CN109303919B - Application of Akt inhibitor in preparation of anti-liver cancer active drug for enhancing lycorine - Google Patents

Abstract

The invention discloses an application of an Akt inhibitor in preparing a medicament for enhancing the anti-liver cancer activity of lycorine. The invention provides an application of an Akt inhibitor in preparing a product for enhancing the anti-liver cancer activity or anti-liver cancer cell activity of lycorine. Experiments prove that the liver cancer cell apoptosis rate of the Akt inhibitor and lycorine combined drug is higher than the sum of the liver cancer cell apoptosis rate of the Akt inhibitor singly taken and the liver cancer cell apoptosis rate of the lycorine singly taken, the Akt inhibitor and the lycorine generate a synergistic effect in the aspect of resisting liver cancer, and the Akt inhibitor enhances the liver cancer resisting activity of the lycorine.

Description

Application of Akt inhibitor in preparation of anti-liver cancer active drug for enhancing lycorine

Technical Field

The invention relates to application of an Akt inhibitor, in particular to application of an Akt preparation in preparing a liver cancer resistant active medicament for enhancing lycorine.

Background

Hepatocellular carcinoma (HCC) is one of the most common aggressive tumors worldwide. Liver cancer is a highly fatal tumor, with median survival rates remaining below 1 year after diagnosis. Liver cancer is a heterogeneous disease derived from chronic liver diseases such as chronic inflammation and cirrhosis. To date, therapeutic strategies for liver cancer have primarily included resection, transplantation, local ablation, and chemotherapy. Since early symptoms of liver cancer are not obvious, most liver cancer patients are diagnosed at a late stage, the chance of operation is lost, and most of the patients who have undergone the operation in time still have relapse and metastasis. Among the chemotherapeutic agents, sorafenib is a small molecule multi-kinase inhibitor, considered an active therapeutic approach, and is the only approved systemic drug to prolong life span of patients with advanced liver cancer. However, the life expectancy of patients with sorafenib-treated advanced liver cancer is only 8-11 months. In the past decades, despite significant progress in conventional treatment regimens for patients with liver cancer, it remains one of the most fatal malignancies worldwide due to limited treatment, poor prognosis, and high recurrence rate. New effective and promising therapeutic strategies still need to be explored extensively on a global scale.

Natural products having various biological activities and mechanisms are generally excellent drugs found as cancer preventive and anticancer drugs. Lycorine (structure shown as a in figure 1), an active alkaloid of common folk medicines, has been explored, and comprises various biological activities such as anticancer, antiviral, antimalarial, antibacterial and anti-inflammatory activities. Although the target or mechanism of lycorine is not defined, the major biological activity and low cytotoxicity of lycorine make it a potential clinical or potential drug. For example, it is widely regarded as a promising anticancer agent by cervical cancer, leukemia, prostate cancer and multiple myeloma. However, the precise molecular mechanism is still unclear.

Akt, also known as protein kinase b (pkb), is a serine/threonine-specific protein kinase that plays an important role in a variety of cellular processes including glucose metabolism, apoptosis, cell proliferation transcription, and cell migration.

Disclosure of Invention

The invention aims to solve the technical problem of how to enhance the anti-liver cancer activity or anti-liver cancer cell activity of lycorine.

In order to solve the technical problems, the invention provides an application of an Akt inhibitor in preparing a product for enhancing the anti-liver cancer activity or anti-liver cancer cell activity of lycorine.

The invention also provides the application of the Akt inhibitor and lycorine in preparing products (such as medicines, vaccines, health products and/or foods) for resisting liver cancer or liver cancer cells.

In the application, the Akt inhibitor can be LY294002, and the structural formula is shown as formula 1.

In the above applications, the ratio of the Akt inhibitor to the lycorine can be determined by those skilled in the art according to the effect of resisting liver cancer or liver cancer cells, for example, the ratio of the Akt inhibitor to the lycorine can be specifically 1 μmol of the Akt inhibitor: and 2 mu mol of lycorine.

The invention also provides products (such as medicines, vaccines, health products and/or foods) for resisting liver cancer or liver cancer cells, wherein the products contain Akt inhibitors and lycorine.

In the product for resisting liver cancer or liver cancer cells, the Akt inhibitor and the lycorine can be packaged separately.

In the product for resisting liver cancer or liver cancer cells, the ratio of the Akt inhibitor to the lycorine can be determined by those skilled in the art according to the effect of resisting liver cancer or liver cancer cells, for example, the ratio of the Akt inhibitor to the lycorine can be specifically 1 μmol of the Akt inhibitor: and 2 mu mol of lycorine.

In the product for resisting liver cancer or liver cancer cells, the active ingredients of the product can be the Akt inhibitor and lycorine, the active ingredients of the product can also contain other ingredients, and the other active ingredients of the product can be determined according to the effect of resisting liver cancer by a person skilled in the art.

In the anti-liver cancer or anti-liver cancer cell product, the product can also contain a carrier or an excipient. The carrier material includes, but is not limited to, water-soluble carrier materials (e.g., polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), sparingly soluble carrier materials (e.g., ethyl cellulose, cholesterol stearate, etc.), enteric carrier materials (e.g., cellulose acetate phthalate, carboxymethyl cellulose, etc.).

In the anti-hepatoma or anti-hepatoma cell product, the Akt inhibitor may be LY 294002.

In the above application or the above anti-liver cancer or anti-liver cancer cell product, the liver cancer may be hepatocellular carcinoma.

In the application, the anti-liver cancer activity, the anti-liver cancer cell activity or the anti-liver cancer cell activity may specifically be promoting apoptosis of the liver cancer cell and/or promoting autophagy of the liver cancer cell and/or reducing the activity of the liver cancer cell.

Experiments prove that the liver cancer cell apoptosis rate of the Akt inhibitor and lycorine combined drug is higher than the sum of the liver cancer cell apoptosis rate of the Akt inhibitor singly taken and the liver cancer cell apoptosis rate of the lycorine singly taken, the Akt inhibitor and the lycorine generate a synergistic effect in the aspect of resisting liver cancer, and the Akt inhibitor enhances the liver cancer resisting activity of the lycorine.

Drawings

FIG. 1 shows that lycorine inhibits the growth of hepatocellular carcinoma.

In FIG. 1, a is the structural formula of lycorine; b is the cell viability assay result; c is the single cell clone formation assay result; d-f is the result of detecting the tumorigenicity of the xenograft tumor, g is the relative change of proliferation in the tumor growth by carrying out immunohistochemical detection through Ki67 staining, and h is the result of detecting the HE staining immunohistochemistry of the heart, the liver, the spleen, the lung and the kidney.

FIG. 2 shows that lycorine promotes autophagy of hepatocellular carcinoma.

In FIG. 2, a is the expression level of p62 and LC-3B in Western blot analysis HepG2 and SMMC-7721; b, analyzing the expression quantity of Atg5, Atg7 and Atg12 in HepG2 and SMMC-7721 by western blot; c is transmission electron microscopy analysis, d and e are autophagy flux analysis; f is the expression quantity of p62 and LC-3B in the liver cancer of the liver cells analyzed by Western blot; g is the expression quantity of LC-3B in the immunohistochemical detection of liver cancer cells.

FIG. 3 shows that inhibition of autophagy can further promote lycorine-induced apoptosis of hepatoma cells.

In FIG. 3, a, c and f are Western blot analysis of the expression levels of p62, LC-3B, cleared Caspase-3, PARP1/2 and Actin in each treated cell. b. d and e are cell viability and apoptosis rate of each treated cell.

In FIG. 3, from left to right in each of the pictures of a and b are control-treated cells, lycorine-alone treated cells, 3-MA-alone treated cells, and lycorine-and 3-MA-combined treated cells, respectively. In each of the pictures of c and d, from left to right, cells were treated with control siRNA alone 1, lycorine and control siRNA in combination 1, LC-3B-1 alone, and lycorine and LC-3B-1 in combination, respectively. e and f, from left to right, for control siRNA treated cells 2 alone, lycorine and control siRNA treated cells 2 in combination, LC-3B-2 treated cells alone, lycorine and LC-3B-2 treated cells in combination, respectively.

FIG. 4 shows that lycorine promotes hepatoma cell apoptosis and autophagy through TCRP1/Akt/mTOR signaling pathway.

In FIG. 4, a is the TCRP1 protein level in Western blot analysis HepG2 and SMMC-7721; b is real-time quantitative PCR analysis of TCRP1 mRNA levels in HepG2 and SMMC-7721 and Western blot analysis of TCRP1 protein levels in HepG2 and SMMC-7721; c is a western blot analysis result which shows that the overexpression of TCRP1 obviously prevents the inhibitory action of lycorine on the Akt/mTOR pathway; d, a western blot analysis result shows that the overexpression of TCRP1 blocks the lycorine-induced apoptosis and autophagy-related protein expression; e is a cell viability detection result; f is autophagy flux analysis; g is that the over-expression of TCRP1 strongly blocks the inhibition effect of lycorine on colony formation; h is that lycorine treatment reduced the protein levels of TCRP1, Akt, 4-EBP1 and p70S6K in HepG2 xenograft tumors.

FIG. 5 is an immunohistochemical assay for phosphorylated Akt and TCRP1 levels.

a and b are much stronger positive staining for phosphorylated Akt and TCRP1 in lycorine (10mg/kg) treated xenograft tumors; c and d were immunohistochemical measurements of phosphorylated Akt and TCRP1 levels in two liver cancer patients.

FIG. 6 shows that Akt inhibitors enhance the anti-hepatoma activity of lycorine. In both pictures, from left to right, cells treated with control, lycorine alone, LY294002 alone, and lycorine and LY294002 in combination were treated.

FIG. 7 shows that siRNA targeting TCRP1 enhances the anti-hepatoma activity of lycorine. In both pictures, from left to right, control siRNA alone treated cells, lycorine and control siRNA combined treated cells, siRNA_TCRP1Treatment of cells, lycorine and siRNA alone_TCRP1The cells are treated in combination.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The materials and methods in the following examples are as follows:

cell culture and cell treatment: human HCC cell lines HepG2, SMMC-7721, HuH-7 and Chang hepatocytes were obtained from the ATCC at month 4 of 2015. The used cells were revived within 1 month. Cell lines were identified by short tandem repeat analysis of PCR amplification. In the presence of 5% CO₂In a humidified atmosphere, cells were maintained at 37 ℃ in DMEM or RPMI-1640 supplemented with 10% FBS and 1% penicillin/streptomycin. Expression vectors for human TCRP1 and Akt were designed and purchased from servicebio technologies (wuhan, china). Lycorine (Lycorine) (purity)>98%) was purchased from shanghai source leaf biotechnology (shanghai, china). Dissolving lycorine with dimethyl sulfoxide (DMSO) to obtain 20mM stock solution, storing at-20 deg.C in aliquots, and diluting the culture medium for use when treating cells; when mice were injected, they were diluted to the corresponding concentrations with PBS. 3-MA, MG-132 and LY294002 were purchased from Selleck (London, ON, Canada). MTT (3- (4, 5-dimethyl-2-thiazolyl) -2, 5-diphenyl-2-H-tetrazolium bromide) reagent was purchased from Sigma-Aldrich (St. Louis, MO, USA). 3-MA was dissolved in dimethyl sulfoxide (DMSO) to prepare a 20mM stock solution, and the medium was used after dilution when treating the cells. LY294002 was prepared as a 20mM stock solution by dissolving in dimethyl sulfoxide (DMSO), and the medium was used after dilution when treating the cells.

Tissue sample: 45 human liver cancer and its matched adjacent non-tumor tissue arrays were purchased from Shanghai biochip Inc. (Shanghai, China). All of these samples are de-identified and the cohort has clinical outcome information.

Cell viability by MTT method: MTT solution was added to the test cells and incubated at 37 ℃ for 4 hours. The medium was then removed. 100ul of DMSO was added, and the absorbance at 570nm was measured using a microplate reader.

Apoptosis assay: apoptosis was measured by staining cells using Muse TM Annexin V and dead Cell assay kit (Millipore, Billerica, MA, USA), and Cell counting, Cell cycle and apoptosis analyses were performed in a bench-top Muse Cell Analyzer (Millipore, Billerica, MA, USA) according to the manufacturer's instructions.

Monoclonal formation experiments (colony formation assay): tumor cells were seeded into 6-well plates and cultured overnight. Cells were then treated with the indicated concentrations of the agents for 48 hours. After rinsing with fresh medium, colonies were formed, fixed with 4% paraformaldehyde, stained with 0.1% crystal violet, and then counted over the indicated time.

Immunofluorescence assay: cells were fixed with 4% paraformaldehyde for 30 min and incubated with 0.5% Triton X-100 in PBS and blocked with 5% BSA for 30 min. Slides were stained with anti-LC-3B antibody overnight at 4 ℃ followed by 1 hour at room temperature with Alexa-Fluor 488 conjugated goat anti-rabbit IgG antibody. Nuclei were stained with 2.5. mu.g/mL DAPI (Invitrogen, Carlsbad, CA, USA) and visualized by an inverted fluorescence microscope (Carl Zeiss, Oberkochen, Germany).

Immunohistochemical detection: tissue sections were excised, formalin fixed, paraffin embedded, and then incubated with anti-Ki 67, anti-cleaved caspase 3, anti-PARP, anti-LC-3B, and anti-TCRP 1 antibodies overnight at 4 ℃, followed by biotinylated secondary antibodies. Immunoreactivity was visualized using the vectasain Elite ABC kit (Vector Laboratories, Burlingame, CA, USA). Known positive controls were included in each experiment, and negative controls were obtained by omitting the primary antibody.

Western blot analysis: the antibodies used were as follows: SQSTM1/p62(D5E2) antibody, Atg5(D5F5U) antibody, Atg7(D12B11) antibody, Atg12(D88H11) antibody, anti-LC-3B (D11) antibody, phospho-p 70S6 kinase (Thr-389) antibody, total p70S6K antibody, phospho-Akt (Ser-473) antibody, total Akt antibody, phospho-4-EBP 1(Thr-37/46) antibody, total 4-EBP1 antibody and Cleaved Caspase 3(D175) antibody are products of CST corporation (Danvers, MA, USA). anti-PARP 1/2(H250) and TCRP1(E-13) antibodies are products of Santa Cruz Biotechnology (Santa Cruz, Calif., USA). The anti- β -actin (a5441) antibody was Sigma-Aldrich (st. louis, MO, USA).

Example 1 autophagy inhibitor enhances anti-hepatoma activity of lycorine (the combination of autophagy inhibitor and lycorine enhances apoptosis of hepatoma cells)

1. Lycorine inhibits growth of hepatocellular carcinoma

Cell viability was determined by MTT assay and lycorine was analyzed against three representative human hepatoma cell lines

(HepG2, SMMC-7721, HuH-7), and a non-tumorigenic human Liver cell line (Chang Liver). When HepG2, SMMC-7721, HuH-7 and Chang Liver were cultured in a medium containing 0, 10, 20, 30, 40 and 50. mu.M of lycorine for 48 hours, respectively, and the cell viability was measured by the MTT method, it was shown that the hepatoma cell viability was significantly decreased by treating 10, 20, 30, 40 and 50. mu.M of lycorine for 48 hours, and at the same time, lycorine did not affect the cell viability of the Chang Liver normal Liver cells at the same dose (b in FIG. 1). The results of monoclonal formation experimental detection of 0, 10 and 20 μ M lycorine-treated HepG2, SMMC-7721 and HuH-7 for 48 hours show that the cell proliferation of HepG2, SMMC-7721 and HuH-7 is significantly inhibited by 10 and 20 μ M lycorine-treated HepG2, SMMC-7721 and HuH-748 hours (C in figure 1). The results of the xenograft tumor tumorigenicity test showed that the tumor size was significantly reduced after treatment with allicin compared to the control group, as shown in d in fig. 1. In agreement with this, the normal group had a much greater weight of tumours than those treated with allicin (fig. 1, e). The growth rate of lycorine-treated xenograft tumors was significantly reduced compared to the control group (f in fig. 1). To further assess the relative change in proliferation in tumor growth, immunohistochemical assays were performed by Ki67 (antigen that labeled the proliferation state of cells) staining, which indicated a significant percentage decrease in tumors in the Lycorine (10mg/kg) and Lycorine (20mg/kg) groups compared to normal group tumors (FIG. 1, g). However, there were no significant differences in cell morphology and body weight of the major target organs (h in fig. 1). Collectively, these results indicate that lycorine inhibits the proliferation of liver cancer in vitro and in vivo.

The detection method for the tumorigenicity of the xenograft tumor comprises the following steps:

HepG2 cells (5 × 10 in 0.2mL PBS)⁶) The resulting mixture was inoculated (by subcutaneous injection) into 7-week-old BALB/c female athymic nude mice (Taconic). When the tumor volume reaches 100mm³At the time, mice were randomly divided into 3 groups: the normal group, Lycorine (10mg/kg) group (Lycorine 10mg/kg) and Lycorine (20mg/kg) group (Lycorine20mg/kg), each group consisting of 8 individuals. And performing intraperitoneal injection of the solution every other day for 33 consecutive days, wherein each Lycorine (10mg/kg) group is injected with 120 μ L Lycorine solution, so that the injection dosage of Lycorine is 10mg/kg body weight/day; each Lycorine (20mg/kg) group is injected with 120 μ L Lycorine solution to make the injection dosage of Lycorine20mg/kg body weight/day; the normal group was injected with an equal volume of PBS. Tumor volume and body weight were monitored every 3 days. After sacrifice of the mice 33 days from the first dose, their tumors were weighed, photographed and fixed in 4% paraformaldehyde for immunohistochemistry.

2. Lycorine promotes autophagy of hepatocellular carcinoma

In addition to apoptosis, autophagy is another mode of programmed cell death in response to cellular stress. First, the effect of lycorine on autophagosome formation was investigated by evaluating LC-3B and p62 (two classical autophagy markers), HepG2, SMMC-7721, 48 hours were cultured in media containing 0, 10, 20, 30 and 40. mu.M lycorine, respectively, and cells were taken for Western blot analysis of the expression levels of p62, LC-3B (D11), Atg5(D5F5U), Atg7(D12B11), Atg12(D88H11) and anti-beta-Actin (A5441) using SQSTM1/p62(D5E2) antibodies, respectively, and P62, LC-3B, Atg5, Atg7, Atg12 and Actin (as internal reference), respectively, indicating that allicin can up-regulate the expression of LC-3B and exhibit a certain dose-dependent expression, while p62 is significantly reduced (FIG. 2 a). Lycorine significantly up-regulated protein expression of Atg5, Atg7 and Atg12 (b in fig. 2). To further verify lycorine-induced autophagy, intracellular morphological changes of liver cancer were studied by transmission electron microscopy. The method comprises the following steps: HepG2, SMMC-772148 hours were incubated with media containing 0 and 40. mu.M, respectively, of lycorine, and the cells were fixed with 4% glutaraldehyde. Ultrathin (50nm) sections were cut in a microtome and stained with 1% uranyl acetate and lead citrate. Finally, the sections were determined by electron microscopy using Hitachi H-7650(Hitachi, Tokyo, Japan). The results show that 40 μ M Lycorine-treated cells (cells denoted by Lycorine in c in fig. 2) had a significant accumulation of double membrane vesicles (containing subcellular material) compared to normal group cells (0 μ M Lycorine-treated cells) (c in fig. 2).

To determine the autophagy flux, cells HepG2, SMMC-7721 were transfected with an autophagy double-targeted adenovirus (mRFP-GFP-LC3) according to the manufacturer's instructions (shanghai gekhae gene chemistry technology). The transfected cells were then cultured with media containing 0 and 40 μ M lycorine, respectively, for 48 hours. Subsequently, the cells were fixed with 4% paraformaldehyde for 10 minutes and washed with PBS. Finally, the GFP/mRFP images were visualized using a laser scanning confocal microscope (Olympus FV1000, Tokyo, Japan). Wherein, mRFP is used for marking and tracking LC3, the weakening of GFP can indicate the fusion of lysosome and autophagosome to form autophagososome, yellow spots appearing after red-green fluorescence Merge are autophagosome, red spots indicate autophagososome, and the strength of autophagy flow can be clearly seen through counting of different color spots. In addition, HepG2, SMMC-772148 hours, were cultured in media containing 0 and 40. mu.M lycorine, respectively, and cells were subjected to nuclear staining with DAPI, and after 10 minutes, immunohistochemical detection was performed on the cells with an anti-LC-3B (D11) antibody. The results show that treatment with concatemeric mRFP-GFP labeled LC-3, with lycorine, resulted in increased LC-3-II conversion, promotion of LC-3 lipidation and spotting, and accumulation of yellow punctate autophagosomes (d and e in FIG. 2). In FIG. 2 d and e, normal group and Control were 0 μ M Lycorine-treated cells and Lycorine was 40 μ M Lycorine-treated cells.

In addition, the effect of lycorine on in vivo hepatoma autophagy was assessed by Western blot assay and immunohistochemical staining HepG2 cells (5 × 10 in 0.2mL PBS)⁶) The resulting mixture was inoculated (by subcutaneous injection) into 7-week-old BALB/c female athymic nude mice (Taconic). When the tumor volume reaches 100mm³In this case, mice were randomly divided into 3 groups, normal group, Lycorine (10mg/kg) group and Lycorine (20mg/kg) group, each group consisting of 8 mice, and were intraperitoneally injected every 33 consecutive days with 120. mu.L of Lycorine (10mg/kg) group at an injection dose of 10mg/kg body weight/day, Lycorine (20mg/kg) group at an injection dose of 20mg/kg body weight/day, and normal group was injected with PBS of equal volume every 3 days to monitor tumor volume and body weight, after mice were sacrificed 33 days from the first administration, tumors were fixed in 4% paraformaldehyde, tumor cells were immunohistochemically tested with anti-LC-3B (D11) antibody, and SQ cells were analyzed with Lylyp 1/62 (D5E 5) antibody, LC-3B (D595E 5) antibody, and Lycorine (D545) antibody (D5464- β) antibody as well as shown by increasing levels in the results of expression level of normal protein in STM 1/10 mg/kg (PG) group, as shown in the results of normal group (10 mg/kg-10 mg/kg) and in the results of normal group (PG 3, as shown in the results of Western blot (No. 20mg/kg) and in the results of STM 20 mg/kg-10 mg/kg) group, and in the results of PCR (No. 20mg/kg) group, and in the case of normal group, and in the case of the same time of PBS (20mg/kg) and in the mice).

In line with this, a similar trend was observed by immunohistochemical staining of LC-3B. In lycorine-treated tumors, the number of autophagic cells positively stained for LC-3B was significantly higher than in the normal group (g in FIG. 2). Taken together, these results indicate that lycorine promotes autophagy in hepatocellular carcinoma.

3. Inhibiting autophagy can further promote lycorine-induced liver cancer cell apoptosis

3.1 autophagy inhibitor 3-MA enhances the anti-liver cancer activity of lycorine, and 3-MA and lycorine generate synergistic effect in the aspect of anti-liver cancer

Although apoptosis and autophagy are two distinct modes of programmed cell death, they have complex interconnections to maintain intracellular homeostasis. To study the effect of lycorine on autophagy and apoptosis of hepatoma cells, 3-methyladenine (3-MA), an autophagy inhibitor (blocking autophagy), and hepatoma cells induced by lycorine with and without 3-MA treatment, respectively, were selected.

The experiment was carried out with 4 treatments, namely control treatment, lycorine single treatment, 3-MA single treatment, and lycorine and 3-MA combined treatment. The specific experimental method is as follows:

and (4) comparison treatment: HepG2, SMMC-7721 were cultured in DMEM medium containing 10% FBS and 1% penicillin/streptomycin at 37 ℃ for 50 hours, respectively, and the cells were collected to obtain control-treated cells.

Lycorine alone treatment: culturing HepG2 and SMMC-7721 in DMEM medium containing 10% FBS and 1% penicillin/streptomycin at 37 deg.C for 2 hr, adding lycorine to the culture medium until the content of lycorine is 40 μ M, culturing at 37 deg.C for 48 hr, and collecting cells to obtain lycorine-treated cells.

3-MA treatment alone: HepG2, SMMC-7721 was cultured at 37 ℃ for 50 hours in a medium containing 3-MA (a medium obtained by adding 3-MA to a DMEM medium containing 10% FBS and 1% penicillin/streptomycin to a content of 3mM of 3-MA), and the cells were collected to obtain 3-MA-treated cells alone.

Treating lycorine and 3-MA in combination: after culturing HepG2, SMMC-7721 in a medium containing 3-MA (medium obtained by adding 3-MA to 3mM of 3-MA in DMEM medium containing 10% FBS and 1% penicillin/streptomycin) at 37 ℃ for 2 hours, respectively, adding lycorine to the medium until the content of lycorine is 40 μ M, culturing at 37 ℃ for 48 hours, respectively, and collecting cells to obtain lycorine-3-MA combined treated cells.

Western blot analysis of p62, LC-3B, Cleaved Caspase-3, PARP and Actin (as internal parameters) expression in control-treated cells, cells treated with lycorine alone, cells treated with 3-MA alone and cells treated with combinations of lycorine and 3-MA, using SQSTM1/p62(D5E2) antibody, LC-3B (D11) antibody, Cleaved Caspase 3(D175) antibody, anti-PARP 1/2(H250) antibody and anti- β -Actin (A5441) antibody, showed that 3-MA significantly attenuated lycorine-induced autophagy in hepatoma cells (HepG2 and SMMC-7721) (a in FIG. 3). Interestingly, 3-MA further increased lycorine-induced apoptosis by activating CleavedCaspase 3 and PARP (a in fig. 3).

PARP (poly ADP-ribose polymerase) is a cleavage substrate for caspase (caspase), a core member of apoptosis.

Cell viability was determined by the MTT method using control treated cells, lycorine alone treated cells, 3-MA alone treated cells and lycorine and 3-MA combined treated cells, apoptosis was measured by staining cells using Muse TM Annexin V and dead cell assay kit (Millipore, Billerica, MA, USA), and the results are shown in b in FIG. 3, where the apoptosis rate (60.18% + -1.02%) of lycorine and 3-MA combined treated HepG2 cells was higher than the sum of the apoptosis rate (40.85% + -1.13%) of lycorine alone treated HepG2 cells and the apoptosis rate (7.53% + -0.13%) of 3-MA alone treated HepG2 cells; the apoptosis rate (63.52% + -2.22%) of the SMMC-7721 cells treated by the combination of lycorine and 3-MA is higher than the sum of the apoptosis rate (46.33% + -2.35%) of the SMMC-7721 cells treated by the combination of lycorine and 3-MA (7.21% + -0.11%). The 3-MA is proved to enhance the anti-liver cancer activity of the lycorine, and the 3-MA and the lycorine generate synergistic effect on the anti-liver cancer aspect.

Example 2, LC-3B-targeting siRNA enhanced the anti-hepatoma activity of lycorine, and LC-3B-targeting siRNA and lycorine produced synergistic effect in anti-hepatoma

NCBI Reference Sequence of human microtubule associated protein1light chain 3beta (Homo sapiens microtubular associated protein1light chain 3beta, MAP1LC3B, LC-3B for short) gene NM-022818.4 (Update Date: 10-JUL-2017).

Random double-stranded RNA was selected as a negative control siRNA in this experiment, and the name of the random double-stranded RNA was siNC, the nucleotide sequence from the 5 'end to the 3' end of one strand of the siNC was (5'-uucuccgaacgugucacguTT-3'), and the nucleotide sequence from the 3 'end to the 5' end of the other strand of the siNC was (3 '-TTaagaggcuugcacagugca-5').

Two siRNAs targeting human LC-3B are selected in the experiment and named as LC-3B-1 and LC-3B-2 respectively, wherein the nucleotide sequence from 5 'end to 3' end of one chain of LC-3B-1 is (5'-gcucuucuagaauuguuuaTT-3') (sequence 1 in the sequence table), and the nucleotide sequence from 3 'end to 5' end of the other chain of LC-3B-1 is (3 '-TTcgagaagaucuuaacaaau-5' (sequence 2 in the sequence table)). The nucleotide sequence from 5 'end to 3' end of one strand of LC-3B-2 is (5'-guagaagauguccgacuuaTT-3' (SEQ ID NO: 3) in the sequence Listing), and the nucleotide sequence from 3 'end to 5' end of the other strand of LC-3B-2 is (3 '-TTcaucuucuacaggcugaau-5' (SEQ ID NO: 4) in the sequence Listing).

In the sequences of the three siRNAs, u, c, g, a and T are respectively uracil ribonucleotide, cytosine ribonucleotide, guanine ribonucleotide, adenine ribonucleotide and thymine deoxyribonucleotide.

The experiment is designed with 8 treatments, namely control siRNA single treatment 1, control siRNA single treatment 2, lycorine and control siRNA combined treatment 1, lycorine and control siRNA combined treatment 2, LC-3B-1 single treatment, lycorine and LC-3B-1 combined treatment, LC-3B-2 single treatment and lycorine and LC-3B-2 combined treatment. The specific experimental method is as follows:

the experimental methods of the control siRNA treatment alone 1 and the control siRNA treatment alone 2 were the same, the combined treatment with lycorine and control siRNA 1 and the combined treatment with lycorine and control siRNA 2 were the same, the control siRNA treatment alone 1 and the combined treatment with lycorine and control siRNA 1 served as the control of LC-3B-1, and the control siRNA treatment alone 2 and the combined treatment with lycorine and control siRNA 2 served as the control of LC-3B-2.

Control siRNA treatment 1 alone and control siRNA treatment 2 alone HepG2 and SMMC-7721 cells were seeded into 6-well plates with 4 × 10 cells per well, respectively⁵And culturing the cells in a DMEM medium containing 10% FBS and 1% penicillin/streptomycin at 37 ℃ for 12h, then transfecting the siNC by using Lipofectamine RNAi Max, performing secondary transfection after 12h, replacing fresh DMEM medium containing 10% FBS and 1% penicillin/streptomycin after secondary transfection of the siNC24h, respectively continuing culturing for 48 hours at 37 ℃, collecting the cells, and obtaining control siRNA single-treated 1 cells and control siRNA single-treated 2 cells. Wherein the content of the first and second substances,the concentration of siNC was 30nM for each transfection.

Combined treatment of lycorine and control siRNA 1 and combined treatment of lycorine and control siRNA 2, cells of HepG2 and SMMC-7721 were inoculated into 6-well plates, and each well was inoculated with 4 × 10 cells⁵And culturing the cells in a DMEM medium containing 10% FBS and 1% penicillin/streptomycin at 37 ℃ for 12h, then transfecting the siNC by using Lipofectamine RNAi Max, performing secondary transfection after 12h, replacing the culture medium containing 40 mu M of lycorine after the secondary transfection of the siNC for 24h, respectively culturing the cells at 37 ℃ for 48 h, and collecting the cells to obtain 1 cell treated by the combination of the lycorine and the control siRNA and 2 cell treated by the combination of the lycorine and the control siRNA. The concentration of siNC was 30nM for each transfection.

LC-3B-1 Single treatment HepG2 and SMMC-7721 cells were seeded separately in 6-well plates 4 × 10 cells per well⁵And culturing the cells in a DMEM medium containing 10% FBS and 1% penicillin/streptomycin at 37 ℃ for 12h, then transfecting LC-3B-1 by using Lipofectamine RNAi Max, performing secondary transfection after 12h, replacing fresh DMEM medium containing 10% FBS and 1% penicillin/streptomycin after secondary transfection of LC-3B-124h, and respectively continuing culturing for 48 h at 37 ℃, and collecting the cells to obtain LC-3B-1 single-treatment cells. The concentration of LC-3B-1 in each transfection was 30 nM.

Combined treatment of lycorine and LC-3B-1 by inoculating HepG2 and SMMC-7721 cells into 6-well plates, 4 × 10 cells per well⁵And culturing the cells in a DMEM medium containing 10% FBS and 1% penicillin/streptomycin at 37 ℃ for 12h, then transfecting LC-3B-1 by using Lipofectamine RNAi Max, performing secondary transfection after 12h, replacing a medium containing 40 mu M of lycorine after the secondary transfection of LC-3B-124h (adding the lycorine into the DMEM medium containing 10% FBS and 1% penicillin/streptomycin until the content of the lycorine is 40 mu M), culturing at 37 ℃ for 48 h respectively, and collecting the cells to obtain the cells treated by the combination of the lycorine and the LC-3B-1. The concentration of LC-3B-1 in each transfection was 30 nM.

LC-3B-2 treatment alone HepG2 and SMMC-7721 cells were seeded separately in 6-well plates 4 × 10 cells per well⁵And culturing the cells in a DMEM medium containing 10% FBS and 1% penicillin/streptomycin at 37 ℃ for 12h, then transfecting LC-3B-2 by using Lipofectamine RNAi Max, performing secondary transfection after 12h, replacing fresh DMEM medium containing 10% FBS and 1% penicillin/streptomycin after secondary transfection of LC-3B-224h, and respectively continuing culturing for 48 h at 37 ℃, and collecting the cells to obtain LC-3B-2 single-treatment cells. The concentration of LC-3B-2 in each transfection was 30 nM.

Combined treatment of lycorine and LC-3B-2 by inoculating HepG2 and SMMC-7721 cells into 6-well plates, 4 × 10 cells per well⁵And culturing the cells in a DMEM medium containing 10% FBS and 1% penicillin/streptomycin at 37 ℃ for 12h, then transfecting LC-3B-2 by using Lipofectamine RNAi Max, performing secondary transfection after 12h, replacing a medium containing 40 mu M of lycorine after the secondary transfection of LC-3B-224h (adding the lycorine into the DMEM medium containing 10% FBS and 1% penicillin/streptomycin until the content of the lycorine is 40 mu M), culturing at 37 ℃ for 48 h respectively, and collecting the cells to obtain the cells treated by the combination of the lycorine and the LC-3B-2. The concentration of LC-3B-2 in each transfection was 30 nM.

Western blot analysis of the expression levels of p62, LC-3B, Cleveland Caspase-3, PARP and Actin (as internal reference) in cells treated with SQSTM1/p62(D5E2) antibody, LC-3B (D11) antibody, Cleaved Caspase 3(D175), anti-PARP 1/2(H250) antibody and anti- β -Actin (A5441) antibody in cells treated with control siRNA alone 1 cells, control siRNA alone 2 cells, lycorine and control siRNA combined 1 cells, lycorine and control siRNA combined 2 cells, LC-3B-1 alone cells, lycorine and LC-3B-2 combined cells, and results show that the expression levels of both LC-3B-1 and LC-3B-2 targeting siRNA LC-3B significantly decreased (liver cancer pG2 and SMMC-7721) in cells treated with LC-3B (Heishi liver cancer 7721) Autophagy (c and f in fig. 3). Both LC-3B-1 and LC-3B-2 siRNA targeting LC-3B reduced the content of LC-3B in lycorine-induced HepG2 and SMMC-7721 cells. Indicating that siRNA knockdown of endogenous LC-3B significantly reduced lycorine-induced autophagy.

Cell viability was determined by MTT method using control siRNA alone in1 cell, control siRNA alone in 2 cells, lycorine and control siRNA in combination in1 cell, lycorine and control siRNA in combination in 2 cells, LC-3B-1 alone in cells, lycorine and LC-3B-1 in combination in cells, LC-3B-2 alone in cells, and lycorine and LC-3B-2 in combination in cells, apoptosis was measured by staining cells using Muse TM Annexin V and dead cell assay kit (Millipore, Billerica, MA, USA), and as a result, shown in d and e in FIG. 3, the rate of apoptosis (68.32% + -1.53%) of HepG2 cells treated with combinations of lycorine and LC-3B-1 was higher than the sum of the rate of apoptosis (47.73% + -1.81%) of HepG2 cells treated with combinations of lycorine and control siRNA and the rate of apoptosis (3.12% + -0.38%) of HepG2 cells treated with LC-3B-1 alone; the apoptosis rate (57.62% + -4.28%) of HepG2 cell treated with the combination of lycorine and LC-3B-2 was higher than the sum of the apoptosis rate (34.25% + -2.92%) of HepG2 cell treated with the combination of lycorine and control siRNA and the apoptosis rate (6.23% + -2.35%) of HepG2 cell treated with LC-3B-2 alone. The apoptosis rate (64.39% + -1.62%) of SMMC-7721 cells treated by the combination of lycorine and LC-3B-1 is higher than the sum of the apoptosis rate (42.33% + -3.85%) of SMMC-7721 cells treated by the combination of lycorine and control siRNA and the apoptosis rate (4.04% + -0.29%) of SMMC-7721 cells treated by LC-3B-1 alone; the apoptosis rate (58.98% + -0.33%) of SMMC-7721 cells treated with lycorine and LC-3B-2 was higher than the sum of the apoptosis rate (38.66% + -4.30%) of SMMC-7721 cells treated with lycorine and control siRNA and the apoptosis rate (3.14% + -0.32%) of SMMC-7721 cells treated with LC-3B-2 alone. The results show that LC-3B-1 and LC-3B-2 target LC-3B siRNAs enhance the anti-liver cancer activity of lycorine, and the LC-3B target siRNA and lycorine generate synergistic effect in the anti-liver cancer aspect. These results strongly suggest that inhibition of autophagy can further promote lycorine-induced apoptosis of hepatoma cells.

Example 3 Akt inhibitors potentiate the anti-hepatoma activity of lycorine (the combination of Akt inhibitors and lycorine potentiate apoptosis of hepatoma cells)

1. Lycorine promotes hepatoma carcinoma cell apoptosis and autophagy through TCRP1/Akt/mTOR signaling pathway

The experiment was carried out according to the experimental method described in example 1.

Tongue cancer resistance-associated protein 1(Tongue cancer resistance-associated protein1, TCRP1) has been reported to promote tumorigenesis in radiation-resistant oral squamous cell carcinoma by selectively activating the PI3K/Akt and NF-KB pathways. As shown in a in FIG. 4, lycorine treatment significantly reduced the expression of the protein level of TCRP1 in hepatoma cells. However, lycorine treatment did not significantly alter the mRNA level of TCRP1 (b in fig. 4), indicating that TCRP1 protein is largely translocated through proteasomal degradation. Subsequently, treatment with MG-132, a proteasome inhibitor, significantly abolished the inhibitory effect of lycorine on the protein level of TCRP1 in hepatoma cells (fig. 4 b). Taken together, these results indicate that lycorine may reduce TCRP1 protein levels by promoting the TCRP1 protein degradation pathway.

Next, it was found that overexpression of TCRP1 in hepatoma cells significantly prevented the inhibitory effect of lycorine on the Akt/mTOR pathway (c in FIG. 4). Furthermore, TCRP1 overexpression also blocked lycorine-induced apoptosis and autophagy-related protein expression, cell death (d-f in fig. 4). Furthermore, TCRP1 overexpression strongly blocked the inhibitory effect of lycorine on colony formation (g in figure 4). In agreement, lycorine treatment reduced the protein levels of TCRP1, Akt, 4-EBP1 and p70S6K in HepG2 xenograft tumors (h in fig. 4). Thus, immunohistochemical staining demonstrated much stronger positive staining of phosphorylated Akt and TCRP1 in lycorine-treated xenograft tumors compared to control xenograft tumors (a and b in figure 5). Next, clinical relevance between Akt and TCRP1 in tissues from liver cancer patients was evaluated. As expected, TCRP1 expression was positively correlated with p-Akt (c and d in fig. 5), which supports results in cultured cells and mouse tumor models. In conclusion, the research result shows that lycorine promotes the apoptosis and autophagy of the hepatoma cells through a TCRP1/Akt/mTOR signaling pathway and plays a key role.

2. Akt inhibitor for enhancing anti-liver cancer activity of lycorine (combination of Akt inhibitor and lycorine for enhancing apoptosis of liver cancer cell)

The Akt inhibitor adopted in the experiment is LY294002, and the structural formula is shown as formula 1.

The experiment was carried out with 4 treatments, namely control treatment, lycorine alone treatment, LY294002 alone treatment, lycorine and LY294002 combined treatment. The specific experimental method is as follows:

and (4) comparison treatment: HepG2, SMMC-7721 were cultured in DMEM medium containing 10% FBS and 1% penicillin/streptomycin at 37 ℃ for 50 hours, respectively, and the cells were collected to obtain control-treated cells.

Lycorine alone treatment: culturing HepG2 and SMMC-7721 in DMEM medium containing 10% FBS and 1% penicillin/streptomycin at 37 deg.C for 2 hr, adding lycorine to the culture medium until the content of lycorine is 40 μ M, culturing at 37 deg.C for 48 hr, and collecting cells to obtain lycorine-treated cells.

LY294002 treated alone: HepG2, SMMC-7721 was cultured at 37 ℃ for 50 hours with a culture medium containing LY294002 (culture medium obtained by adding LY294002 to LY294002 in an amount of 20. mu.M to DMEM medium containing 10% FBS and 1% penicillin/streptomycin), respectively, and the cells were collected to obtain LY294002 alone.

Combined treatment of lycorine and LY 294002: after HepG2, SMMC-7721 was cultured with a culture medium containing LY294002 (a culture medium obtained by adding LY294002 to LY294002 at 20. mu.M to a DMEM medium containing 10% FBS and 1% penicillin/streptomycin) at 37 ℃ for 2 hours, lycorine was further added to the culture medium to a content of lycorine of 40. mu.M and the culture was continued at 37 ℃ for 48 hours, respectively, and the cells were collected to obtain lycorine and LY294002 combined-treated cells.

Cells treated with control, lycorine alone, LY294002 alone in combination with lycorine and LY294002 were measured for apoptosis by staining the cells using the Muse TM Annexin V and dead cell assay kit (Millipore, Billerica, MA, USA), and the results indicated that the rate of apoptosis (62.82% ± 3.82%) of HepG2 cells treated with lycorine and LY294002 in combination was greater than the sum of the rate of apoptosis (37.66% ± 3.35%) of HepG2 cells treated with lycorine alone and the rate of apoptosis (4.85% ± 0.34%) of HepG2 cells treated with LY294002 alone; the apoptosis rate (60.59% + -4.68%) of SMMC-7721 cells treated with lycorine and LY294002 in combination was higher than the sum of the apoptosis rate (39.19% + -4.16%) of SMMC-7721 cells treated with lycorine alone and the apoptosis rate (3.99% + -0.28%) of SMMC-7721 cells treated with LY294002 alone (FIG. 6). Shows that LY294002 enhances the anti-liver cancer activity of lycorine, and LY294002 and lycorine generate synergistic effect in the aspect of anti-liver cancer.

Example 4 siRNA targeting TCRP1 enhances anti-hepatoma activity of lycorine (agent inhibiting TCRP1 expression combined with lycorine enhances apoptosis of hepatoma cells)

Random double-stranded RNA was selected as a negative control siRNA in this experiment, and the name of the random double-stranded RNA was siNC, the nucleotide sequence from the 5 'end to the 3' end of one strand of the siNC was (5'-uagaccuugguacucgacgaucuuuTT-3'), and the nucleotide sequence from the 3 'end to the 5' end of the other strand of the siNC was (3 '-TTaucuggaaccaugagcugcuagaaa-5').

In the experiment, an siRNA targeting TCRP1 is selected and named as siRNA_TCRP1The nucleotide sequence from 5 'end to 3' end of one strand of (a) is (5'-uagucccaguuaugcuccagaguuuTT-3'), siRNA_TCRP1The nucleotide sequence from the 3 'end to the 5' end of the other strand of (3 '-TTaucagggucaauacgaggucucaaa-5').

In the sequences of the two siRNAs, u, c, g, a and T are respectively uracil ribonucleotide, cytosine ribonucleotide, guanine ribonucleotide, adenine ribonucleotide and thymine deoxyribonucleotide.

The experiment is designed with 4 treatments, namely single treatment of control siRNA, combined treatment of lycorine and control siRNA, and siRNA_TCRP1Individual treatment, lycorine and siRNA_TCRP1And (4) performing combined treatment. The specific experimental method is as follows:

control siRNA treatment alone HepG2 and SMMC-7721 cells were seeded separately in 6-well plates 4 × 10 cells per well⁵The cells were cultured in DMEM medium containing 10% FBS and 1% penicillin/streptomycin at 37 ℃ for 12 hours, then transfected into sinC by Lipofectamine RNAi Max, and after 12 hours, the secondary transfection was performed, and after 24 hours, the sinC was replaced by fresh DMEM medium containing 10% FBS and 1% penicillin/streptomycinThe medium was further cultured at 37 ℃ for 48 hours, respectively, and the cells were collected to obtain control siRNA-treated cells alone. The concentration of siNC was 30nM for each transfection.

Combined treatment with lycorine and control siRNA HepG2 and SMMC-7721 cells were seeded in 6-well plates 4 × 10 cells/well⁵And culturing the cells in a DMEM medium containing 10% FBS and 1% penicillin/streptomycin at 37 ℃ for 12h, then transfecting the siNC by using Lipofectamine RNAi Max, performing secondary transfection after 12h, replacing a culture medium containing 40 mu M of lycorine after 24h of secondary transfection of the siNC (adding the lycorine into the DMEM medium containing 10% FBS and 1% penicillin/streptomycin until the content of the lycorine is 40 mu M), and culturing at 37 ℃ for 48 h respectively, and collecting the cells to obtain the cells treated by the combination of the lycorine and the control siRNA. The concentration of siNC was 30nM for each transfection.

siRNA_TCRP1Separate treatment HepG2 and SMMC-7721 cells were seeded separately in 6-well plates 4 × 10 per well⁵Cells were cultured in DMEM medium containing 10% FBS and 1% penicillin/streptomycin at 37 ℃ for 12h and then transfected with siRNA using Lipofectamine RNAi Max_TCRP1And performing secondary transfection after 12h, and performing secondary transfection on siRNA_TCRP1After 24h, fresh DMEM medium containing 10% FBS and 1% penicillin/streptomycin is replaced to continue culturing at 37 ℃ for 48 hours respectively, and cells are collected to obtain siRNA_TCRP1The cells were treated individually. Wherein, siRNA of each transfection_TCRP1The concentrations were all 30 nM.

Lycorine and siRNA_TCRP1Combined treatment HepG2 and SMMC-7721 cells were seeded separately in 6-well plates 4 × 10 cells per well⁵Cells were cultured in DMEM medium containing 10% FBS and 1% penicillin/streptomycin at 37 ℃ for 12h and then transfected with siRNA using Lipofectamine RNAi Max_TCRP1And performing secondary transfection after 12h, and performing secondary transfection on siRNA_TCRP1Changing culture medium containing lycorine 40 μ M after 24 hr (adding lycorine into DMEM medium containing 10% FBS and 1% penicillin/streptomycin to reach lycorine content of 40 μ M), culturing at 37 deg.C for 48 hr, respectively, collecting cells to obtain lycorine and siRNA_TCRP1The cells are treated in combination. Wherein, siRNA of each transfection_TCRP1The concentrations were all 30 nM.

Treating cells with control siRNA alone, treating cells with lycorine and control siRNA in combination, and treating siRNA with control siRNA_TCRP1Treatment of cells, lycorine and siRNA alone_TCRP1Combined treatment of cells apoptosis was measured by staining cells using Muse TM Annexin V and dead cell assay kit (Millipore, Billerica, MA, USA), and the results indicated that lycorine and siRNA_TCRP1The apoptosis rate (61.22% + -1.49%) of the HepG2 cell treated by the combination is higher than that (36.90% + -3.88%) of the HepG2 cell treated by the combination of lycorine and the control siRNA and the siRNA_TCRP1Sum of apoptosis rates (5.19% ± 0.31%) of HepG2 cells treated alone; lycorine and siRNA_TCRP1The apoptosis rate (58.24% + -4.37%) of the combined SMMC-7721 cell is higher than that (35.55% + -2.96%) of the SMMC-7721 cell and siRNA of the combined lycorine and control siRNA_TCRP1Sum of apoptosis rates (4.04% ± 0.35%) of SMMC-7721 cells treated alone (fig. 7). The siRNA targeting TCRP1 is shown to enhance the anti-liver cancer activity of lycorine, and the siRNA targeting TCRP1 and lycorine generate synergistic effect in the aspect of anti-liver cancer.

<110> Tianjin Chinese medicine university

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Claims (9)

1. The application of the Akt inhibitor in preparing the product for enhancing the anti-liver cancer activity of lycorine is LY 294002.
2. 2. Use according to claim 1, characterized in that: the liver cancer is hepatocellular carcinoma.
3. The application of the Akt inhibitor in preparing the product for enhancing the anti-hepatoma cell activity of lycorine is LY 294002.
4. 4. Use according to claim 3, characterized in that: the liver cancer is hepatocellular carcinoma.
5. The application of the Akt inhibitor and lycorine in preparing the anti-liver cancer product is LY 294002.
6. 6. Use according to claim 5, characterized in that: the anti-liver cancer product is an anti-liver cancer cell product.
7. 7. An anti-hepatoma product, characterized in that: the product contains an Akt inhibitor and lycorine, wherein the Akt inhibitor is LY 294002.
8. 8. The product of claim 7, wherein: the product for resisting liver cancer is a product for resisting liver cancer cells.
9. 9. The product according to claim 7 or 8, characterized in that: the liver cancer is hepatocellular carcinoma.

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