CN112336867A

CN112336867A - Composition of PKD inhibitor and anticancer drug and application thereof

Info

Publication number: CN112336867A
Application number: CN202011069659.4A
Authority: CN
Inventors: 陈娇; 陈红利; 崔博淼; 吕蝶; 康颖竹; 张平; 冯云
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2020-09-30
Filing date: 2020-09-30
Publication date: 2021-02-09

Abstract

The present invention provides a composition comprising a protein kinase D inhibitor and an anti-cancer agent. The research of the invention finds that the combined treatment mode can improve the sensitivity and specificity of cancer treatment, provide a brand new visual angle for clinical treatment and play a more important role, thereby having wide application value.

Description

Composition of PKD inhibitor and anticancer drug and application thereof

Technical Field

The invention belongs to the technical field of oral squamous cell carcinoma treatment, and relates to an application of a PKD (PKD) small molecule inhibitor in blocking drug resistance of oral squamous cell carcinoma cells.

Background

Oral Squamous Cell Carcinoma (OSCC) is one of the most common malignant tumors of the head and neck, accounting for about 90% or more, with about 500000 new cases of OSCC per year, and the incidence rate thereof has been increasing year by year in recent years. The 5-year survival rate of late-stage OSCC has been only 40% -50% over the last 30 years.

Paclitaxel (PTX), a cytotoxic agent that stabilizes tubulin, is widely used to treat a variety of tumors. Unlike traditional antitumor drugs, PTX acts as an antitumor agent by interfering with the stability of tubulin and inhibiting division and proliferation of tumor cells.

The multi-drug resistance (MDR) of tumor refers to the phenomenon that tumor cells generate cross-resistance to other anti-tumor drugs with completely different structures and action mechanisms while generating resistance to one anti-tumor drug, and is one of the main reasons for hindering the success of tumor therapy and also an important factor for causing the recurrence of tumor therapy.

Cancer treatment has been a worldwide problem, and anti-tumor drugs have been continuously developed. Cytotoxic drugs are still the subject of tumor treatment drugs at present, but have the problems of poor curative effect on solid tumors, large adverse reaction, easy generation of drug resistance and the like.

Disclosure of Invention

The drug resistance phenomenon exists in more than 90% of cancer deaths, and the invention provides a solution for drug-resistant tumors aiming at the great problem of tumor resistance in clinical tumor treatment.

In recent years, small molecule inhibitors targeted to cancers as a new treatment means are becoming research and development of tumor treatment drugs. Several new small molecule Inhibitors targeting Protein Kinase D (PKD) have recently been developed, including CRT0066101, CRT5, CID755673 and kb-NB142-70(Evans IM, Bagherzadeh A, Charles M, Raynham T, Ireon C, Boakes A et al, Characterisation of the Biological effects of a novel protein kinase D inhibitor in biochemical cells [ J ]. Biochem J, 2010; 429:565-572.George KM, Frantz MC, Bravo-Altamirano K, Lavalle CR, Tandon M, Leimgruber S et al. Design, Synthesis, and Biological Evaluation of PKD Inhibitors [ J ]. pharmaceuticals 228; 3: 2011.2011.). These compounds show an effect of inhibiting PKD activity in vitro cell experiments. At present, no application report of the PKD small molecule inhibitor in inhibiting oral squamous cell carcinoma drug-resistant cells exists.

The present invention provides a composition comprising a protein kinase D inhibitor and an anti-cancer agent.

The compatible dosage of the protein kinase D inhibitor and the anticancer drug can be appropriately adjusted by the specific use environment, for example, the mass ratio of the protein kinase D inhibitor to the anticancer drug can be 20-60:1, and further can be 35-45:1, specifically 40: 1. 41:1, 42:1, 43:1, etc., are not limited to the above ranges.

The invention also provides application of the composition in preparing a product for treating squamous carcinoma.

Wherein the squamous cell carcinoma is a drug-resistant squamous cell carcinoma.

The squamous cell carcinoma is selected from paclitaxel or docetaxel, or structural analogues or pharmaceutically acceptable salts of paclitaxel or docetaxel.

Wherein the squamous carcinoma is selected from oral squamous carcinoma.

Further, the cancer cells can be selected from tongue squamous carcinoma.

The invention also provides application of the composition in preparing a drug-resistant cancer cell product.

Further, the drug-resistant cancer cell is selected from cancer cells resistant to drugs selected from paclitaxel or docetaxel, or structural analogs or pharmaceutically acceptable salts of paclitaxel or docetaxel.

Wherein the cancer cell is selected from squamous carcinoma.

The invention also provides application of the protein kinase D inhibitor and the anti-cancer drug in preparing a combined drug for treating squamous cell carcinoma or anti-squamous cell carcinoma.

The invention also provides the use of a protein kinase D inhibitor in a product for treating oral squamous cell carcinoma.

Wherein the protein kinase D inhibitor is selected from one or more of CRT006610, CRT5, CID755673 and kb-NB 142-70.

Wherein the anticancer drug is selected from paclitaxel or docetaxel, or structural analogues or pharmaceutically acceptable salts of paclitaxel or docetaxel.

The composition or the product of the invention also comprises pharmaceutically acceptable auxiliary materials or auxiliary components.

The auxiliary materials are general names of all additional materials except the main medicine in the medicinal preparation, and the auxiliary materials have the following properties: (1) no toxic effect on human body and few side effects; (2) the chemical property is stable and is not easily influenced by temperature, pH, storage time and the like; (3) has no incompatibility with the main drug, and does not influence the curative effect and quality inspection of the main drug; (4) does not interact with the packaging material.

The auxiliary component has certain physiological activity, but the addition of the component does not change the dominant position of the composition in the disease treatment process, but only plays an auxiliary effect, and the auxiliary effects are only utilization of the known activity of the component and are auxiliary treatment modes which are conventional in the field of medicine. If the auxiliary components are used in combination with the composition of the present invention, the protection scope of the present invention should still be included. Such as taurine.

Pharmaceutically acceptable adjuvants, such as cellulose and its derivatives (such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), and emulsifiers (such as propylene glycol, glycerol, mannitol, sorbitol, etc.)

) Wetting agents (e.g., sodium lauryl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, and the like.

The invention finds that a PKD small molecule inhibitor CRT0066101 can inhibit the phosphorylation level of protein kinase D2(PKD2), enhance the sensitivity of oral squamous cell carcinoma drug-resistant cells to chemotherapy drugs and induce apoptosis, thereby effectively inhibiting the generation of oral squamous cell carcinoma drug-resistant cells. On the basis, the combined treatment mode can improve the sensitivity and specificity of OSCC treatment, provides a brand-new visual angle for clinical treatment and plays a more important role, thereby having wide application value.

The small molecule inhibitor CRT0066101 did not show any signs of distress in the animal xenograft model (indicating no toxic side effects) and no side effects on normal tissue structure function (i.e. effects on catheter function) were found.

Drawings

Fig. 1 is a flowchart of an application method of a small molecule inhibitor CRT0066101 in blocking drug resistance of oral squamous cell carcinoma.

FIG. 2 is a flow chart of a method for establishing the human oral squamous carcinoma drug-resistant cell line Cal-27/PTX provided by the embodiment of the invention.

FIG. 3 is a graph of the inhibition rate of the chemotherapeutic drug Paclitaxel (PTX) on Cal-27/PTX cells at various concentrations provided by the practice of the present invention.

FIG. 4 is a schematic representation of PKD expression in Cal-27/PTX cells provided by an embodiment of the present invention.

Figure 5 is a graph of the effect of the small molecule inhibitor CRT0066101 of different concentrations of the present invention on PKD2 phosphorylation activity.

FIG. 6 is a graph showing the results of small molecule inhibitor CRT0066101 to enhance the sensitivity of Cal-27/PTX cells to PTX.

Wherein, figure 2 shows that the invention successfully establishes the human oral squamous cell carcinoma Cal-27/PTX drug-resistant cell strain by adopting a drug continuous contact and concentration increasing induction method and inducing the Cal-27 cells for 10 months by PTX.

FIG. 3 shows that IC50 for Cal-27/PTX increased from 2.999ng/ml to 57.77ng/ml in parental cells, indicating that Cal-27/PTX cells developed resistance to PTX with an RI of 19.26 being intermediate (RI >20 being high, 5> RI >15 being intermediate, RI <5 being low).

Fig. 4 shows that protein kinases PKD1, PKD2, PKD3 are differentially expressed in Cal-27/PTX resistant cells and parental cells Cal-27, where PKD1 and PKD3 were not significantly differentially expressed in resistant cells and parental cells, whereas PKD2 was significantly elevated in resistant cells (P × <0.0001), suggesting that PKD2 has an important role in oral squamous cell carcinoma cell resistant cells.

Figure 5 shows that the extent of activation of PKD2 phosphorylation was significantly reduced after 24 hours of CRT0066101 treatment of Cal-27/PTX resistant cells at different concentrations, whereas there was essentially no difference in PKD2 expression, with a significant reduction in PI (P x 0.0001) of Cal-27/PTX resistant cells treated with CRT0066101 at a concentration of 3 μ M for 24 hours. The small molecule inhibitor CRT0066101 can inhibit the phosphorylation level of PKD2 kinase, and the CRT0066101 with the concentration of 3 mu M can be used as the optimal concentration of the subsequent experiment.

Fig. 6 shows that the drug-resistant cell apoptosis is obviously increased after 24 hours of combined action of CRT with the concentration of 3 muM and paclitaxel with the concentration of 30ng/ml, which shows that after the PKD small-molecule inhibitor is treated by inhibition, the phosphorylation level of PKD2 kinase of the drug-resistant cell is obviously inhibited, and the sensitivity of the drug-resistant cell to the chemotherapeutic drug PTX is obviously increased.

Detailed Description

In the specific embodiment of the invention, the PKD small molecule inhibitor is CRT 0066101; the oral squamous cell carcinoma drug-resistant cells are oral squamous cell carcinoma cells resistant to the targeted drug; the targeted drug is selected from paclitaxel; the oral squamous carcinoma drug-resistant cells comprise paclitaxel-resistant cells Cal-27/PTX. The oral squamous carcinoma cell Cal-27 is frozen and stored in a laboratory, and is cultured in RPMI-1640 complete culture solution (containing 10 percent (volume fraction) of calf serum and 10 percent of penicillin)⁵U/L, streptomycin 100mg/L]。

Example 1

The application method of the PKD small molecule inhibitor provided by the invention in blocking the drug resistance of oral squamous cell carcinoma cells comprises the following steps (as shown in figure 1):

s101, establishing a human oral squamous carcinoma drug-resistant cell line Cal-27/PTX;

s102, CCK8 testing the cell inhibition rate of different concentrations of PTX to Cal-27/PTX

S103, detecting the PKD expression condition of the Cal-27/PTX cells by using Western blot;

s104, detecting the phosphorylation activation expression condition of the CPD small molecule inhibitor CRT0066101 with different concentrations on Cal-27/PTX cell PKD2 by Western blot;

s105, detecting the apoptosis rate of Cal-27/PTX cells under the combined action of CRT0066101 and Paclitaxel (PTX) by flow cytometry.

The method for establishing the human oral squamous carcinoma drug-resistant cell line Cal-27/PTX comprises the following steps:

culturing Cal-27 cells in logarithmic growth phase to 70-80% of adherent cells, continuously inducing for 24 hours by using paclitaxel with the concentration of 20ng/ml as an initial concentration, withdrawing the culture medium containing the medicine, replacing the culture medium without the medicine, changing the culture medium every day to remove dead cells until the cells recover to grow, continuously passaging until the state of the cells is good, repeating the operation for three times, gradually increasing the concentration of the paclitaxel, and repeating the operation until the final paclitaxel induction concentration is 400ng/ml, wherein the total period is 10 months. Finally obtaining the drug-resistant cell strain Cal-27/PTX.

A method for detecting the cytostatic rate of different concentrations of PTX on Cal-27/PTX by CCK8 comprises the following steps:

step one, plate preparation: collecting Cal-27 and Cal-27/PTX cells in logarithmic growth phase, performing trypsinization centrifugation, collecting, counting with culture medium heavy suspension cells, and making into 3 × 10⁴Each cell suspension was seeded in a 96-well plate at 100. mu.l per well.

Step two, preparing PTX with different concentrations: PTX stock solutions were diluted with medium to 10 concentration gradients of 5ng/ml, 10ng/ml, 20ng/ml, 40ng/ml, 50ng/ml, 100ng/ml, 200ng/ml, 300ng/ml, 400ng/ml, 500ng/ml, respectively, and 5 replicate wells were set for each concentration gradient.

Step three, drug treatment: adherent Cal-27 and Cal-27/PTX cell culture media were aspirated off, 200 μ l of media was added to each well, with different concentrations of PTX-containing media added to the dosed group, and no-dosed media added to the blank and negative control groups.

Step four, detecting by CCK 8: after 72 hours of co-culture of the drug and the cells, the medium in the wells was aspirated, 100. mu.l of fresh medium (containing 10. mu.l of CCK8 solution) was added thereto, the culture was continued for 4 hours, the absorbance at 450nm (i.e., OD value) was measured with a microplate reader, a curve was drawn, and IC of the cells to PTX was calculated using Graphpad Prism software₅₀And calculating the drug resistance index RI.

RI ═ drug resistant cell IC₅₀Parental cell IC₅₀

The method for detecting the PKD expression condition of the oral squamous cell carcinoma drug-resistant cells by Western blot comprises the following steps:

step one, preparing a protein sample: cal-27, Cal-27/PTX cells at 1X 10⁶Cells were cultured in 6-well plates to 90% density, then trypsinized and harvested. After washing the cells twice with pre-cooled phosphate buffered saline PBS, 100. mu.l of lysine buffer (50mM Tris-HCl, pH 8.0; 5mM EDTA; 150mM NaCl; 0.5% Nonidet P-40; 0.5mM PMSF; and 0.5mM DTT) was added to lyse the cells at 4 ℃ for 30 minutes, the lysate was transferred to a new Ep tube, centrifuged at 4 ℃ for 12500g for 20 minutes, and the supernatant was collected and transferred to another new Ep tube. A small amount of supernatant was taken and protein content was measured using BCA protein content assay kit. 50 μ g of the mixture was added to a 5 Xnodding buffer and mixed, boiled in a water bath at 100 ℃ for 5 minutes (to denature the protein) and stored at-20 ℃ for further use.

Step two, SDS-PAGE electrophoresis: the concentration of the separation gel used in the invention is 8%, the concentration of the concentration gel is 5%, the 80V constant pressure is 30 minutes, and the 110V constant pressure is 1 hour-2 hours.

Step three, film transferring: after electrophoresis, the gel was taken out, soaked in a transfer buffer (25mM Tris base, 0.2M glycine, 20% methanol pH 8.5) for 30 minutes, and at the same time, a PVDF membrane having an area slightly larger than that of the gel was soaked in methanol for 5 minutes (for activating the membrane), and then soaked in the transfer buffer for 20 minutes. The membrane is clamped between two layers of filter paper by glue, and is placed in a semi-dry electric transfer tank after being pressed and fixed, and the membrane is transferred for 1 hour at a constant voltage of 20V.

Step four, antigen blocking: after transfer, PVDF membrane at 37 degrees C blocking 1 hours (blocking solution: 5% skimmed milk powder and containing 0.1% Tween 20 TBS), or 4 degrees C blocking overnight, TBST (1X TBS, 0.1% Tween-20) washing membrane for 10 minutes.

Step five, adding a primary antibody: adding 5% of skimmed milk powder respectively to dilute to primary antibody with proper concentration: PKD1 antibody (1: 1000), PKD2 antibody (1: 1000), PKD3 antibody (1: 1000), β -actin antibody (1: 1000), incubated at room temperature for 2 hours or overnight at 4 ℃, and membrane washed with TBST for 15 minutes × 3 times.

Step six, adding a secondary antibody: 5% skimmed milk powder was added to dilute to a suitable concentration of horseradish peroxidase-labeled secondary goat-anti-rabbit antibody (1:2000) and secondary goat-anti-mouse antibody (1:2000), respectively, and the mixture was incubated at 37 ℃ for 1 hour and washed with TBST for 15 minutes and 3 times.

Step seven, chemiluminescence: the images were developed, exposed, developed, and collected by Chemicoc XRS (BioRad) using ECL luminophore reagent (EasyECL Western blot kit).

A method for detecting the phosphorylation activation expression of a PKD2 in Cal-27/PTX cells by using a PKD small molecule inhibitor CRT0066101 with different concentrations through Western blot, which comprises the following steps:

step one, preparing a protein sample: Cal-27/PTX cells at 1X 10⁶After culturing cells to 70% density in a 6-well plate, adding different concentrations of small molecule inhibitor CRT0066101 (1. mu.M, 2. mu.M, 3. mu.M, 5. mu.M) for 24 hours, digesting with pancreatin and collecting cells. After washing the cells twice with pre-cooled phosphate buffered saline PBS, 100. mu.l of lysine buffer (50mM Tris-HCl, pH 8.0; 5mM EDTA; 150mM NaCl; 0.5% Nonidet P-40; 0.5mM PMSF; and 0.5mM DTT) was added to lyse the cells at 4 ℃ for 30 minutes, the lysate was transferred to a new Ep tube, centrifuged at 4 ℃ for 12500g for 20 minutes, and the supernatant was collected and transferred to another new Ep tube. A small amount of supernatant was taken and protein content was measured using BCA protein content assay kit. 50 μ g of the mixture was added to a 5 Xnodding buffer and mixed, boiled in a water bath at 100 ℃ for 5 minutes (to denature the protein) and stored at-20 ℃ for further use.

Step five, adding a primary antibody: adding 5% of skimmed milk powder respectively to dilute to primary antibody with proper concentration: PKD2 antibody (1: 1000), p-PKD2 antibody (1: 1000), β -actin antibody (1: 1000), incubated at room temperature for 2 hours or overnight at 4 ℃ and TBST washed 15 minutes X3 times.

Step seven, chemiluminescence: the images were developed with ECL luminophore reagent (EasyECL Western blot kit), exposed, developed, and collected by Chemicoc XRS (BioRad), and the Phosphorylation Index (PI) was calculated.

PI＝Density of phospho-PKD2 band/density of total PKD2 band

A method for detecting the apoptosis rate of Cal-27/PTX cells co-acted by CRT0066101 and Paclitaxel (PTX) by flow cytometry, comprising:

step one, preparing a cell sample: Cal-27/PTX cells at 1X 10⁶Density in 6-well plates, after culturing cells to 70% density, 3 μ M of CRT0066101 and 30ng/ml paclitaxel were added separately/co-acted for 24 hours, trypsinized without EDTA and the cells were harvested.

And step two, washing the collected cells twice by using PBSF (PBS + 5% FBS), adding 500 mu l of Binding Buffer in a Kjeldahl apoptosis kit to resuspend the cells, adding 5 mu l of Annexin V-FITC and 5 mu l of PI, incubating for 15 minutes at room temperature in a dark place, and detecting by using a flow cytometer FC 500.

The experimental results are as follows:

cytostatic Rate of different concentrations of PTX to Cal-27/PTX

Referring to FIG. 3, Cal-27/PTX cells were aligned at 3X 10³Density seeds are cultured in a 96-well plate overnight, PTX (5 ng/ml, 10ng/ml, 20ng/ml, 40ng/ml, 50ng/ml, 100ng/ml, 200ng/ml, 300ng/ml, 400ng/ml and 500ng/ml) with different concentrations is added for respectively acting for 72 hours, and IC of Cal-27 and Cal-27/PTX cells is detected by a CCK8 method₅₀And a resistance index RI. Specifically, IC50 of Cal-27/PTX increased from 2.999ng/ml of parental cell to 57.77ng/ml, and the final resistance index RI was 19.26 (RI)>20 is highly resistant, 5>RI>15 moderate drug resistance, RI<5 is low drug resistance), so the strain Cal-27/PTX cell is moderately resistant to PTX.

PKD expression in drug-resistant cells Cal-27/PTX

Referring to FIG. 4, Cal-27/PTX cells were aligned at 1X 10⁶Cells were cultured in 6-well plates to 90% density, then trypsinized and harvested. Western blot detection of PKD1, PKD2 and PKD3 expression in two groups of cells. The expression of PKD2 was significantly higher in the Cal-27/PTX cells of the inventive method than in the parent Cal-27 (P.about.. times.<0.0001)。

PKD small molecule inhibitor CRT0066101Cal-27/PTX cell PKD2 phosphorylation activation expression conditions at different concentrations

Referring to FIG. 5, Cal-27/PTX cells were grown at 1X 10⁶After culturing cells to 70% density in a 6-well plate, adding different concentrations of small molecule inhibitor CRT0066101 (1. mu.M, 2. mu.M, 3. mu.M, 5. mu.M) for 24 hours, digesting with pancreatin and collecting cells. Western blot was used to detect the phosphorylation activation and expression of PKD2 in the cells treated differently. Specifically, 3 μ M of CRT0066101 treated Cal-27/PTX cells for 24 hours, the reduction in PI index was most pronounced and the results were statistically significant (P ×) by analysis of variance<0.0001)。

Apoptosis rate of Cal-27/PTX cells by cooperation of CRT0066101 and Paclitaxel (PTX)

Referring to FIG. 6, Cal-27/PTX cells were grown at 1X 10⁶Density in 6-well plates, after culturing cells to 70% density, 3. mu.M CR was addedT0066101 and 30ng/ml PTX (mass ratio of the two after conversion: 1233.9 ng: 30ng) were separately/co-acted for 24 hours, digested with trypsin without EDTA and the cells were collected. Flow cytometry was used to detect the apoptosis rate in the different treatment groups. Specifically, the apoptosis rate (97.27%) of the cells affected by the CRT0066101+ PTX is obviously higher than that of the cells affected by the CRT0066101 (13.61%) or the PTX (23.27%), and the result has statistical significance by variance analysis.

Example 2

Mixing CRT0066101 and PTX with proper filler, and making into capsule.

Example 3

CRT0066101 and PTX are taken, and proper fillers, adhesives and lubricants are taken to prepare granules.

Example 4

Preparing CRT0066101, proper filling agent, adhesive and lubricant into tablet A; preparing PTX, proper filler, adhesive and lubricant into tablet B; tablets a and B are packaged or used in combination.

Example 5

CRT0066101 and PTX are taken, and proper solvent, cosolvent and the like are taken to prepare the injection.

Claims

1. A composition characterized by: it includes protein kinase D inhibitors and anti-cancer drugs.

2. The composition of claim 1, wherein: the protein kinase D inhibitor is selected from one or the combination of more than two of CRT006610, CRT5, CID755673 and kb-NB 142-70; the anticancer drug is selected from paclitaxel or docetaxel, or structural analogues or pharmaceutically acceptable salts of paclitaxel or docetaxel.

3. The composition of claim 2, wherein: the mass ratio of the protein kinase D inhibitor to the anticancer drug is 20-60: 1; further selected from 35-45: 1.

4. Use of a composition according to any one of claims 1 to 3 in the manufacture of a product for the treatment of squamous carcinoma.

5. Use according to claim 4, characterized in that: the squamous cell carcinoma is a drug-resistant squamous cell carcinoma; further, the squamous cell carcinoma is a squamous cell carcinoma which generates drug resistance to paclitaxel or docetaxel, or structural analogues or pharmaceutically acceptable salts of paclitaxel or docetaxel.

6. Use according to claim 4 or 5, characterized in that: the squamous carcinoma is selected from oral squamous carcinoma; further, squamous cell carcinoma of tongue.

7. Use of the composition of any one of claims 1-3 in the preparation of a drug-resistant cancer cell product; further, the drug-resistant cancer cell is selected from cancer cells resistant to drugs selected from paclitaxel or docetaxel, or structural analogs or pharmaceutically acceptable salts of paclitaxel or docetaxel.

8. Use according to claim 7, characterized in that: the cancer cell is selected from squamous carcinoma.

9. Use of a protein kinase D inhibitor and an anti-cancer agent in the preparation of a combination for the treatment of squamous cell carcinoma or anti-squamous cell carcinoma.

10. Use of a protein kinase D inhibitor in a product for the treatment of oral squamous cell carcinoma.