT5 is a miracle and poorly characterized sirtuins protein. SIRT5 lacks powerful deacetylase activity and has the primary function of removing succinic, malonic and glutaraldehyde modifications from target proteins, mitochondria and whole cell lysines, thereby regulating multiple metabolic pathways, affecting a variety of proteins involved in cellular metabolism and other processes. SIRT5 may also be involved in the process of tumor development; research shows that SIRT5 can regulate the activity of K280 of SHMT2, so as to strengthen serine metabolism of tumor cell and promote proliferation of tumor cell; additional studies have shown that SIRT5 can promote survival of melanoma cells through chromatin modification, is essential for proliferation and survival of cutaneous melanoma as well as uveal melanoma, while SIRT 5-dependent genes include MITF and c-MYC, with MITF proved to be a critical lineage-specific survival oncogene in melanoma. Thus, SIRT5 may be a key target for malignancy treatment.

The traditional Chinese medicine is an attractive topic in the medical field for treating tumors. The traditional Chinese medicine monomer has the advantages of clear molecular formula, clear action mechanism and the like, and gradually becomes a new pet for treating various diseases. The tetrandrine is a natural product separated and purified from tetrandrine of Fangji family, and has antiallergic, antiinflammatory, and immunity regulating effects. However, there is no report on what kind of connection exists between tetrandrine and mitochondrial SIRT5, and the specific mechanism of tetrandrine in anti-tumor is not clear.

Disclosure of Invention

First, the technical problem to be solved

The invention provides an application of tetrandrine as a mitochondrion SIRT5 targeted inhibitor in preparing an anti-tumor drug, which clearly discloses an internal connection of the tetrandrine as the mitochondrion SIRT5 targeted inhibitor, and provides a theoretical basis for the anti-tumor effect of the tetrandrine, so that the tetrandrine can be popularized from the effects of anti-sensitization, anti-inflammation, organism immunity regulation and the like to the treatment of any malignant tumor related to the expression of mitochondrion SIRT5, and the application range of the tetrandrine is widened.

(II) technical scheme

The technical scheme of the invention comprises the following steps:

the invention provides an application of tetrandrine serving as a mitochondrion SIRT5 targeted inhibitor in preparing antitumor drugs.

Preferably, tetrandrine binds directly to mitochondrial SIRT5 protein to inhibit its expression, induce the development of cancer cell blocking autophagy, and thereby inhibit the progression of malignancy.

Preferably, the antitumor drug takes tetrandrine as the only effective component or is combined with other antitumor drugs.

Preferably, the tumor includes, but is not limited to, melanoma, gastric cancer, or cervical cancer. The tumour may be any tumour regulated by mitochondrial SIRT5 expression.

(III) beneficial effects

The invention provides an application of tetrandrine as a direct targeting SIRT5 inhibitor in preparing antitumor drugs. Experiments prove that tetrandrine can be directly combined with mitochondrial SIRT5 protein to inhibit the expression of the tetrandrine and induce the occurrence of autophagy; meanwhile, tetrandrine can be used as an autophagy blocking agent after autophagy occurs, so that the way of autophagy degradation is cut off, and autophagy blocking of cancer cells is induced, thereby inhibiting tumor progression. Therefore, tetrandrine can be used as an effective ingredient for treating malignant tumors related to mitochondrial SIRT5 expression.

Drawings

FIG. 1 shows the detection of the binding of tetrandrine to SIRT5 protein (a: biotin Pull Down test; b: cell thermal shift detection; c: isothermal titration calorimetry detection).

FIG. 2 is a graph showing the effect of tetrandrine on SIRT5 protein expression.

FIG. 3 shows the detection of the drug effect (a: tetrandrine structural formula; b: morphological analysis of added 10. Mu.M tetrandrine in melanoma cells A375 and MV 3).

FIG. 4 shows autophagy (a: electron microscopy of 10. Mu.M of tetrandrine-treated melanoma cells after 48 hours; b: western Blot autophagy; c: immunofluorescence autophagy flow)

FIG. 5 is an in vivo inhibition of melanoma by SIRT 5-overexpressing reversible tetrandrine.

FIG. 6 shows the inhibitory effect of tetrandrine on gastric cancer (SGC) and cervical cancer (Hela) in vitro and in vivo.

Detailed Description

The invention will be better explained by the following detailed description of the embodiments with reference to the drawings.

Example 1

This example demonstrates that tetrandrine TET can target SIRT5 protein in captured cells.

The experimental method is as follows:

(1) The biotin probe-labeled tetrandrine (supplied by Shanghai Huaying Co.) was purchased.

(2) Preparation of cell lysate: the grown A375 or MV3 cells in a 10cm dish were scraped off using the cells and added to a 5mL centrifuge tube and centrifuged at 800rpm for 5min on a centrifuge at 4℃and washed with pre-chilled PBS, the cells were transferred to a 1.5mL centrifuge tube and centrifuged again at 800rpm for 5min. After centrifugation, PBS was removed, 500. Mu.L of IP lysate containing protease inhibitors was added for lysis, and after 30min, centrifugation was performed at 12000rmp for 10min at 4℃to remove the pellet to obtain a cell lysate.

(3) According to the principle that Streptavidin (strepitavidin) can bind to Biotin. The biotin probe-labeled tetrandrine was mixed with cell lysate, and DMSO without TET was used as a blank in the experiment. As shown in a of fig. 1: streptavidin (strepavidin) was able to capture the Biotin-labeled TET, but only the lanes to which cell lysates were added detected a protein band and identified by mass spectrometry as SIRT5. The Pull-down experiment result shows that tetrandrine has interaction with SIRT5.

Furthermore, figure 1 b shows that performing a cell thermal shift assay experiment determines the effect of tetrandrine on SIRT5 stability, as shown by the lower and lower relative band intensity signal of SIRT5 in cells of tetrandrine TET treated group as the temperature increases from 46 ℃ to 60 ℃; as also shown in FIG. 1 c, the change in enthalpy of SIRT5 was inversely proportional to the molar ratio of tetrandrine as demonstrated using isothermal titration calorimetry, and the binding coefficient between the two was modeled by using software with a KD of 24.3e-6+763e-9. This suggests that SIRT5 has a strong interaction with tetrandrine TET.

Example 2

This example demonstrates the effect of tetrandrine on expression of melanoma cell SIRT5 protein. The experimental method is as follows: taking human melanoma cells A375 and MV3 in logarithmic growth phase, inoculating into 96-well culture plate with cell density of 1×10 ⁴ Cells/well, 100 μl per well. Placing the 96-well plate at 37deg.C and 5% CO ₂ The culture was carried out in an incubator for 12 hours, and then the original medium was discarded, and tetrandrine solution (dissolved in DMSO) was added to each group at concentrations of 5. Mu.M, 10. Mu.M, and 15. Mu.M, respectively, to add an equal volume of MDSO group as a blank.

Preparing the cell lysate of the human melanoma cells A375 and MV 3: cells were collected with a cell scraper and then lysed with RIPA lysis buffer (bi yun, shanghai china) for 20min, and 1mM PMSF (bi yun, shanghai china) was added. After centrifugation of the sample (14,000 rpm,4 ℃) for 15min, the supernatant was collected and denatured at 100℃for 30min. Standard curves were drawn using BCA protein assay kit to determine protein concentration. Expression of SIRT5 protein was detected by Western Blot assay. Meanwhile, TRIzol reagent is used for extracting total RNA of cells, and the expression quantity of SIRT5 mRNA is detected through RT-qPCR test.

The experimental results are shown in FIG. 2. Experimental results show that tetrandrine inhibits the expression of SIRT5 protein in melanoma cells A375 and MV3, and the inhibition effect has a certain concentration dependence (as shown in a of figure 2). At the same time, see FIG. 2 b, the mRNA level of SIRT5 is not decreased, but remains unchanged or slightly increased (especially in the 15. Mu.M treated group).

The experimental results show that: tetrandrine achieves the effect of inhibiting SIRT5 protein expression mainly by affecting the stability of melanoma cell SIRT5 protein.

Example 3

Since studies have shown that SIRT5 can promote melanoma cell survival through chromatin modification. The inventors therefore treated human melanoma cells a375 and MV3 in vitro with Tetrandrine (TET) to observe the cell morphology after human melanoma cell treatment.

The experimental method is as follows:

taking human melanoma cells A375 and MV3 in logarithmic growth phase, inoculating into 96-well culture plate with cell density of 1×10 ⁴ Cells/well, 100 μl per well. Placing the 96-well plate at 37deg.C and 5% CO ₂ The culture was carried out in an incubator for 12 hours, and then the original medium was discarded, and tetrandrine solution (dissolved in DMSO) was added to each group at concentrations of 5. Mu.M, 10. Mu.M, and 15. Mu.M, respectively, to add an equal volume of MDSO group as a blank. After 48 hours of treatment, the cell morphology was observed under an optical microscope at various multiples, and as shown in fig. 3 and fig. 4 a, it was found that cells of the tetrandrine-treated group had a large number of vacuoles generated, and autophagy was presumed to occur.

To further investigate the type of autophagy, immunofluorescence assays were performed on each of the above groups of cells. During the experiment, the melanoma cells after TET treatment were detected by using an autophagy double-standard system (GFP-RFP-LC 3B), nuclei were stained with DAPI, and combined for fluorescent microscopy, and the experimental results are shown in fig. 4 c: tetrandrine TET induced autophagy flow blocking in melanoma cell lines.

Preparing the cell lysate of the human melanoma cells A375 and MV 3: cells were collected with a cell scraper and then lysed with RIPA lysis buffer (bi yun, shanghai china) for 20min, and 1mM PMSF (bi yun, shanghai china) was added. After centrifugation of the sample (14,000 rpm,4 ℃) for 15min, the supernatant was collected and denatured at 100℃for 30min. Standard curves were drawn using BCA protein assay kit to determine protein concentration. The expression levels of the markers associated with autophagy (LC 3B, ULK1, P62, syncaxin 17) in each treatment group were continuously examined by Western Blot, and the experimental results are shown in fig. 4 b: at treatment concentrations of 5 μΜ, 10 μΜ, 15 μΜ of TET, the expression of the autophagy-related marker (LC 3B, ULK 1) in cells is significantly increased. At the same time, P62 expression was increased and Syntaxin17 expression was decreased, further suggesting autophagy tumor blocking.

These experimental results show that tetrandrine can induce blocking autophagy of melanoma cells.

Example 4

This example uses a melanoma mouse model to verify the effect of TET on the oncologic effect of melanoma cells that overexpress SIRT5. The experimental method is as follows:

(1) And (3) molding: taking human melanoma cell A375 in logarithmic growth phase and A375 over expressing SIRT5, culturing to cell density of 1×10 ⁶ 100 μl/mL was taken and injected into 8-week-old BALB/c-nu mice, and tumors were formed subcutaneously in the mice to obtain a common melanoma mouse model and a SIRT 5-overexpressing melanoma mouse model.

(2) The melanoma mice model was divided into 4 groups of 3 mice each, which were intraperitoneally injected 1 time every 2 days for 30 days as follows:

v+tet group: the ordinary mouse model is injected with TET solution (dissolved in DMSO) daily with the injection dosage of 120mg/Kg/day;

sirt5+tet group: mice models over-expressing SIRT5 were daily injected with TET solution (120 mg/Kg/day) in solution (in DMSO);

sirt5+dmso group: mice models that overexpressed SIRT5 were injected daily with DMSO (equal volume to the upper group);

v+dmso group: the normal mouse model was injected daily with DMSO (equal volume to the upper group).

(3) Tumors were excised subcutaneously from mice for the following assays.

The experimental results are shown in a and b of fig. 5, and the volume and weight of melanoma in the sirt5+dmso group are the greatest; this suggests that overexpression of SIRT5 may promote melanoma progression. Whereas the v+tet group had a decrease in melanoma volume and weight compared to the v+dmso group, indicating that TET inhibited melanoma progression. Meanwhile, compared with the single tetrandrine (V+TET) group, the tumor volume and the weight of the tetrandrine combined over-expression SIRT5 group (SIRT5+TET) group are increased, which shows that the over-expression SIRT5 can reverse the inhibition effect of the tetrandrine on melanoma in vivo to a certain extent.

Subsequently, levels of Ki-67 (characterizing tumor cell proliferation activity) and SIRT5 protein expression in xenograft tumors (e.g., c of FIG. 5) were examined by immunohistochemistry, wherein the number of Ki-67 and SIRT5 positive cells was greatest in the SIRT5+DMSO treated group and higher than the blank group of DMSO, while the number of Ki-67 and SIRT5 positive cells was least in the V+TET treated group, but the number of Ki-67 and SIRT5 positive cells was increased in the combination of tetrandrine over-expressing SIRT5 group (SIRT5+TET group). This also demonstrates that overexpression of SIRT5 reverses the inhibitory effect of TET on melanoma, consistent with previous experimental results.

Example 5

Based on the existing research conclusion, SIRT5 can regulate the activity of K280 of SHMT2, so that serine metabolic capacity of tumor cells is enhanced to promote proliferation of the tumor cells, and then the proliferation of the tumor cells can be inhibited by inhibiting SIRT5. The following experiments prove that TET can inhibit proliferation of gastric cancer SGC and cervical cancer cell Hela cells in vitro and in mice.

The experimental protocol was as follows:

(1) Taking gastric cancer cell SGC and cervical cancer cell Hela in logarithmic growth phase, and SGC cell and Hela cell over expressing SIRT5, inoculating into 96-well culture plate with cell density of 1×10 ⁴ Cells/well, 100 μl per well. Placing the 96-well plate at 37deg.C and 5% CO ₂ The culture was carried out in an incubator for 12 hours, and then the original medium was discarded, and each group was treated as follows:

DMSO group: respectively adding an equal volume of blank DMSO into gastric cancer cell SGC and cervical cancer cell Hela;

TET group: respectively adding 10 mM/well TET solution (dissolved in DMSO) into gastric cancer cell SGC and cervical cancer cell Hela;

SIRT5 group, adding equal volume blank DMSO into SGC cells and HeLa cells which over express SIRT 5;

sirt5+tet group: 10 mM/well TET solution (in DMSO) was added to SIRT 5-overexpressing SGC cells and HeLa cells.

OD values per well on day 0,1,3,5,7 of treatment were examined to characterize the change in cell number in each well. As shown in fig. 6 a: OD values for SIRT5 and DMSO groups continued to increase with increasing treatment days, and were always at maximum in all groups; the OD of the TET group was minimal and tended to be more stable or decreasing with increasing treatment days, whereas the cell OD of the sirt5+tet group was higher than that of the TET group and increased with increasing treatment days. Therefore, the TET can inhibit proliferation of gastric cancer cells and cervical cancer cells, and simultaneously, after SIRT5 is overexpressed, the inhibition effect of the TET on the gastric cancer cells and the cervical cancer cells is reversed.

(2) And (3) molding: culturing gastric cancer cell SGC and cervical cancer cell Hela in logarithmic growth phase to cell density of 1×10 ⁶ 100 mu L of 100-mL BALB/c-nu mice with the age of 8 weeks are taken and injected, and subcutaneous tumor formation is carried out on the mice, so as to obtain a gastric cancer mouse model and a cervical cancer mouse model.

(3) The gastric cancer mouse model and the cervical cancer mouse model were respectively divided into 2 groups of 3 mice each, and injected 1 time every 2 days for 30 days as follows:

TET group: the mice model was intraperitoneally injected daily with TET solution (dissolved in DMSO) at a dose of 120mg/Kg/day;

DMSO group: mice model were intraperitoneally injected daily with DMSO (equal volume to the upper group);

(4) Tumors were excised subcutaneously from mice for measurement volume and weighing.

As shown in fig. 6 b-e, the volume and weight of the stomach and cervical cancer tumors in the TET group mice were much smaller than in the DMSO group. Animal model experiments prove that tetrandrine TET has very good inhibition effect on gastric cancer and cervical cancer tumors in vivo.

In summary, the invention confirms that tetrandrine is a natural compound small molecule which directly targets SIRT5, when the tetrandrine is combined with SIRT5 in cells, the tetrandrine can induce autophagy of the cells, and simultaneously, the tetrandrine can be used as an autophagy blocking agent after autophagy occurs, so that the way of autophagosome degradation is cut off, and autophagy blocking of cancer cells is induced, thereby inhibiting the progress of tumors. Therefore, the tumors regulated by SIRT5 can be treated by adopting tetrandrine as an inhibiting drug, and particularly has good inhibiting effect on melanoma, gastric cancer, cervical cancer and the like.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Claims (5)

1. The tetrandrine is used as a mitochondrion SIRT5 targeted inhibitor in the preparation of antitumor drugs.

2. The use according to claim 1 wherein tetrandrine binds directly to mitochondrial SIRT5 protein to inhibit its expression, induces autophagy in cancer cells, and acts as an autophagy blocking agent after autophagy occurs, thereby inhibiting malignant progression.

3. The use according to claim 1, wherein the antitumor drug comprises tetrandrine as the only active ingredient or in combination with other antitumor drugs.

4. The use of claim 1, wherein the tumour is a tumour regulated by mitochondrial SIRT5 expression.

5. The use according to claim 4, wherein the tumor includes, but is not limited to, melanoma, gastric cancer or cervical cancer.