CN114504568A - Application of small molecule compound in preparing medicine for inducing immunogenic cell death - Google Patents

Abstract

The invention relates to the technical field of biological medicines, in particular to application of a small molecular compound in preparation of a medicine for inducing immunogenic cell death. The small molecular compound comprises piceatannol, the piceatannol activates autophagy through regulating TFEB/TFE3, further synergistically promotes oxaliplatin-induced immunogenic cell death, and generates a T cell reaction with systemic anti-tumor effect, so that the combined treatment is achieved for enhancing the anti-tumor treatment effect, the persistent anti-tumor immune response is favorably induced to reduce the possibility of relapse, and the small molecular compound is suitable for being widely applied to activating the potential treatment effect of TFEB/TFE3 in various diseases.

Description

Application of small molecule compound in preparing medicine for inducing immunogenic cell death

Technical Field

The invention relates to the technical field of biological medicines, in particular to application of a small molecular compound in preparation of a medicine for inducing immunogenic cell death.

Background

Some chemotherapy drugs can induce tumor cell death and simultaneously induce tumor cell immunogen, so that some molecules with immunogenicity are expressed on the surface of tumor cells to activate the anti-tumor immune response of the organism and induce cytotoxic T Cells (CTL) to kill the tumor cells more effectively, thereby achieving a durable and ideal anti-tumor effect, and the process is defined as Immunogenic Cell Death (ICD). Recently, it has been discovered that various chemotherapeutic drugs, such as anthracyclines (anthracyclines), Oxaliplatin (Oxaliplatin), Mitoxantrone (MTX), photodynamic therapy, radiotherapy, etc., can induce not only apoptosis of tumor cells, but also ICD. These clinically applied ICD inducers generally have better efficacy in tumor therapy than non-ICD inducers.

The key events of ICDs are tumor cell expression and release of damage-associated molecular patterns (DAMPs) that recruit antigen presenting cells, recognize and phagocytose associated cell antigens, and present them to T cells, thereby activating adaptive immune responses, recognizing and eliminating tumor antigens, and generating long-lasting anti-tumor effects. Therefore, compared with a medicine for simply killing tumors, the ICD inducer applied to tumor treatment has great significance for improving the prognosis of cancer patients and prolonging the life cycle of the patients. At present, it is generally considered that 3 marker events of immunogenic death of tumor cells are respectively the release of ATP (adenosine triphosphate) in tumor cells to the outside of cells; endoplasmic reticulum stress-associated chaperones such as Calreticulin (CALR) translocate from the endoplasmic reticulum to the surface of cell membranes; and high-mobility group box 1 (HMGB 1) is released extracellularly from the nucleus. In the absence of any of these conditions, it is difficult for chemotherapeutic drugs to induce immunogenic cell death. ATP, as an important trending factor, recruits various immune effector cells such as dendritic cells or immature dendritic cells to the vicinity of dead tumor cells and promotes differentiation of the immature dendritic cells into mature dendritic cells, thereby effectively performing an antigen presenting function. Further, ATP released from tumor cells binds to a purine receptor P2Y2 on the surface of monocytes, thereby recruiting monocytes to apoptotic tumor cell sites and exerting effective antigen presentation. CALR is an endoplasmic reticulum protein, which plays an important role in quality control of proteins and cell proliferation. The route of CALR translocation from the endoplasmic reticulum to the cell surface during ICD depends on endoplasmic reticulum stress, in particular PERK (also known as EIF2AK 3) mediated phosphorylation of eukaryotic initiation factor 2 α (EIF 2 α). HMGB1 is a cell-localized chromosome-binding protein that is released from the nucleus to the cytoplasm during ICD. HMGB1 is a cytokine associated with inflammatory responses that aggregates multiple receptors, such as Toll-like receptor 4 (TLR 4) and TLR 2. The HMGB1 released outside the cell can be combined with TLR4 on dendritic cells to promote the maturation of the dendritic cells through a MYD88 pathway and present tumor antigens to CTL so as to play a role in killing tumors.

Autophagy is a conserved signal pathway in cells, and is a main mode for lysosome-mediated degradation of macromolecules such as long half-life proteins and protein aggregates in cells and damaged organelles. Transcription factors EB (Transcription factor EB, TFEB) and E3 (Transcription factor E3, TFE 3) are key molecules of autophagy, which activate a variety of genes that regulate autophagy at the transcriptional level. Activation of TFEB/TFE3 is therefore an effective way to activate autophagy. In addition, there is a variety of evidence that autophagy has a key role in cancer. For example, when autophagy is activated, cytoplasmic components of tumor origin are available for lysosomal hydrolysis, thereby facilitating antigen processing in dying tumor cells. In addition, highly activated autophagy can increase ATP secretion by stressed tumor cells, which acts as an important signal to enhance the effect of ICDs by acting on purine receptors to activate dendritic cells and killer T cells and recruit them to the local tumor microenvironment.

However, there is evidence that many chemotherapeutic drugs such as Oxaliplatin (OXA) and the like induce ICD, but their effects are relatively weak, so it is very urgent to find new small molecule compounds and enhance the action of ICD inducer, thereby exerting anticancer function.

Disclosure of Invention

The application aims to solve the technical problems that in the prior art, when part of chemotherapeutic drugs such as Oxaliplatin (OXA) are used for treating cancers, the induced immune response is weak, the anti-cancer effect is not obvious, and the treatment effect is poor, and provides the application of the small molecular compound in preparing the drug for inducing immunogenic cell death.

In a first aspect, the present application provides the use of a small molecule compound comprising piceatannol in the manufacture of a medicament for inducing immunogenic cell death.

In a second aspect, the application provides a pharmaceutical composition for inducing immunogenic cell death, comprising piceatannol and oxaliplatin, wherein the piceatannol is present in a concentration of 1-40 μ M.

The application of the small molecule compound provided by the first aspect of the application in preparing the medicine for inducing immunogenic cell death includes piceatannol, which activates autophagy of cells by controlling TFEB/TFE3, and further synergistically promotes immunogenic cell death induced by oxaliplatin, and generates T cell reaction with systemic anti-tumor effect, so as to enhance anti-tumor treatment effect, achieve combined treatment, facilitate induction of durable anti-cancer immune response, and reduce recurrence possibility, and the small molecule compound is suitable for being widely applied to activating potential treatment effect of TFEB/TFE3 in various diseases.

In the pharmaceutical composition for inducing immunogenic cell death provided by the second aspect of the application, the small molecular compounds of piceatannol and oxaliplatin are included in the pharmaceutical composition, so that piceatannol and oxaliplatin act synergistically, TFEB/TFE3 is activated by piceatannol to induce autophagy of cells, and immunogenic cell death reaction induced by oxaliplatin is enhanced, so as to exert higher-effect therapeutic action.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

Wherein:

FIG. 1 is an analysis graph of piceatannol in conjunction with OXA-induced ATP release.

FIG. 2 is a graph of the cell membrane translocation analysis of piceatannol in combination with OXA-induced CALR.

FIG. 3 is a graph of a release analysis of piceatannol in combination with OXA induced HMGB 1.

FIG. 4 is a graph showing the nuclear transport analysis of piceatannol-promoted TFEB and TFE 3.

FIG. 5 is a graph of the analysis of piceatannol promoting autophagy.

FIG. 6 is an analysis chart showing that piceatannol promotes autophagy dependent on TFEB and TFE 3.

FIG. 7 is a graph of piceatannol in conjunction with OXA-induced ATP release dependent on TFEB/TFE3 mediated autophagy.

FIG. 8 is a graph showing the analysis of piceatannol on endoplasmic reticulum stress.

FIG. 9 is a graph of the endoplasmic reticulum stress assay of piceatannol-enhanced OXA-induced cell membrane translocation.

FIG. 10 is a graph of the analysis of piceatannol promoting autophagy, increasing endoplasmic reticulum stress and simultaneously coordinating with OXA-induced ICD in MCA205 osteosarcoma cells.

FIG. 11 is a graph showing the effect of piceatannol in increasing OXA-inhibited tumors in immunized normal mice.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

In a first aspect, the embodiments herein provide the use of a small molecule compound comprising piceatannol in the manufacture of a medicament for inducing immunogenic cell death.

The application of the small molecule compound provided by the first aspect of the embodiment of the application in preparing the medicine for inducing immunogenic cell death includes piceatannol, which activates autophagy of cells through controlling TFEB/TFE3, and further synergistically promotes immunogenic cell death induced by oxaliplatin, and generates T cell reaction with systemic anti-tumor effect, so as to enhance anti-tumor treatment effect, achieve combined treatment, facilitate induction of durable anti-cancer immune response to reduce recurrence possibility, and be widely applied to activating potential treatment effect of TFEB/TFE3 in various diseases.

In some embodiments, the piceatannol promotes nuclear translocation that activates transcription factor TFEB and transcription factor TFE 3.

In some embodiments, the piceatannol promotes autophagy by modulating nuclear translocation of TFEB/TFE 3-activated transcription factor TFEB and transcription factor TFE3, increasing the level of autophagy marker protein LC 3-II.

In some embodiments, the piceatannol increases expression of transcription factor 4 activated by a marker protein of endoplasmic reticulum stress, of transcription factor 6-C/EBP homologous protein, and increases phosphorylation level of eukaryotic initiation factor 2 of endoplasmic reticulum stress to promote endoplasmic reticulum stress.

In some embodiments, the medicament further comprises oxaliplatin. The provided oxaliplatin can induce immune response, and can enhance the synergistic effect of oxaliplatin on inducing immunogenic cell death to play an anti-tumor role through the synergistic effect with piceatannol.

In some embodiments, the piceatannol promotes extracellular ATP and HMGB1 release by the oxaliplatin.

In some embodiments, the piceatannol promotes oxaliplatin-induced membrane calreticulin membrane translocation and PERK-mediated endoplasmic reticulum stress.

In some embodiments, the cellular membrane translocation of CALR, release of HMGB1, and release of ATP are induced due to the synergistic effect of piceatannol with oxaliplatin, confirming that piceatannol induces promotion of immunogenic cell death in combination with oxaliplatin.

In some embodiments, the piceatannol induces promotion of immunogenic cell death in conjunction with the oxaliplatin.

In some embodiments, the piceatannol increases the anti-tumor effect of the oxaliplatin.

In a second aspect, the embodiments of the present application provide a pharmaceutical composition for inducing immunogenic cell death, the pharmaceutical composition includes piceatannol and oxaliplatin, wherein the piceatannol is at a concentration of 1-40 μ M.

According to the pharmaceutical composition for inducing immunogenic cell death provided by the second aspect of the embodiment of the application, the small molecule compounds of piceatannol and oxaliplatin are included in the pharmaceutical composition, so that the piceatannol and the oxaliplatin act synergistically, TFEB/TFE3 is activated by the piceatannol to induce autophagy of cells, and an immunogenic cell death reaction induced by the oxaliplatin is enhanced, so as to exert a higher therapeutic effect.

In some embodiments, a pharmaceutical composition for inducing immunogenic cell death comprises piceatannol and oxaliplatin, wherein the piceatannol is at a concentration including, but not limited to, 1 μ M, 5 μ M, 10 μ M, 15 μ M, 20 μ M, 25 μ M, 30 μ M, 35 μ M, 40 μ M.

In some embodiments, the pharmaceutical composition further comprises pharmaceutically acceptable adjuvants to facilitate its preparation into dosage forms that satisfy various routes of administration.

In some embodiments, the pharmaceutical formulation is one of a tablet, a pill, a capsule, a granule, a powder, a liquid, an emulsion, a suspension, an ointment, an injection, a skin patch.

In some embodiments, the auxiliary agent includes one or more of a filler, a disintegrant, a binder, an emulsifier, a lubricant, a glidant, a colorant, but is not limited thereto.

In some embodiments, the pharmaceutical composition is used to enhance anti-tumor effects based on promotion of immunogenic cell death, wherein tumors include, but are not limited to, malignant melanoma, liver cancer, lung cancer, breast cancer, colon cancer, nasopharyngeal cancer, bladder cancer, cervical cancer, esophageal cancer, gastric cancer, prostate cancer, and lymphoma.

The following description will be given with reference to specific examples.

Example 1

A pharmaceutical composition for inducing immunogenic cell death, comprising piceatannol and oxaliplatin, wherein the piceatannol is present at a concentration of 10 μ M.

Example 2

A pharmaceutical composition for inducing immunogenic cell death, comprising piceatannol and oxaliplatin, wherein the piceatannol is present at a concentration of 20 μ M.

Example 3

A pharmaceutical composition for inducing immunogenic cell death, comprising piceatannol and oxaliplatin, wherein the piceatannol is present at a concentration of 40 μ M.

Determination of Properties

1. Cell experiments prove that piceatannol enhances ICD induced by Oxaliplatin (OXA)

(1) Effect of piceatannol in combination with Oxaliplatin (OXA) on Adenosine Triphosphate (ATP) release

After treating the cells with 40. mu.M piceatannol, low concentration (200. mu.M) of OXA, piceatannol (40. mu.M) in combination with OXA (200. mu.M) and high concentration of OXA (200. mu.M) as a control for 24h in U2OS and MCA205 cells, the change in intracellular ATP content was detected. Intracellular ATP content was detected by staining cells by quinacrine (quinacrine) incubation for 30 min and then by fluorescence microscopy, and its extracellular release was reflected by a decrease in intracellular ATP.

(2) Detecting the influence of piceatannol cooperating with OXA on the release of calreticulin CALR and the mechanism thereof

After 48 hours of transfection of CALR-RFP plasmid into U2OS cells in U2OS and MCA205 cells, cells were treated with 40 μ M piceatannol, low concentration of OXA (200 μ M), piceatannol (40 μ M) in combination with low concentration of OXA (200 μ M), and high concentration of OXA (400 μ M) as controls for 24 hours, 4% paraformaldehyde was fixed and the location of CALR-RFP was finally examined by fluorescence microscopy.

Meanwhile, in U2OS, after the cells were treated in the same manner as above, the influence of piceatannol in cooperation with OXA on the translocation of endogenous CALR cell membranes was also examined by flow cytometry. After the treatment of the cells is finished, digesting the cells by using an enzyme-free digestive juice, collecting cell suspension, washing the cell suspension for 3 times by using PBS (phosphate buffer solution), staining the cells for 5 minutes by using PI (polyimide), and fixing the cells for 5 minutes by using 0.25% paraformaldehyde; blocking with 2% donkey serum for 30 min, adding 1: after 30 min incubation with cells, the CALR antibody was diluted 200, washed 3 times with PBS, and blocked at 1: FITC-488 conjugated secondary antibody was diluted 500 and incubated with cells for 30 min; after 3 times of PBS washing, the CALR translocation was detected on a flow cytometer.

(3) Testing the Effect of piceatannol in combination with OXA on the Release of HMGB1

After treating the cells with 40. mu.M piceatannol, low concentration of OXA (200. mu.M), low concentration piceatannol (40. mu.M), combined OXA (200. mu.M), and high concentration OXA (400. mu.M) as controls for 24h in U2OS and MCA205 cells, the cells were fixed with 4% paraformaldehyde and then labeled with an antibody against HMGB1 (1: 200) for fluorescent antibody localization, and HMGB1 localization was detected by fluorescence microscopy. Meanwhile, a cell culture medium is collected, and the content of the HMGB1 outside the cells is detected by using western blot so as to further react to release HMGB1 outside the cells.

2. Detection of piceatannol-activated TFEB/TFE 3-induced autophagy

After treating the cells with piceatannol (10. mu.M, 20. mu.M and 40. mu.M) at different concentrations for 24 hours by transfecting Flag-TFEB and GFP-TFE3 plasmids into U2OS cells for 48 hours, the entry of TFEB and TFE3 from the cytoplasm into the nucleus was detected by fluorescence microscopy, the cytoplasm and the nucleus were separated at the same time, and the contents of TFEB and TFE3 in the cytoplasm and the nucleus were detected by Western blot. It was further confirmed that it promotes nuclear transport of TFEB and TFE 3.

To determine whether piceatannol activated autophagy, U2OS cells were treated with varying concentrations of piceatannol (10 μ M, 20 μ M and 40 μ M) for 24 hours, the cells were harvested, proteins were extracted, and expression of the autophagy marker LC3-II was detected by Western blot using an antibody against LC 3. Meanwhile, after treating the cells with piceatannol (40. mu.M), Chloroquine (CQ) (50. mu.M), piceatannol (40. mu.M) and CQ (50. mu.M), intracellular proteins were extracted, and expression of LC3-II was detected by Western blot using an antibody against LC 3. CQ was an autophagy inhibitor used for control. It is expected that treatment of cells with piceatannol, in combination with CQ, will increase the induction of LC 3-II. The effect of small molecules on autophagy flow was then examined using the fluorescent reporter system mRFP-GFP-LC 3. When the probe is positioned in an autophagosomal or autophagosomal body, the probe will emit both red and green light. When lysosomes fuse with autophagosomes, resulting in a decrease in PH, resulting in quenching of the GFP signal, RFP, which is PH insensitive, will be detected as red fluorescence. Thus, the individual red dot-like structures represent autophagosomes. The mRFP-GFP-LC3 plasmid was also transfected into U2OS cells, and after treating the cells with piceatannol (40. mu.M) for 24 hours, the cells were fixed and examined by fluorescent microscopy.

To determine whether piceatannol-induced autophagy was dependent on TFEB and TFE3, piceatannol (40 μ M) was added to control and treated groups 24 hours after TFEB and TFE interference with siRNA 348 hours, and changes in LC3-II were detected using Western blot to determine whether piceatannol-induced autophagy was dependent on TFEB and TFE 3.

3. Determination of whether piceatannol in combination with OXA-induced ATP Release is dependent on TFEB/TFE 3-mediated autophagy

In U2OS cells, after knocking down the autophagy key protein ATG5 (autophagy-related protein 5) with siRNA or knocking down TFEB and TFE 348 hours, and then adding 40 μ M piceatannol to treat the cells for 24 hours, piceatannol (40 μ M) was combined with OXA (200 μ M), and the change of intracellular ATP was detected by quinacrine staining, thereby determining whether the ATP release induced by piceatannol in combination with OXA depends on TFEB/TFE3 mediated autophagy.

4. In vivo experiment detection of chemotherapeutic anti-cancer effect of piceatannol on enhancing oxaliplatin

First, establishing a mouse tumor model and dosing

To verify whether small molecule drugs can effectively induce immunogenic cellular responses, we used the in vivo tumor cell vaccination experiments in mice to evaluate the efficacy of drug-induced ICD. We inoculated MCA205 mouse fibrosarcoma carcinoma cells in immunocompetent C57BL/6 mice and started intraperitoneal injections of piceatannol, OXA and a combination of both at the time of tumor formation. Meanwhile, the change of the body weight and the size of the tumor of the mouse is recorded by regular measurement, the mouse is dissected within a certain time, the tumor is taken out, and the size and the weight of the tumor are measured.

Second, the change of tumor infiltrating lymphocytes in tumor tissues is detected by flow cytometry

The mice were dissected, tumor tissue removed and digested into single cell suspensions. The cells were incubated with fluorescent labeled antibodies against CD3, CD8, CD4, CD25 for 30 minutes at four degrees, washed three times with FACS buffer, fixed with IC fixative for 20 minutes at room temperature, permeabilized with permeabilizing solution for 10 minutes, and centrifuged at 400-600 x g for 5 minutes. In the permeabilization solution, FOXP3 antibody was added and incubated for 30 minutes at room temperature, and FACS buffer was washed three times for flow cytometry analysis. We will analyze and compare the ratio of killer T lymphocytes (CD 3+ CD8 +) to regulatory T cells (Treg, CD4+ FOXP3+ CD25 +) expressed in tumors of the model group and Ctrl mice.

Analysis of results

1. Piceatannol in conjunction with Oxaliplatin (OXA) -induced release of Adenosine Triphosphate (ATP)

Extracellular ATP release is one of the key signals for Immunogenic Cell Death (ICD), and we will determine whether piceatannol, in conjunction with OXA, promotes intracellular ATP release. As shown in A of FIG. 1 and B of FIG. 1, the immunofluorescent staining results showed that piceatannol (40. mu.M) increased the intracellular ATP release induced 24 hours after low concentration of OXA (200. mu.M) treated cells.

2. Piceatannol in conjunction with OXA to induce CALR cell membrane translocation

Cell membrane translocation of cell membrane CALR (calreticulin) is also a key signal for Immunogenic Cell Death (ICD), and it was further examined whether piceatannol promotes OXA-induced CALR cell membrane translocation. As shown in A of FIG. 2 and B of FIG. 2, the immunofluorescent staining results showed that piceatannol (40. mu.M) increased the cell membrane translocation of CALR induced 24 hours after low concentration of OXA (200. mu.M) treated cells. Meanwhile, as shown in C of FIG. 2, the flow-through results further confirmed that piceatannol (40. mu.M) increased cell membrane translocation of CALR induced by low concentration of OXA (200. mu.M). These results indicate that piceatannol in combination with OXA induced cell membrane translocation of CALR.

3. Piceatannol in combination with OXA induced release of HMGB1

The release of extracellular HMGB1 is also a key signal for Immunogenic Cell Death (ICD). It was further determined whether piceatannol promotes OXA-induced release of HMGB1 from the cytoplasm to the outside of the cell. As shown in A of FIG. 3, the immunofluorescent staining results showed that piceatannol (40. mu.M) increased the release of intracellular HMGB1 from the nucleus induced by low concentrations of OXA (200. mu.M). As shown in fig. 3B, the simultaneous collection of extracellular medium also showed that piceatannol facilitated the release of HMGB 1. These results indicate that piceatannol synergizes OXA-induced release of HMGB1 cells.

4. Piceatannol promotes nuclear translocation of TFEB and TFE3

TFEB and TFE3 are key molecules that regulate the autophagy lysosomal pathway, which promotes autophagy by transcriptionally regulating a variety of autophagy-related genes. To examine whether piceatannol activates TFEB and TFE3, we treated U2OS cells transfected with Flag-TFEB and GFP-TFE3 plasmids with piceatannol for 24 hours, as shown in a of fig. 4 and B of fig. 4, and immunofluorescence results showed that various concentrations of piceatannol (10, 20, 40 μ M) significantly promoted the transport of TFEB from the cytoplasm into the nucleus. After the cytoplasm and the nucleus were separated at the same time, as shown in C of fig. 4, the change in expression of TFEB in the cytoplasm and the nucleus was detected by Western blot, and it was further confirmed that, 24 hours after the cells were treated with piceatannol (40 μ M), the expression of TFEB in the cytoplasm became small and the expression level in the nucleus was significantly increased, indicating that piceatannol promotes the transfer of TFEB from the cytoplasm to the nucleus. Similarly, immunofluorescence (D in FIG. 4 and E in FIG. 4) and Western blot results (F in FIG. 4) also show that piceatannol (40 μ M) treated cells promoted nuclear transport of TFE3 for 24 hours. The results indicate that piceatannol promotes the accumulation of TFEB and TFE3 nuclei.

5. Piceatannol for promoting cell autophagy

Since TFEB and TFE3 are molecules of autophagy, it was further examined whether nuclear translocation of TFEB and TFE3 by piceatannol activated autophagy. As shown in A of FIG. 5, the Western blot results showed that piceatannol (10, 20, 40. mu.M) treated cells increased the content of autophagy marker protein LC3-II 24 hours later; meanwhile, as shown in B of fig. 5, the content of LC3-II was also significantly increased after 3, 6, 9, 12, 24 hours after 40 μ M treatment of the cells. And as shown in C of fig. 5 and D of fig. 5, the increase of LC3-II was further promoted after piceatannol (40 μ M) treated cells for 24 hours on the basis of lysosomal inhibitor CQ (chloroquine), indicating that piceatannol promotes autophagy flow. In FIG. 5E and FIG. 5F, immunofluorescence results show that piceatannol (40 μ M) promotes autophagosomal formation 24 hours after treatment of U2OS cells expressed on the GFP-RFP-LC3 plasmid. These results indicate that piceatannol significantly promotes autophagy.

6. Piceatannol promotes autophagy dependent on TFEB and TFE3

To confirm whether piceatannol-induced autophagy was dependent on TFEB and TFE3, TFEB (a in fig. 6) and TFE3 (B in fig. 6) were first knocked down by siRNA interference. The results show that after knockdown of TFEB and TFE3 simultaneously, autophagy flow induced 24 hours after piceatannol (40 μ M) treatment of cells was significantly attenuated (C of fig. 6), indicating that piceatannol-induced autophagy is dependent on TFEB and TFE 3.

7. Release of ATP by piceatannol in conjunction with OXA is dependent on TFEB/TFE 3-mediated autophagy

To determine whether piceatannol in combination with OXA induced ATP release was dependent on TFEB/TFE3 mediated autophagy, siRNA knockdown induced ATP release after autophagy critical gene ATG5 (autophagy-associated protein 5) (fig. 7 a) significantly inhibited piceatannol (40 μ M) in combination with low concentration OXA (200 μ M) for 24 hours (fig. 7B and fig. 7C). Since TFEB and TFE3 have been demonstrated to be key molecules for piceatannol to regulate autophagy, inhibition of piceatannol after simultaneous knockdown of TFEB and TFE3 resulted in significant inhibition of piceatannol (40 μ M) in conjunction with low concentration OXA (200 μ M) treatment of cells for 24 hours induced release of ATP (fig. 7D and fig. 7E). These results indicate that piceatannol in concert with OXA-induced ATP release is dependent on TFEB/TFE 3-mediated autophagy.

8. Piceatannol for promoting endoplasmic reticulum stress

Endoplasmic reticulum stress is a key event leading to ICD is an important factor in CALR cell membrane translocation, and since piceatannol was found to promote OXA leading to CALR cell membrane translocation, it was determined whether this process was associated with endoplasmic reticulum stress. First, it was examined whether piceatannol promotes endoplasmic reticulum stress. Western blot results showed that piceatannol (10, 20, 40 μ M) treated U2OS cells for 24 hours increased phosphorylation of eIF2 α (eukaryotic initiation factor 2), a key molecule of endoplasmic reticulum stress, without affecting the overall level of eIF2 α (fig. 8 a and fig. 8B). Meanwhile, the expression of ATF4 (Activating transcription factor 4 ) (C in figure 8) and CHOP (Activating transcription factor 6-C/EBP homologous protein) (D in figure 8) which are marker proteins of endoplasmic reticulum stress of U2OS cells treated by piceatannol (10, 20, 40 mu.M) for 24 hours is obviously increased. These results indicate that piceatannol promotes endoplasmic reticulum stress.

9. Piceatannol-enhanced OXA-induced CALR cell membrane translocation associated with endoplasmic reticulum stress

To determine whether piceatannol-induced CALR cell membrane translocation is associated with endoplasmic reticulum stress, PERK, a key protein to endoplasmic reticulum stress, was knocked down (fig. 9 a and 9B), and the results showed that piceatannol-induced endoplasmic reticulum stress was inhibited after PERK deletion (fig. 9B). It also inhibited CALR cell membrane translocation (FIG. 9C and FIG. 9D) caused by piceatannol (40. mu.M) in cooperation with OXA (200. mu.M). These results show that piceatannol enhances OXA-induced CALR cell membrane translocation and PERK-mediated endoplasmic reticulum stress.

10. Piceatannol in cooperation with OXA-induced ICD (immunogenic cell death) in MCA205 osteosarcoma cells

To further confirm that piceatannol synergistically enhanced OXA-induced ICD, further experiments were performed in mouse MCA205 osteosarcoma carcinoma cells. Similar to the findings of U2OS, the level of autophagy critical protein LC3-II was promoted 24 hours after piceatannol (10, 20, 40 μ M) treatment in MCA205 (fig. 10 a), while piceatannol (40 μ M) treatment increased autophagy lysosomes 24 hours (fig. 10B), indicating that piceatannol also promoted autophagy in MCA205 cells. While piceatannol (40 μ M) induced ATP release 24 hours after co-treatment with OXA (200 μ M) on cells (FIG. 10C and FIG. 10D). Piceatannol (40 μ M) also promoted induction of HMGB1 release 24 hours after OXA (200 μ M) treatment of cells (fig. 10E and fig. 10F). Piceatannol (40 μ M) also promoted induction of CALR cell membrane translocation 24 hours after OXA (200 μ M) treatment of cells (fig. 10G and fig. 10H). These results indicate that piceatannol synergizes with OXA-induced ICD (immunogenic cell death) in MCA205 osteosarcoma cells.

11. Piceatannol increases the tumor-inhibiting effect of OXA in immunocompromised mice

To determine whether piceatannol promotes the effects and molecular mechanisms of OXA tumor suppression, normal immunized C57BL/6 mice were inoculated with MCA205 cells and were treated with piceatannol (10 mg/kg, administered every other day), OXA (30 mg/kg) and combinations thereof (10 mg/kg piceatannol and 30 mg/kg) after tumors grew to a macroscopic size. The results showed that piceatannol significantly enhanced the growth and size of OXA-inhibited tumors (fig. 11B, fig. 11C, fig. 11D) without affecting the body weight of the mice (fig. 11 a). Meanwhile, flow cytometry analysis results showed that piceatannol in cooperation with OXA increased the ratio of killer T cells in tumors (E in fig. 11), and further increased the ratio of killer T cells to regulatory T cells (tregs) to exert their antitumor effects (F in fig. 11 and G in fig. 11). These results indicate that piceatannol enhances OXA-induced ICD and thereby exerts an anti-tumor effect.

In summary, the provided small molecule compound is applied to the preparation of drugs for inducing immunogenic cell death, wherein the small molecule compound comprises piceatannol, the piceatannol activates autophagy of cells by regulating TFEB/TFE3, and further synergistically promotes immunogenic cell death induced by oxaliplatin, and generates T cell reaction with systemic anti-tumor effect, so as to enhance anti-tumor treatment effect, achieve combined treatment, facilitate the induction of durable anti-cancer immune response to reduce the possibility of relapse, and is suitable for being widely applied to the potential treatment effect of activating TFEB/TFE3 in various diseases.

The above disclosure is only for the purpose of illustrating the preferred embodiments of the present invention, and it is therefore to be understood that the invention is not limited by the scope of the appended claims.

Claims (9)

1. Use of a small molecule compound in the manufacture of a medicament for inducing immunogenic cell death, wherein the small molecule compound comprises piceatannol, wherein the piceatannol promotes nuclear translocation that activates transcription factor TFEB and transcription factor TFE 3.

2. The use of the small molecule compound of claim 1 for the preparation of a medicament for inducing immunogenic cell death, wherein piceatannol activates autophagy by modulating the nuclear translocation of TFEB/TFE 3-activated transcription factor TFEB and transcription factor TFE3, increases the level of autophagy marker protein LC3-II, and promotes autophagy.

3. The use of a small molecule compound according to claim 1 for the manufacture of a medicament for inducing immunogenic cell death, wherein piceatannol increases expression of transcription factor 4, transcription factor 6-C/EBP homologous protein activated by a marker protein of endoplasmic reticulum stress, and increases phosphorylation level of eukaryotic initiation factor 2 of endoplasmic reticulum stress to promote endoplasmic reticulum stress.

4. Use of a small molecule compound according to claim 1 for the manufacture of a medicament for inducing immunogenic cell death, wherein said medicament further comprises oxaliplatin.

5. Use of a small molecule compound according to claim 4 in the manufacture of a medicament for inducing immunogenic cell death, wherein said piceatannol promotes the release of extracellular ATP and HMGB1 by said oxaliplatin.

6. The use of a small molecule compound according to claim 4, wherein said piceatannol promotes oxaliplatin-induced membrane calreticulin membrane translocation and PERK-mediated endoplasmic reticulum stress in the manufacture of a medicament for inducing immunogenic cell death.

7. Use of a small molecule compound according to claim 4 in the manufacture of a medicament for inducing immunogenic cell death, wherein said piceatannol in combination with said oxaliplatin induces a promotion of immunogenic cell death.

8. Use of a small molecule compound according to claim 4 in the manufacture of a medicament for inducing immunogenic cell death, wherein said piceatannol increases the anti-tumor effect of said oxaliplatin.

9. A pharmaceutical composition for inducing immunogenic cell death is characterized by comprising piceatannol and oxaliplatin, wherein the concentration of piceatannol is 1-40 mu M.

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