CN113893234A - Application of erianin in preparation of bladder cancer resistant medicine for targeted inhibition of AKT activation - Google Patents

Abstract

The invention provides application of erianin in preparation of an anti-bladder cancer medicament for targeted inhibition of AKT activation. The erianin can reduce the expression of P-Akt and AKT and up-regulate the expression of cell cycle related proteins P27 and P21, so that the cell cycle is blocked in the G1 stage, thereby inhibiting the proliferation of the T24 cell of bladder cancer. In the experiment, the erianin is found to have an inhibiting effect on the T24 cells of the bladder cancer, and show good time and concentration dependence, so that a theoretical basis is provided for further researching the action mechanism of the erianin on the bladder cancer, a new treatment scheme is provided for treating the bladder cancer, and a new candidate drug is provided for treating the bladder cancer.

Description

Application of erianin in preparation of bladder cancer resistant medicine for targeted inhibition of AKT activation

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to application of erianin in preparation of an anti-bladder cancer medicine for targeted inhibition of AKT activation.

Background

AKT, also known as Protein Kinase B (PKB), is a member of the serine/threonine protein kinase family discovered in 1991, and is present in mammals and is translated into three subtypes from three different genes: akt1, Akt2, and Akt3, play a crucial role in controlling cell survival and apoptosis. Its function is involved in cell cycle regulation, apoptosis initiation and other cell survival regulation, as well as in many important physiological and pathological processes such as angiogenesis, telomerase activity and cell invasiveness. It has been found that AKT is an important signaling molecule for tumorigenesis, and its subtype AKT2 is a 481 amino acid protein, and when its 308 th serine and 473 th threonine are phosphorylated, its kinase activity is increased, and it plays a role in promoting cell proliferation and inhibiting apoptosis. In the aspect of cell cycle regulation, activated AKT can directly phosphorylate down-stream target mTOR to activate the activated AKT, and the cell cycle is started. P21 and P27 are negative regulators of the cell cycle, and can stop the cell cycle and block proliferation. AKT negatively regulates the expression of P21 and P27, inactivates the P21 and P27 through phosphorylation, and inhibits the cell cycle retardation, thereby promoting the G1 stage development and accelerating the cell proliferation and differentiation. Because AKT plays an important role in gene expression regulation and apoptosis, AKT is used as a target for various tumor therapies.

Erianin is bibenzyl compound separated from herba Dendrobii, and has chemical name of 2-methoxy-5- [2- (3,4, 5-trimethoxy-phenyl) -ethyl ] -phenol. The study finds that the erianin can increase the accumulation of intracellular drugs, play the role of antitumor multidrug resistance, inhibit the growth of tumors in a plurality of ways and cause irreparable DNA damage and the like. The erianin not only has an inhibitory effect on osteosarcoma, but also can promote apoptosis of breast cancer cells by activating JNK/c-Jun signal pathways, and finally achieves the purpose of inhibiting cell proliferation. In recent years, erianin has attracted much attention as an antitumor agent having a high anticancer effect.

Although erianin, which has few toxic side effects, has been studied more extensively in the inhibition of many cancers, it has been studied less extensively in the anti-bladder cancer cell T24. Currently, it is not clear whether erianin can effectively target AKT activation, and thus inhibit the proliferation of bladder cancer cell T24.

Disclosure of Invention

The invention aims to solve the technical problems and provides application of erianin in preparation of a medicine for treating bladder cancer by targeted inhibition of AKT.

The technical scheme of the invention is as follows:

application of erianin in preparation of bladder cancer resistant drugs for targeted inhibition of AKT activation.

The erianin can reduce the expression of P-Akt and AKT and up-regulate the expression of cell cycle related proteins P27 and P21, so that the cell cycle is blocked in the G1 stage, thereby inhibiting the proliferation of the T24 cell of bladder cancer.

As a preferred technical scheme, the invention provides application of erianin in preparing a medicament for blocking the cell cycle of bladder cancer.

As a preferable technical scheme, the invention provides the application of erianin in preparing a medicament for up-regulating the expression level of cell cycle related proteins of bladder cancer, wherein the cell cycle related proteins are P27 or/and P21.

As a preferable technical scheme, the invention provides application of erianin in preparing a medicament for inhibiting the proliferation of T24 cells of bladder cancer.

As a preferable technical scheme, the invention provides application of erianin in preparing a medicament for inhibiting the clone formation of T24 cells of bladder cancer.

It is another object of the present invention to provide a product.

The product provided by the invention contains erianin as an active ingredient, and the application of the product is at least one of the following (1) to (4):

(1) targeted inhibition of AKT activation;

(2) blocking the bladder cancer cell cycle;

(3) up-regulating the expression level of bladder cancer cell cycle related protein;

(4) inhibiting proliferation of bladder cancer T24 cells;

(5) inhibiting the clone formation of the T24 cell of the bladder cancer.

Due to the adoption of the technical scheme, the invention has the beneficial effects that:

according to the invention, through research, erianin is found to have good in-vitro anti-tumor effect on bladder cancer cells T24, have obvious inhibition effect on bladder cancer cell T24 proliferation, and enhance the inhibition effect along with the increase of the drug concentration; the T24 cell can be blocked at the G0/G1 stage; when the concentration of the erianin is 15.6-125ng/mL, the expression content of the P-Akt protein can be reduced, the inhibition effect on the expression content of the Akt protein can be generated when the concentration is higher than 250ng/mL, and the expressions of cell cycle related proteins P21 and P27 can be up-regulated. The invention provides the targeted inhibition of the expression of AKT by the erianin which is an effective component of dendrobium, widens the medical application of the erianin, and provides a new treatment scheme for treating bladder cancer.

Drawings

FIG. 1 is a chemical structural diagram of erianin;

FIG. 2 is a graph of the results of an IC50 experiment; wherein A is an IC50 result chart of 24 h; b is a 48h IC50 result graph;

FIG. 3 is a graph of the inhibitory effect of erianin on bladder cancer T24 cell proliferation; wherein A is a cell viability experiment result graph; b is a result graph of cell proliferation inhibition rate;

FIG. 4 is a graph of the inhibitory effect of erianin on the clonogenic formation of T24 cells from bladder cancer; wherein A is a crystal violet staining result graph; b is a result graph of inhibition rate of different concentrations of erianin on T24 cells to clone formation;

FIG. 5 is a graph of the effect of erianin on the cell cycle of bladder cancer T24; wherein A is a flow cytometer detection result graph; b is a cell cycle statistical analysis result graph;

FIG. 6 is a graph showing the effect of erianin on the expression of T24 cyclin-associated protein in bladder cancer; wherein A is an immunoblotting experimental result diagram of the expression of cell cycle related protein by erianin; b is a result graph of quantized gray scale values of p21, p27, p-Akt and AKT.

Detailed Description

The specific embodiments and effects of the present invention will be illustrated by the following examples, but the scope of the present invention is not limited thereto.

Examples

Materials and methods

1. A cell line.

Human bladder cancer T24 cell line was purchased from the cell resource center of Shanghai bioscience research institute of Chinese academy of sciences.

2. Drugs and reagents.

Erianin, chemically 2-methoxy-5- [2- (3,4, 5-trimethoxy-phenyl) -ethyl ] -phenol, having the chemical formula shown in figure 1, available from Shanghai-derived leaf Biotech, Inc., cat #: b20844, CAS No.: 95041-90-0.

And the DMSO reagent is used for dissolving the erianin, and after the erianin is dissolved, filtered and sterilized, the erianin is stored at room temperature in a dark place for later use.

The reagents are commercially available in retail markets.

3. The main instrument is provided.

311 gas jacket type CO₂Incubator, Thermo Fisher Scientific, usa; infine M200 pro Nano Quant grating type continuous wavelength microplate reader, TECAN, Switzerland; elix3+30L +3YNERGY ultrapure water system, Millipore corporation, usa; allegra X-22R refrigerated centrifuge, Beckmann Kulter, USA; ChemiDoc XRS + gel imaging System, Bio-Rad, USA.

CCK-8 experiments.

(1) Taking bladder cancer T24 cells in logarithmic growth phase, and washing for 2-3 times by using D-hanks liquid at room temperature;

(2) after cells are normally digested by 0.25% pancreatin, the pancreatin is neutralized by a DMEM medium containing 10% FBS, and after cell suspension is collected, the cells are centrifuged at 2000rpm for 5 min;

(3) blowing and beating with serum-free DMEM medium to resuspend the cells with the density of 2 multiplied by 10³Per mL minuteUniformly distributing single cell suspension;

(4) inoculating into 96-well cell culture dish, and culturing at 37 deg.C with 5% CO₂An incubator;

(5) 24h after adherent growth of the cells, each group was intervened with different concentrations of erianin (7.8, 15.6, 31.2, 62.5, 125ng/mL) and the control group was given an equal volume of DMSO (i.e., 0. mu.g.L)^-1Erianin treated cells), cells were cultured for 2h,1d,2d,3d,4d, and 5 d;

(6) taking each pore plate cell, discarding the old culture medium, adding 100 mu L of CCK-8 solution into each pore, continuously culturing for 2h, and detecting the light absorption value at 450nm of each pore by using an enzyme-labeling instrument;

(7) the effect of different doses of erianin on stem prognosis of T24 cells on inhibition of cell proliferation was compared. Cell proliferation inhibition (%) (1-drug group OD450nm value/control group OD450nm value) × 100%. (ii) a

5. Clone formation experiments.

(1) After the cells were digested conventionally with pancreatin at room temperature, they were blown into a uniformly distributed single cell suspension in a DMEM medium at 2X 10 cells per well³Inoculating the density of each cell in a 6-hole plate, and continuously culturing in a constant-temperature incubator;

(2) adding erianin after culturing for 6 days to make final concentration 15.6, 31.2, 62.5, 125, 250ng/mL, replacing erianin with equal volume DMSO as control group, and setting 3 multiple wells at the same concentration;

(3) continuously culturing for 6 days, removing the upper layer culture medium, and gently washing the plate with PBS for 2 times;

(4) fixing with 4% paraformaldehyde for 15min, and discarding paraformaldehyde;

(5) dyeing the crystal violet solution for 15min, and repeatedly soaking and washing with clear water;

(6) drying in the air at room temperature, collecting and storing images;

(7) adding 2mL of 10% glacial acetic acid solution into each well to dissolve crystal violet, taking 100 mu L of each well and placing the well into a 96-well plate, and detecting the absorbance value of each well at the wavelength of 560nm by using an enzyme-labeling instrument. The clone inhibition (%) was (1-drug OD560nm value/control OD560nm value) × 100%.

6. And (4) analyzing the cell cycle.

(1) GetBladder cancer in logarithmic growth phase T24 cells, T24 cells at 5X 10 per well⁵The concentration of (2) is inoculated in a 6-hole plate, and each hole is 2 mL;

(2) after 24h of culture, the intervention is carried out by adding erianin with the concentration of 125ng/mL, and the control group is added with DMSO with the same volume for dissolving 125ng of erianin. Collecting cells by trypsinization after 24 h;

(3) washing cells for 2 times by using precooled PBS, then re-suspending the cells into single cell suspension by using the precooled PBS, slowly dripping precooled ethanol, slightly blowing, uniformly mixing, and then fixing at 4 ℃ overnight;

(4) taking out cells, centrifuging at 1000rpm for 10min, removing supernatant, washing with precooled PBS for 2 times, and resuspending cells with PBS;

(5) adding RNase A solution, and carrying out water bath at 37 ℃ for 30 min; centrifuging at 1000r for 10min, and discarding the supernatant;

(6) adding PI staining solution to resuspend cells, and incubating for 30min at 4 ℃ in a dark place;

(7) the excitation wavelength is 488nm through detection on a computer, and cell cycle distribution is analyzed by using FlowJo software.

7. And (4) performing western blotting.

7.1 extraction of cellular proteins.

(1) Culturing human bladder cancer T24 cells to logarithmic growth phase, removing the culture medium, and washing with PBS 3 times;

(2) precooling at 4 ℃, and then centrifuging for 10min at 12000rpm of a 4 ℃ centrifuge;

(3) adding RIPA cell lysate to an ice box, standing for 30min at 4 ℃, and collecting the cell lysate;

(4) taking 2 mu L of supernatant, and determining the protein concentration by using a BCA protein quantitative kit;

(5) adding the sample buffer solution into the protein sample, mixing, performing metal bath at 95 deg.C for 10min, standing on ice for 10min, and storing in a refrigerator at-20 deg.C.

7.2SDS-PAGE gel electrophoresis.

Separating gel 12%, concentrating gel 5%, and gel thickness 1.0mm, and performing vertical plate electrophoresis with discontinuous buffer system.

7.3 Membrane transfer, antibody incubation, and visualization

(1) Transferring the protein to an NC membrane by a semi-dry transfer method;

(2) placing the NC membrane after membrane conversion into ponceau working solution, and dyeing for 5 min;

(3) washing the dyed film with deionized water, and sequentially cutting the target protein and the internal reference protein according to the position of a protein Marker;

(4) washing ponceau @ on the membrane with TBST, and adding 5% skimmed milk powder prepared from TBST;

(5) sealing with 5% skimmed milk powder (prepared by TBST) at room temperature for 1 hr;

(6) incubating the beta-actin primary antibody at 4 ℃ overnight, and recovering the primary antibody the next day;

(7) washing membrane with TBST for 5 times, each time for 10 min;

(8) incubating the secondary antibody at room temperature for 1.5h, washing the membrane for 5 times by TBST, and each time for 10 min;

(9) preparing a proper amount of luminous liquid, and mixing the liquid A and the liquid B in equal volume;

(10) absorbing the redundant TBST on the NC membrane, dripping the prepared luminescent liquid, and reacting for 2-3 min;

(11) exposing, developing and fixing the X-ray film;

(12) beta-actin is used as protein contrast, and a Gel-Pro Analyzer image analysis system is adopted to calculate and analyze the absorbance value of the X-ray plate and analyze the result.

Experimental data processing

All data were statistically analyzed using SPSS18.0 software, data were expressed as mean ± standard deviation (X ± s), and mean comparisons among multiple samples were analyzed by one-way ANOVA for group comparisons. P <0.05 is statistically significant for differences.

Second, result in

Inhibition of T24 cell proliferation by erianin

The result is shown in figure 2, and the CCK-8 experiment result shows that erianin can obviously inhibit the proliferation of the bladder cancer cell T24, the IC50 at 24h is 217.6 +/-0.03 ng/mL, and the IC50 at 48h is 160 +/-0.04 ng/mL. As shown in figure 3, the intervention of different concentrations of erianin in cultured T24 cells for 2h,1d,2d,3d,4d and 5d respectively is determined by a CCK-8 method, all dose groups have obvious inhibition effects after the 3 rd day of intervention and show a dose effect dependent relationship, wherein the proliferation inhibition rate is the highest and the inhibition effect is the most obvious at the 5 th day.

Effect of (di) Erianin on T24 cell clonogenic

The results are shown in FIG. 4. The result after crystal violet staining can be shown in figure 4-A, and the positive staining is gradually reduced along with the gradual increase of the concentration of the erianin, which indicates that the inhibition effect of the erianin is gradually enhanced; compared with a control group through the result of OD value at 560nm, the erianin with 15.6, 31.2, 62.5, 125 and 250ng/mL has obvious inhibition effect on clone formation (p is less than 0.05). FIG. 4-B shows the inhibition of clone formation by erianin at various concentrations on T24 cells, and it was found that the inhibition of clone formation by erianin increases with increasing concentration on T24 cells.

Effect of (III) Erianin on the cell cycle

The results are shown in FIG. 5. Flow cytometry results and cell cycle detection analysis results show that the proportion of G1 cells is increased in the administration group compared with the control group, the proportion of S-phase and G2 cells is obviously reduced, and the difference has statistical significance (P < 0.05).

Effect of (tetra) Erianin on cell cycle-related protein expression

The result of the immunoblotting experiment of erianin on the expression of cell cycle related protein is shown in figure 6-A, and the result shows that after adding erianin stem with different concentrations, the expressions of cell cycle related protein P27 and P21 are gradually increased along with the increase of the concentration of the drug, and the protein expression of P-Akt is gradually reduced along with the increase of the concentration of the drug. The quantitative gray value results of P21, P27, P-Akt and AKT are shown in FIG. 6-B, and the results indicate that the expression of P21 and P27 proteins is up-regulated and the protein expression level of P-Akt is down-regulated after erianin intervenes in T24 cells; the AKT protein expression level has no obvious change when the concentration of the erianin is 15.6, 31.2, 62.5 and 125ng/mL, and the AKT protein expression is inhibited when the concentration of the erianin is 250 ng/mL.

According to the experimental result, the expression of p-AKT can be reduced by the erianin after the erianin is used for interfering the T24 cells of the bladder cancer; when the concentration of the erianin is 15.6, 31.2, 62.5 and 125ng/mL, the AKT protein expression content is not obviously changed, and when the concentration of the erianin is 250ng/mL, the AKT protein expression is inhibited, and simultaneously, the expressions of cell cycle related proteins P27 and P21 are up-regulated, so that the cell cycle is blocked in a G1 stage, and the proliferation of the bladder cancer T24 cells is inhibited.

In conclusion, erianin can effectively and targetedly inhibit AKT activation, thereby inhibiting the proliferation of the bladder cancer cell T24.

Finally, it should be noted that the above description is a detailed description of the present invention with reference to specific preferred embodiments, and the technical scope of the present invention is not limited to the description. For those skilled in the art to which the invention relates, various changes and modifications can be made without departing from the spirit of the invention, and several simple deductions or substitutions can be made, which shall be considered to belong to the protection scope of the invention.

Claims (7)

1. Application of erianin in preparation of bladder cancer resistant drugs for targeted inhibition of AKT activation.

2. The use of claim 2, wherein erianin inhibits the proliferation of T24 cells in bladder cancer by decreasing the expression of P-Akt, AKT and by upregulating the expression of cyclin-related proteins P27, P21, thereby arresting the cell cycle at G1.

3. Application of erianin in preparing medicine for blocking cell cycle of bladder cancer is provided.

4. The application of the erianin in preparing the medicine for up-regulating the expression level of the cell cycle related protein of the bladder cancer is characterized in that: the cell cycle related protein is P27 or/and P21.

5. Application of erianin in preparing medicine for inhibiting proliferation of bladder cancer T24 cell is provided.

6. Application of erianin in preparing medicine for inhibiting clone formation of bladder cancer T24 cell is provided.

7. A product, the active ingredient of which is erianin, is characterized in that the product is used as at least one of the following (1) to (4):

(1) targeted inhibition of AKT activation;

(2) blocking the bladder cancer cell cycle;

(3) up-regulating the expression level of bladder cancer cell cycle related protein;

(4) inhibiting proliferation of bladder cancer T24 cells;

(5) inhibiting the clone formation of the T24 cell of the bladder cancer.

Images