Cell penetrating peptide Y119E and its application for inhibiting cisplatin-resistant tumor growth

Abstract

This invention relates to a cell-penetrating peptide Y119E and its application in inhibiting the growth of cisplatin-resistant tumors. The invention is characterized by using the cell-penetrating peptide TAT as a carrier to deliver a cell-penetrating peptide containing PGAM1 phosphorylated at position Y119 into tumor cells. The cell-penetrating peptide containing Y119 phosphorylated peptide (Y119E) has the sequence YGRKKRRQRRR-KIWRRSEDVP. The short cell-penetrating peptide Y119E is used in the drug application of inhibiting the growth of cisplatin-resistant tumors. It can effectively inhibit tumor growth subcutaneously in nude mice.

Description

Application of cell-penetrating peptide Y119E and its inhibition of cisplatin-resistant tumor growth Technical Field

[0001] This invention relates to a cell-penetrating peptide Y119E and its application in inhibiting the growth of cisplatin-resistant tumors, belonging to the medical field. Background Technology

[0002] 1. The anticancer drug cisplatin inhibits tumor cell proliferation by damaging DNA.

[0003] Cisplatin, as a first-generation platinum-based antitumor drug, has a history dating back to the 18th century, when it was used by scientists as an inert electrode due to its chemical inertness. 1,2 It wasn't until Barnett Rosenberg discovered the inhibitory effect of cisplatin on cell proliferation in 1965 that platinum gradually came into the public eye as an anticancer drug, and after clinical trials, it was approved for marketing by the U.S. Food and Drug Administration in 1978. 1,2 It was subsequently widely used in the treatment of malignant tumors such as testicular cancer, ovarian cancer, lung cancer, and head and neck tumors, occupying an important position among chemotherapy drugs. Especially in the treatment of testicular cancer and ovarian cancer, the initial cure rates were approximately 100% and 85%, respectively. 2 After being taken up by cells, cisplatin undergoes hydrolysis and activation, subsequently targeting DNA molecules directly. Cisplatin coordinates with the 7th atom of guanine and adenine within the DNA molecule, forming intra-chain cross-linking products. This causes the DNA to remain unwound or bent, reducing its stability and preventing replication. 1 Furthermore, cisplatin can also induce the production of ROS (reactive oxygen species) in cells, further leading to DNA damage and apoptosis. Cisplatin's simple structure, well-defined mechanism, low toxicity, broad anticancer spectrum, strong efficacy, and synergistic effects with various antitumor drugs have successfully sparked a surge of research into platinum-based drugs, and it remains a first-line treatment for solid tumors in clinical practice.

[0004] 2. DNA damage repair systems increase tumor cell resistance to cisplatin.

[0005] While cisplatin has been very successful in treating cancers such as lung cancer, the development of cisplatin resistance in cells remains a major challenge in platinum-based drug research. Once resistance develops in the human body, the drug's efficacy decreases, requiring increased dosages or replacement of the drug, which increases cisplatin's cytotoxicity and other negative effects. Existing research has explored some mechanisms of resistance in vitro, mainly attributing them to: firstly, the potential inactivation of cisplatin by intracellular proteins or small biomolecules; and more importantly, the repair of cisplatin-DNA cross-linked products by the intracellular DNA damage repair system. 3Most cells possess DNA damage repair mechanisms, meaning that repair proteins exist to repair damaged DNA. Cisplatin's anticancer effect manifests in disrupting the normal structure of DNA. Therefore, to successfully kill cancer cells, it is essential to overcome the repair proteins' resistance to cisplatin-DNA cross-linking products. Consequently, the mechanism of cisplatin resistance in clinical tumors remains a highly valuable scientific question for clinical research. Investigating the mechanism of cisplatin resistance in cells and finding methods to inhibit cellular resistance is the best chemosensitization strategy. Combining cisplatin with resistance reversal drugs will more effectively promote the application of cisplatin.

[0006] 3. Aerobic glycolysis provides the material support and reducing power for DNA repair in tumor cells.

[0007] The continuous division of tumor cells poses a challenge to their metabolism. 4 First, there is a need for a doubling of essential cellular components, i.e., enhanced synthesis of biomolecules; second, a sufficient energy supply is required; and third, sufficient reducing power is needed to maintain intracellular redox balance. To cope with the challenges posed by proliferation, tumor cells undergo metabolic reprogramming to meet the material and other demands of rapid proliferation. Compared to differentiated cells, rapidly proliferating tumor cells, even under oxygen-sufficient conditions, consume large amounts of glucose through glycolysis rather than aerobic oxidation, producing large amounts of lactic acid. This metabolic pathway is known as "aerobic glycolysis." 5-9 This is also known as the "Warburg effect". 5-9Aerobic glycolysis provides the following advantages for the rapid proliferation of tumor cells: First, the glycolytic metabolic pathway has two very important branches: ① the pentose phosphate pathway, which enters through the intermediate product glucose-6-phosphate. Its main metabolites include ribose-5-phosphate (R-5-P) and reduced coenzyme II (NADPH). R-5-P is a major precursor molecule for nucleotide biosynthesis; while NADPH can provide reducing power for the synthesis of biomolecules and can also be used to balance reactive oxygen species generated during rapid cell proliferation; ② the serine synthesis pathway, which enters through the intermediate product 3-phosphoglycerate. Its main metabolites are serine and glycine, which can be used for protein biosynthesis and can also produce one-carbon units during the conversion of serine to glycine for nucleotide biosynthesis. Therefore, the pentose phosphate pathway and the serine synthesis pathway, as important branches of glycolysis, both participate in nucleotide synthesis, providing indispensable material guarantees for DNA replication and damage repair. In addition, NADPH produced in the pentose phosphate pathway can effectively combat ROS and reduce ROS-induced DNA oxidative damage. All of the above evidence indicates that tumor metabolic reprogramming is involved in regulating cellular DNA damage repair and is likely to further participate in regulating the process of radiotherapy and chemotherapy resistance related to DNA damage repair.

[0008] 4. PGAM1 is a key regulatory enzyme that coordinates aerobic glycolysis and lateral metabolism.

[0009] Aerobic glycolysis is regulated by metabolic enzymes. Studies have shown that changes in the expression or activity of glycolytic metabolic enzymes are closely related to rapid tumor proliferation, metastasis, immune escape, or clinical drug resistance, potentially serving as therapeutic targets for cancer. Phosphoglycerate mutase 1 (PGAM1) is one of the metabolic enzymes in the glycolytic pathway, catalyzing the interconversion of 3-phosphoglycerate (3-PG) and 2-phosphoglycerate (2-PG). Because it catalyzes a reversible reaction, PGAM1 has long been overlooked. However, recent studies have shown that PGAM1 expression is negatively regulated by the tumor suppressor TP53, and it is widely highly expressed and significantly activated in various tumors, such as lung cancer, breast cancer, cholangiocarcinoma, clear cell renal cell carcinoma, prostate cancer, and glioma. 10,11 Clinical data analysis revealed that PGAM1 expression levels were negatively correlated with cancer patient prognosis and positively correlated with tumor grade and severity, playing a crucial role in tumor progression. The underlying mechanism is that PGAM1 not only ensures the smooth progress of glycolysis, but more importantly, its substrate 3-PG is a competitive inhibitor of G6PD, a key enzyme in the pentose phosphate pathway, while its product 2-PG is an activator of PHGDH, a key enzyme in the serine synthesis pathway. Activation of PGAM1 leads to downregulation of 3-PG levels, relieving inhibition of the pentose phosphate pathway, while simultaneously upregulating 2-PG levels, activating the serine synthesis pathway. 10,11Therefore, although PGAM1 is not a rate-limiting enzyme, it can simultaneously regulate the branching pathways of glycolysis, promote DNA damage repair, resist oxidative stress damage, and is closely related to processes such as tumor proliferation and metastasis, making it an attractive target for cancer treatment.

[0010] 5. The activity of PGAM1 is positively correlated with cisplatin resistance in non-small cell lung cancer.

[0011] Recent studies have shown that high expression of PGAM1 in non-small cell lung cancer (NSCLC) patients is significantly associated with poor prognosis. The PGAM1 inhibitor HKB99 effectively inhibits the proliferation and migration of NSCLC and induces apoptosis. As mentioned above, PGAM1 can enhance two branches of aerobic glycolysis metabolism: the pentose phosphate pathway and the serine synthesis pathway. These pathways provide material support and reducing power for tumor cell DNA repair, potentially reversing resistance to chemotherapeutic drugs such as cisplatin. This establishes a link between high PGAM1 expression in NSCLC and cisplatin resistance. Our previous results showed that PGAM1 is highly expressed and upregulated in NSCLC A549 cells and tissues, promoting the production of nucleotides and NADPH. Inhibiting PGAM1 expression or reducing PGAM1 activity increases the DNA damage caused by cisplatin to A549 / DDP cells, thus enhancing the antitumor effect of cisplatin on A549 / DDP cells.

[0012] Cell transmembrane peptides (CPPs) are a large class of sequences consisting of 10–30 amino acids, also known as protein transduction domains or transduction peptides. To date, discovered CPPs include Pep-1, pVEC, MAP, pAntp, Transportan, Polyarginines, and TAT. Among these, TAT is a classic CPP with high transmembrane penetration efficiency. 12,13 TAT contains 11 amino acids with the amino acid sequence YGRKKRRQRRR. TAT can be located in both the cell nucleus and cytoplasm and is currently widely used as a delivery tag for biological macromolecules. 14 . Summary of the Invention

[0013] The purpose of this invention is to provide a cell-penetrating peptide Y119E and its application in inhibiting the growth of cisplatin-resistant tumors. Based on the molecular mechanism of PGAM1-PKM2 binding, a cell-penetrating peptide (Y119E) that mimics the phosphorylation of Y119 was developed based on the sequence of PGAM1. It is known that TAT itself has no effect on cell proliferation and survival, which provides a guarantee for the drug of cell-penetrating peptide. Furthermore, the cell-penetrating peptide can effectively inhibit the interaction of PGAM1-PKM2, thereby inhibiting the growth of cisplatin-resistant tumors.

[0014] The technical solution of the present invention is implemented as follows: a cell permeation peptide Y119E, characterized in that: based on the sequence of PGAM1, the cell permeation peptide TAT is used as a carrier to transport the cell permeation peptide containing the Y119 phosphorylation site into the cell, the cell permeation peptide (Y119E) containing Y119 phosphorylation has the sequence YGRKKRRQRRR-KIWRRSEDVP.

[0015] The aforementioned cell-penetrating peptide Y119E inhibits the growth of cisplatin-resistant tumors.

[0016] The cell-penetrating peptide Y119E inhibits tumors including non-small cell lung cancer.

[0017] The positive effect of this invention is that it can effectively inhibit the interaction between PGAM1 and PKM2, thereby inhibiting the growth of cisplatin-resistant tumors. Intraperitoneal injection of the cell-penetrating peptide Y119E significantly inhibited the growth rate of cisplatin-resistant tumor cells under the skin of nude mice.

[0018] PGAM1 pY119 phosphorylation, as a hallmark modification of tumors, is specifically present in tumors such as lung cancer, breast cancer, gastric cancer, and acute T-lymphoblastic leukemia, and can be considered a potential target for tumor therapy. Therefore, we selected non-small cell lung cancer resistant to cisplatin chemotherapy for testing. Specific experimental results are as follows:

[0019] (1) Harvest 5 × 10⁵ A549 / DDP non-small cell lung cancer cells (A549 / DDP cells) in good growth condition that are resistant to cisplatin-based chemotherapy. 6 (each) and resuspended in 100 μL PBS;

[0020] (2) Take 18 female nude mice (BALB / c-nude) aged 6-8 weeks and inject the cell suspension into the armpit (subcutaneous) of the nude mice respectively;

[0021] (3) Tumor-bearing mice were randomly divided into experimental group and control group (nine nude mice in each group). The experimental group mice were injected intraperitoneally with cell-penetrating peptide Y119E every other day; the peptide containing only TAT was injected intraperitoneally into the control group mice as a negative control.

[0022] (4) Periodically measure the long axis (L) and short axis (W) of tumors in nude mice, according to the formula (LW). 2 / 2 Calculate the volume of the tumor;

[0023] (5) After 20 days, the tumor was removed from the subcutaneous tissue of the mouse, and the tumor volume growth curve was plotted. The specific experimental data and statistical results are as follows:

[0024]

[0025] (7) Tumor weight and statistical results are as follows:

[0026]

[0027] The above results indicate that, compared with the cell-penetrating peptide TAT, Y119E effectively inhibits the subcutaneous growth of cisplatin-resistant non-small cell lung cancer cells in nude mice. Attached Figure Description

[0028] Figure 1 shows that cisplatin-resistant A549 / DDP cells do not respond to cisplatin-induced ROS production.

[0029] Figure 2 shows that cisplatin-resistant A549 / DDP cells do not respond to cisplatin-induced DNA damage.

[0030] Figure 3 shows that cisplatin-resistant A549 / DDP cells do not respond to cisplatin-induced apoptosis.

[0031] Figure 4 shows the phosphorylation level of PGAM1 Y119 in cisplatin-resistant A549 / DDP cells.

[0032] Figure 5 shows the construction of A549 / DDP. WT and A549 / DDP Y119F Stable cell lines.

[0033] Figure 6 shows A549 / DDP. Y119F Cells resume responding to cisplatin-induced ROS production.

[0034] Figure 7 shows A549 / DDP. Y119F Cells re-respond to cisplatin-induced DNA damage.

[0035] Figure 8 shows A549 / DDP. Y119F Cells re-respond to cisplatin-induced apoptosis.

[0036] Figure 9 shows the amino acid sequence of the cell permeation peptide Y119E.

[0037] Figure 10 shows how the cell-penetrating peptide Y119E inhibits the interaction between PGAM1 and PKM2.

[0038] Figure 11 shows the administration pattern of subcutaneous tumor-bearing mice and cell-penetrating peptide Y119E.

[0039] Figure 12 shows that the cell-penetrating peptide Y119E inhibits tumor growth in nude mice.

[0040] Figure 13 shows that the cell-penetrating peptide Y119E inhibits tumor growth (volume) in nude mice.

[0041] Figure 14 shows that the cell-penetrating peptide Y119E inhibits tumor growth (mass) in nude mice. Detailed Implementation

[0042] The present invention will be further described below with reference to the accompanying drawings and embodiments: As shown in Figures 1-14; a cell permeation peptide Y119E, characterized in that: based on the sequence of PGAM1, the cell membrane penetration peptide TAT is used as a carrier to transport the cell permeation peptide containing the Y119 phosphorylation site into the cell, the cell permeation peptide (Y119E) containing Y119 phosphorylation has the sequence YGRKKRRQRRR-KIWRRSEDVP.

[0043] The aforementioned cell-penetrating peptide Y119E inhibits the growth of cisplatin-resistant tumors.

[0044] The cell-penetrating peptide Y119E inhibits tumors including non-small cell lung cancer.

[0045] Example 1

[0046] Materials and Methods

[0047] 1. Cell Culture

[0048] Before conducting cell experiments, the laminar flow hood should be wiped clean with 75% alcohol swabs. Place the necessary reagents and materials in the laminar flow hood, turn on UV mode, and sterilize for at least 30 minutes before use. A549 and A549 / shPGAM1 cells should be cultured in F12 medium. A549 / DDP and A549 / DDP / PGAM1 cells should be cultured in... WT and A549 / DDP / PGAM1 Y119F Cells were cultured in 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution, and incubated at 37°C in a constant temperature incubator containing 5% CO2.

[0049] Prepare the F12 and 1640 culture media, PBS buffer, and trypsin required for cell culture according to Tables 1, 2, 3, and 4.

[0050] Table 1. F12 culture medium formulation (pH 7.2-7.4)

[0051]

[0052] Table 2. 1640 culture medium formulation (pH 7.2-7.4)

[0053]

[0054] Table 3. 1×PBS formulation (pH 7.2-7.4)

[0055]

[0056]

[0057] Table 4 10× Trypsin formulation

[0058]

[0059] 2. Cell protein extraction

[0060] (1) Digest the cells to be lysed with 1× trypsin and collect them into the corresponding 1.5mL EP tube. Centrifuge at 300g for 5min at 4℃.

[0061] (2) Prepare LSB cell lysis buffer according to Table 5.

[0062] (3) Remove the supernatant, add 200 μL of LSB cell lysis buffer and 1 μL of nuclease, and pipette until the solution becomes clear and no longer viscous. Then let it stand at room temperature for 1 hour to allow it to fully lyse.

[0063] (4) Boil the sample in a metal bath at 100°C for 10 minutes, then immediately separate it, dispense it into portions, and store it at -80°C for later use.

[0064] Table 5 1×LSB buffer formulation

[0065]

[0066] 3. Protein immunoblotting

[0067] (1) Prepare a 10% separating gel according to Table 6. After mixing, immediately add it to the gel plate up to 2.5 cm from the top, then add 1 mL of anhydrous ethanol and press it to the surface of the gel. Let it stand for more than 30 minutes, and after solidification, pour off the anhydrous ethanol.

[0068] Table 6 10% Separating Gel Formulation

[0069]

[0070] (2) Prepare a 5% stacking gel according to Table 7. After mixing, immediately add it to the gel plate and insert the comb, being careful not to create air bubbles. Let it stand for 30 minutes. Remove the comb and rinse the gel thoroughly to prevent any gel fragments from interfering with sample loading. Place the cleaned gel in the electrophoresis tank.

[0071] Table 7 5% Concentrated Gum Formulation

[0072]

[0073] (3) Prepare 10× electrophoresis solution and 10× transfer solution according to Tables 8 and 9 respectively.

[0074] Table 8 10× Electrophoresis Solution Formulation

[0075]

[0076] Table 9 10× Transfer Solution Formulation

[0077]

[0078] (4) Prepare 1× electrophoresis buffer: Mix 1800mL DDW and 200mL 10× electrophoresis buffer and pour into the electrophoresis tank until the gel is submerged; Prepare 1× transfer buffer: Mix 700mL DDW, 200mL methanol and 100mL 10× transfer buffer and store at -20℃ for later use.

[0079] (5) Add the sample and protein molecular weight standard (Marker) to the loading wells, and fill the remaining wells with 1×LoadingBuffer.

[0080] (6) Cover the electrophoresis tank, turn on the switch, perform constant voltage electrophoresis, set the voltage to 80V, and wait until the marker produces red stripes and the stripes are horizontal and neat, then change the voltage to 120V.

[0081] (7) When the color indicator of the Loading Buffer reaches the bottom of the electrophoresis tank, stop electrophoresis, remove the gel plate and wash it.

[0082] (8) Transfer: Based on the molecular weight of the marker, cut out the corresponding target protein band and place it in the old transfer buffer. Cut out a PVDF membrane of the same size as the protein band, activate it in methanol solution for 1 min, and then place it in the transfer buffer. Place the gel and PVDF membrane in the transfer clamp in the order of "black gel, white membrane" (sponge-filter paper-membrane-gel-filter paper-sponge) and place it in the transfer tank. Pour in pre-cooled 1× transfer buffer, add ice cubes, and perform constant current transfer at 275 mA for 75 min.

[0083] (9) Sealing: Place the PVDF membrane in the sealing solution and seal it at room temperature for about 1 hour. Prepare the sealing solution and 10×TBS buffer solution according to Tables 10 and 11 respectively.

[0084] Table 10 Blocking Fluid Formulation

[0085]

[0086] Table 11 10×TBS Formulation

[0087]

[0088] (10) Incubation of primary antibody: Mix 900 mL DDW, 100 mL TBS and 1 mL Tween to make a 1×TBST solution. Dilute the primary antibody with 1×TBST at a ratio of 1:1000. Place the membrane in the primary antibody dilution solution and incubate on a horizontal shaker for 1 h or overnight at 4°C.

[0089] (11) Incubation with secondary antibody: Wash the membrane 3 times with 1×TBST, 5 min each time. Dilute the secondary antibody with 1×TBST at a ratio of 1:5000 or 1:10000, place the membrane in the secondary antibody dilution solution, incubate on a horizontal shaker for 50 min, and then wash 3 times with 1×TBST, 5 min each time.

[0090] (12) Exposure and development: Place the PVDF film in an appropriate position on the developing plate, use filter paper to absorb the residual liquid, and evenly add ECL developer for exposure.

[0091] 4. Total RNA extraction

[0092] (1) Culture cells in 6-well plates and extract total RNA when the cell density reaches 90%.

[0093] (2) Discard the old culture medium, gently rinse the cells twice with PBS, add 1 mL of Trizol to cover the cell surface at once, gently blow the cells off with a regular pipette tip, and repeat the blowing until there is no sticky feeling. Transfer the cells to a 1.5 mL EP tube and let them stand at room temperature for 10 min to allow the cells to lyse completely.

[0094] (3) Add 200 μL of chloroform using an RNA-grade pipette tip, shake vigorously for about 30 seconds, and let stand at room temperature for 5 minutes.

[0095] (4) Place in a pre-cooled centrifuge and centrifuge at 4°C and 12,000 rpm for 15 min. After centrifugation, transfer 400 μL of supernatant to an RNA-grade EP tube, add 400 μL of isopropanol, mix thoroughly, gently invert 8 to 10 times, and let stand at room temperature for 20 min.

[0096] (5) Centrifuge at 4°C, 12000 rpm for 10 min, and aspirate the supernatant, being careful not to aspirate the precipitate at the bottom of the EP tube.

[0097] (6) Add 1 mL of 75% alcohol prepared with DEPC water along the tube wall to wash the RNA precipitate.

[0098] (7) Centrifuge at 4℃, 12000 rpm for 5 min, and collect the supernatant.

[0099] (8) Repeat steps (6) and (7).

[0100] (9) After opening the lid, dry at room temperature for about 5 minutes, add 10 μL DEPC water to the sedimentation position, soak for 3 minutes, and then gently blow and mix.

[0101] (10) Detect the concentration of RNA, pay attention to the values ​​at A260 and A280 and the position of the absorption peak, and then place the RNA on ice for subsequent reverse transcription.

[0102] 5. Reverse transcription

[0103] (1) Prepare the genomic DNA removal reaction solution according to Table 12. Mix the reaction solution with a pipette and incubate it in a preheated 42°C metal bath for 2 minutes to remove genomic DNA.

[0104] (2) Prepare the reverse transcription reaction solution according to Table 13, mix well and incubate in a preheated 37°C water bath for 15 min, then transfer to an 85°C metal bath for 5 s to obtain cDNA.

[0105] (3) Detect cDNA concentration. The obtained cDNA sample can be stored in a -80℃ freezer for a long time.

[0106] Table 12 Genomic DNA Removal Reaction Solution

[0107]

[0108] Table 13 Reverse Transcription Reaction Solution

[0109]

[0110] 6. Construction of wild-type plasmids

[0111] (1) Primer design: Primers were designed using Primer 5.0.

[0112] (2) Agarose gel electrophoresis for recovery of the target fragment: First, prepare the PCR reaction solution according to the PCR reaction system shown in Table 14, and perform the PCR reaction according to the procedure. Second, mix the above PCR product with 10× Loading Buffer at a ratio of 9:1 and terminate the reaction. Prepare a 1% agarose gel for electrophoresis, and set the electrophoresis conditions to 120V for 30min. Then, place the agarose gel in a scanning gel imaging system, locate the target band under UV light, cut it out and place it in a 5mL EP tube. Finally, recover the DNA according to the agarose gel DNA recovery kit, and determine the fragment concentration using a spectrophotometer.

[0113] Table 14 PCR Reaction Solution

[0114]

[0115] (3) Double enzyme digestion and product recovery: Prepare the double enzyme digestion reaction solution according to Table 15 (using EcoRI and BamHI as examples). The digestion products were recovered using a standard DNA product purification kit, and the concentration was finally determined using a spectrophotometer.

[0116] Table 15 Double Enzyme Digestion Reaction System

[0117]

[0118] (4) Connection: Prepare the connection reaction solution as shown in Table 16, and react at 16°C overnight.

[0119] Table 16 Connection of Reaction Solution

[0120]

[0121]

[0122] (5) Transformation and plating: First, add the ligation product to 100 μL LDH5α competent cells and incubate on ice for 30 min. Heat shock in a 42℃ water bath for 90 s, then incubate on ice for 5 min. Next, add 900 μL of antibiotic-free LB medium and incubate at 37℃ with shaking at 160 rpm for 45 min to 1 h. Then, centrifuge at 12000 rpm for 1 min, remove part of the supernatant, and resuspend the precipitate in the remaining 100 μL of liquid. Finally, add the suspension to LB plates, spread evenly with a spreader, and then incubate upside down in a 37℃ incubator for 12–16 h.

[0123] (6) Picking and shaking: Select single colonies of suitable size and shape, pick them out with a small pipette tip and add them to 5 mL of LB liquid medium containing resistance. Incubate overnight at 37℃ and 160 rpm with shaking for 14-16 h.

[0124] (7) Plasmid extraction: Before plasmid extraction, the bacterial strain was preserved. In a clean bench, 500 μL of 40% glycerol and 500 μL of bacterial culture were mixed and then stored at -80°C. The remaining bacterial culture was extracted according to the instructions of the Kangwei endotoxin-free plasmid mini-extraction kit, the concentration was determined, and then stored at -20°C.

[0125] (8) Plasmid identification: The plasmids were sequenced by Kumei Biotechnology, and the sequencing results were compared using SeqMan software. The bacterial strains with successfully aligned sequences were expanded for later use.

[0126] 7. Construction of mutant plasmids

[0127] (1) Primer design: Primers were designed using Primer 5.0.

[0128] (2) Using the wild-type plasmid constructed in the above steps as a template, a large number of target fragments were obtained by one-step PCR. The one-step PCR system is shown in Table 2.19.

[0129] (3) Add DNP1 and digest at 37°C for 3 hours.

[0130] (4) The subsequent steps are the same as those in 2.2.7.1(5) to (8).

[0131] 8. Lentiviral Packaging

[0132] The cells used for lentiviral packaging are mainly HEK293T cells.

[0133] (1) Cells should be counted using a cell counter 12 hours before transfection, and the cells should be seeded in a 10cm cell culture plate to achieve a cell count of 1×10⁻⁶. 7 Cell transfection was performed when the cell density reached 80%.

[0134] (2) Before transfection, the cells should be changed, the old culture medium should be discarded, the cells should be gently rinsed twice with PBS, and then replaced with 4 mL of serum-free and antibiotic-free culture medium and placed in a cell culture incubator for continued culture.

[0135] (3) When transfecting, the corresponding plasmid and liposome PEI should be diluted first. Prepare the corresponding number of sterile 1.5mL EP tubes and prepare transfection reagents A and B respectively. Add 250μL of antibiotic-free DMEM medium and plasmid to tube A. The mass ratio of packaging plasmid PAX:core plasmid:packaging plasmid PMD should be 3:4:1, that is, 7.5μg of packaging plasmid PAX, 10μg of core plasmid and 2.5μg of packaging plasmid PMD should be added to a 10cm cell culture dish. Add 250μL of antibiotic-free medium and 10μg of PEI to tube B. Gently mix the liquid in the tube with a pipette and let it stand at room temperature for 5min.

[0136] (4) Mix the plasmid and liposome thoroughly and let stand at room temperature for 15 minutes.

[0137] (5) Tilt the culture dish slightly so that the culture medium is all at the bottom. Use a pipette to gently add the plasmid and PEI mixture into the culture dish and continue to put it back into the cell culture incubator for further culture.

[0138] (6) Discard the old culture medium in the cell culture dish 6 hours after transfection and replace it with complete culture medium.

[0139] (7) Viral supernatant can be collected 48 hours after transfection. The supernatant is collected every 24 hours thereafter, for a total of three times. The collected viral supernatant is temporarily stored in a 4°C refrigerator.

[0140] (8) After all the viral supernatant has been collected, centrifuge at 800 rpm for 5 minutes. This step is to remove cell debris from the viral supernatant. Then filter it through a 0.22 μm filter membrane into a new 50 mL centrifuge tube. Add the virus to the ultrafiltration tube in multiple portions. The ultrafiltration tube should be rinsed with PBS beforehand. Centrifuge at 3000 g and 4 °C until no liquid is filtered out. The liquid in the ultrafiltration tube at this point is the concentrated virus solution. Aliquot the virus into 1.5 mL EP tubes, 100 μL per tube, label them, seal them tightly with sealing film, and store them in a -80 °C freezer for later use.

[0141] 9. Lentiviral infection of cells

[0142] (1) Count the cells before infection.

[0143] (2) Take a certain amount of cell suspension to make the cell count 1×10⁻⁶. 3 Add the solution to a 1.5 mL EP tube containing 100 μL of concentrated virus solution, along with 5 μg / mL of polybrene, and finally add an appropriate amount of antibiotic-free F12 medium to make a total volume of 500 μL. Let it stand at room temperature for 15 min.

[0144] (3) Add the incubated virus-cell mixture to a 24-well plate and label it.

[0145] (4) After 24 hours of infection, discard the liquid in the well plate, gently rinse twice with PBS, and replace with complete culture medium. Change the medium for the cells every 24 hours thereafter. A second infection can be performed after 72 hours, following the same steps.

[0146] 10. Detection of intracellular ROS levels

[0147] (1) Cell seeding: The cell density in a 12-well plate was 1.25 × 10⁻⁶ cells / well. 5 / well, cultured with 1mL of complete culture medium for 1 day.

[0148] (2) Treat with or without medication for 48 hours.

[0149] (3) Half an hour before harvesting the cells, add Cell Rox staining solution at a ratio of 1:500. No need to change the solution, continue culturing.

[0150] (4) Discard the old culture medium, digest with 1× trypsin for 2 min, stop digestion with a small amount of serum, transfer the cell suspension to a centrifuge tube, centrifuge at 4℃, 300g, for 5 min to collect the cells.

[0151] (5) Wash the cells twice with PBS, 4℃, 300g, 5min.

[0152] (6) Resuspend the cells in 200 μL PBS and perform flow cytometry analysis.

[0153] 11. Detection of intracellular DNA damage levels (immunofluorescence technique)

[0154] (1) Cell seeding: Pre-place cell slides into 6-well plates and seed cells at a density of 3 × 10⁶ cells / well. 5 / Kong, culture overnight.

[0155] (2) On the second day, cisplatin was added and stimulated for 24 hours. After 24 hours, the cell smears were transferred to a new six-well plate, placed on ice, and washed once with pre-cooled PBS for 2 minutes.

[0156] (3) Wash once with 1 mL of permeation solution 1×CSK for 2 min.

[0157] (4) Wash once with 1 mL of 1×CSK-0.5% Triton for 5 min.

[0158] (5) Wash once again with 1 mL of permeabilizing solution and 1×CSK for 2 min, and then wash once with ice-cold PBS for 2 min.

[0159] (6) Fix with 4% paraformaldehyde at room temperature for 7 min, then wash with PBS at room temperature 3 times, 2 min each time.

[0160] (7) Block with 5% BSA-PBST blocking solution at room temperature for 30 min or at 4°C overnight.

[0161] (8) Incubation with primary antibody: Wash 3 times with PBST for 2 min each time, dilute the primary antibody with blocking buffer at a ratio of 1:200, attach sealing film to the lid of the culture dish, add 50 μL of primary antibody to the sealing film, take out the slide, drain the water, place it with the cell side facing the sealing film, and incubate overnight at 4°C in the dark.

[0162] (9) Incubation of secondary antibody: Wash 3 times with PBST for 2 min each time, dilute the secondary antibody with blocking buffer at a ratio of 1:200, add 50 μL to each slide and incubate at room temperature in the dark for 0.5 h.

[0163] (10) Wash 3 times with PBST for 2 min each time to stain cell nuclei. Dilute DAPI with 1×PBS at a ratio of 1:2000, add 2mA DAPI staining solution to each well, and stain in the dark for 2 min.

[0164] (11) Discard the DAPI diluent, add 2 mL of 1×PBS and wash 3 times, 2 min each time. Take out the slide, wash with DDW for 5 s, and drain the water.

[0165] (12) Mounting: Add 7 μL of Mowiol 488 mounting solution to each slide, and slowly cover the cell side with the Mowiol 488 mounting solution, avoiding the formation of air bubbles. After mounting in the dark for more than 4 hours, fluorescence microscopy can be used for imaging and analysis.

[0166] 12. Detection of intracellular DNA damage levels (flow cytometry)

[0167] (1) Cell seeding: The cell density in a 12-well plate was 1.25 × 10⁻⁶ cells / well. 5 / well, cultured in 1mL of normal complete culture medium for 1 day.

[0168] (2) Treat with or without medication for 48 hours.

[0169] (3) Discard the old culture medium, digest with 1× trypsin for 2 min, stop digestion with an appropriate amount of serum, gently blow the cells up, transfer them to a centrifuge tube, centrifuge to collect the cells, 800 rpm for 5 min.

[0170] (4) Wash the cells twice with 1 mL of 1×PBS, 300 g each time for 5 min.

[0171] (5) Wash once with 1 mL of 1×CSK permeation solution, 300 g for 5 min.

[0172] (6) Permeate on ice with 100 μL of 1×CSK-0.2% Triton for 5 min.

[0173] (7) Wash once with 1 mL of 1×CSK permeation solution, 300 g for 5 min.

[0174] (8) Wash the cells once with 1 mL of ice-cold 1×PBS, 300 g for 5 min each time.

[0175] (9) Discard the supernatant and add 100 μL of 4% paraformaldehyde for 7 min to fix.

[0176] (10) Wash the cells once with 1mL of 1×PBS at room temperature, 300g for 5min each time.

[0177] (11) Discard the supernatant, add 200 μL of 5% BSA-PBST blocking solution, and block at room temperature for 1 h.

[0178] (12) Incubation of primary and secondary antibodies: Dilute the primary antibody with blocking buffer at a ratio of 1:200 and the secondary antibody with blocking buffer at a ratio of 1:500. Incubate 100 μL of the primary and secondary antibody mixture for 1 h.

[0179] (13) Wash the cells once with 1mL of 1×PBS at room temperature, 300g for 5min each time.

[0180] (14) Add 200 μL of 1×PBS, resuspend the cells, and perform flow cytometry analysis.

[0181] 13. Apoptosis detection

[0182] (1) Cell seeding: The cell density in a 12-well plate was 1.25 × 10⁻⁶ cells / well. 5 / well, cultured in 1mL of normal complete culture medium for 1 day.

[0183] (2) Treat with or without medication for 48 hours.

[0184] (3) Collect cells, retain the old culture medium, digest the cells with EDTA-free trypsin for about 5 minutes, stop digestion with a small amount of serum, transfer the cell suspension to a centrifuge tube, and collect the cells by centrifugation at 300g for 5 minutes at 4℃.

[0185] (4) Resuspend the cells in complete culture medium and incubate at 37°C for 30 min.

[0186] (5) Wash the cells twice with pre-cooled PBS, 300g each time for 5min.

[0187] (6) Resuspend cells in 100 μL of 1×Annexin V binding buffer, and stain with 3-5 μL each of YF647-Annexin V and PI in the dark for 10-15 min.

[0188] (7) Resuspend cells in 400 μL PBS and perform flow cytometry analysis.

[0189] 14. Tumor-bearing experiment in nude mice

[0190] (1) Select female nude mice aged 6 to 8 weeks.

[0191] (2) Digest and collect the required cells, and count them. Take 1×10⁻⁶ cells from each of the two cell lines. 6 One cell was resuspended in 100 μL PBS and inoculated into the axilla of the left forelimb of a nude mouse.

[0192] (3) Cisplatin was then injected intraperitoneally every other day at a dose of 2 mg / kg. The tumor volume of each nude mouse was measured, and the corresponding tumor volume was calculated and recorded.

[0193] (4) Two weeks after inoculation, the tumor was removed from the nude mouse, and its volume was measured and recorded.

[0194] result

[0195] It is known that inhibiting PGAM1 expression reduces intracellular DNA synthesis and NADPH production, while increasing reactive oxygen species (ROS) and DNA damage levels. This means that high PGAM1 expression can promote intracellular DNA synthesis, increase reducing NADPH levels to balance ROS levels, reduce DNA damage, and ultimately promote cell proliferation. This phenotype is exactly the opposite of the DNA damage and ROS upregulation phenotype mediated by cisplatin, a commonly used first-line chemotherapy drug. Therefore, we hypothesized that PGAM1 may be involved in cisplatin resistance in non-small cell lung cancer. To this end, we conducted a series of experiments and obtained the following results and conclusions:

[0196] (I) Verification that cisplatin-resistant cells are insensitive to cisplatin treatment, as shown in Figures 1-3. Among them:

[0197] Figure 1. Cisplatin-resistant A549 / DDP cells do not respond to cisplatin-induced ROS production. (A) Non-small cell lung cancer A549 cells and cisplatin-resistant A549 / DDP cells were seeded at appropriate densities into 12-well plates, treated with cisplatin (10 μM) for 48 h, and cells were collected to detect intracellular ROS levels. (B) The results of three independent experiments in A were statistically analyzed. n=3, one-way ANOVA, ****, P<0.0001; ns, P>0.05, no statistical difference.

[0198] Figure 2. Cisplatin-resistant A549 / DDP cells did not respond to cisplatin-induced DNA damage. (A) A549 and A549 / DDP cells were seeded at appropriate densities into 12-well plates, treated with cisplatin (10 μM) for 48 h, and cells were collected to detect intracellular γH2Ax levels, i.e., DNA damage levels. (B) The results of three independent experiments in A were statistically analyzed. n = 3, one-way ANOVA, ****, P < 0.0001; ns, P > 0.05, no statistical difference, scale bar: 20 μm.

[0199] Figure 3. Cisplatin-resistant A549 / DDP cells did not respond to cisplatin-induced apoptosis. (A) A549 and A549 / DDP cells were seeded at appropriate densities into 12-well plates, treated with cisplatin (10 μM) for 48 h, and cells were collected to detect apoptosis levels. (B) The results of three independent experiments in A were statistically analyzed. n=3, one-way ANOVA, ****, P<0.0001; ns, P>0.05, no statistical difference.

[0200] (II) Intracellular PGAM1 Y119 phosphorylation levels in cisplatin-resistant cells contribute to resistance to cisplatin treatment, as shown in Figure 4-8, where:

[0201] Figure 4. Phosphorylation level of PGAM1 Y119 in cisplatin-resistant A549 / DDP cells. (A) A549 and A549 / DDP cells were seeded at appropriate densities into 6-well plates. After stable growth, cells were collected, intracellular total protein was obtained, and intracellular PGAM1 protein was enriched using IP technology. Western blotting was used to detect the expression of PGAM1 and the corresponding phosphorylation modification in A549 and A549 / DDP cells, and quantitative statistical analysis was performed. (B) The results of three independent experiments in A were statistically analyzed. n=3, studentt-test, **, P<0.01.

[0202] Figure 5. Constructing A549 / DDP WT and A549 / DDP Y119F Stable cell lines. (A) Overexpression of PGAM1 in A549 / DDP cells and PGAM1 knockdown (KD) A549 / DDP cells. WT Or PGAM1 Y119F The protein level of PGAM1 was detected and quantified using Western blotting (WB). (B) The results of the three independent experiments in A were statistically analyzed. n=3, student t-test, **, P<0.01; ns, P>0.05, no statistical difference.

[0203] Figure 6. A549 / DDP Y119F Cells re-respond to cisplatin-induced ROS production. (A) A549 / DDP WT and A549 / DDP Y119F Cells were seeded at an appropriate density into 12-well plates, treated with cisplatin (10 μM) for 48 h, and then collected to detect intracellular ROS levels. (B) The results of three independent experiments in A were statistically analyzed. n = 3, one-way ANOVA, *, P < 0.05; ns, P > 0.05, no statistical difference.

[0204] Figure 7. A549 / DDP Y119F Cells re-respond to cisplatin-induced DNA damage. (A) A549 / DDP WT and A549 / DDP Y119F Cells were seeded at an appropriate density into 12-well plates, treated with cisplatin (10 μM) for 48 h, and then collected to detect intracellular γH2Ax levels, i.e., DNA damage levels. (B) The results of the three independent experiments in A were statistically analyzed. n = 3, one-way ANOVA, **, P < 0.01; ns, P > 0.05, no statistical difference.

[0205] Figure 8. A549 / DDP Y119F Cells re-respond to cisplatin-induced apoptosis. (A) A549 / DDP WTand A549 / DDP Y119F Cells were seeded at an appropriate density into 12-well plates, treated with cisplatin (10 μM) for 48 h, and then collected to detect apoptosis levels. (B) The results of three independent experiments in A were statistically analyzed. n = 3, one-way ANOVA, ****, P < 0.0001; ns, P > 0.05, no statistical difference.

[0206] (III) The above results indicate that PGAM1 Y119 phosphorylation specifically exists in cisplatin-resistant lung cancer cells and helps resist the therapeutic effect of cisplatin. We designed the cell-penetrating peptide Y119E to inhibit PGAM1-PKM2 binding at the cellular level and inhibit the growth of cisplatin-resistant lung cancer at the mouse level, as shown in Figure 10-14. Among them:

[0207] Figure 9. Amino acid sequence of cell permeation peptide Y119E.

[0208] Figure 10. Cell permeability peptide Y119E inhibits the interaction between PGAM1 and PKM2. (A) Cisplatin-resistant human non-small cell lung cancer cells A549 / DDP were used. Cells were treated with either TAT or Y119E, and then PGAM1 protein was enriched using antibodies. PKM2 binding and PGAM1 phosphorylation levels were detected by Western blotting. (B) The results of three independent experiments in A were statistically analyzed. n=3, studentt-test, ****, P<0.0001.

[0209] Figure 11. Schematic diagram of drug administration of subcutaneous tumor-bearing mice and cell-penetrating peptide Y119E.

[0210] Figure 12. Cell permeability peptide Y119E inhibits tumor growth in nude mice. Nine pairs of male nude mice aged 6-8 weeks were randomly selected. A549 / DDP cells were transplanted into the axilla of the mice via subcutaneous injection. Then, TAT or Y119E was injected intraperitoneally every other day. On day 20 post-transplantation, the tumors were removed from the mice and photographed for record-keeping.

[0211] Figure 13. Cell permeability peptide Y119E inhibits tumor growth (volume) in nude mice. Tumor volume was calculated periodically for mice treated as shown in Figure 12. n=9, studentt-test, ****P<0.0001, ns, P>0.05, no statistically significant difference.

[0212] Figure 14. Cell permeability peptide Y119E inhibits tumor growth (mass) in nude mice. For mice treated as shown in Figure 12, tumor formation was assessed by weight. n=9, studentt-test, ****P<0.0001.

[0213] 1 Ghosh,S.Cisplatin:The first metal based anticancer drug.BioorgChem88,102925(2019).

[0214] https: / / doi.org / 10.1016 / j.bioorg.2019.102925

[0215] 2 Rottenberg,S.,Disler,C.&Perego,P.The rediscovery of

[0216] platinum-based cancer therapy.NatRev Cancer 21,37-50(2021).

[0217] https: / / doi.org / 10.1038 / s41568-020-00308-y

[0218] 3 Selvakumaran,M.,Pisarcik,D.A.,Bao,R.,Yeung,A.T.&

[0219] Hamilton,T.C.Enhanced cisplatin cytotoxicity by disturbingthenucleotide excision repairpathway in ovarian cancer cell lines.

[0220] CancerRes 63,1311-1316(2003).

[0221] 4 Hanahan,D.&Weinberg,R.A.Hallmarks ofcancer:the next

[0222] generation.Cell 144,646-674(2011).

[0223] https: / / doi.org / 10.1016 / j.cell.2011.02.013

[0224] 5 Koppenol,W.H.,Bounds,P.L.&Dang,C.V.Otto Warburg's

[0225] contributions to current concepts ofcancermetabolism.NatRev

[0226] Cancer 11,325-337(2011). https: / / doi.org / 10.1038 / nrc3038 6 VanderHeiden,M.G.,Cantley,L.C.&Thompson,C.B.

[0227] Understanding the Warburg effect:the metabolic requirements ofcellproliferation.Science 324,1029-1033(2009).

[0228] https: / / doi.org / 10.1126 / science.1160809

[0229] 7 Lunt,S.Y.&Vander Heiden,M.G.Aerobic glycolysis:meeting

[0230] the metabolic requirements ofcell proliferation.Annu Rev CellDevBiol27,441-464(2011).

[0231] https: / / doi.org / 10.1146 / annurev-cellbio-092910-154237

[0232] 8 Cairns,R.A.,Harris,I.S.&Mak,T.W.Regulation ofcancer cell

[0233] metabolism.NatRev Cancer 11,85-95(2011).

[0234] https: / / doi.org / 10.1038 / nrc2981

[0235] 9 DeBerardinis,R.J.,Lum,J.J.,Hatzivassiliou,G.&Thompson,C.

[0236] B.The biology ofcancer:metabolic reprogramming fuels cell

[0237] growth and proliferation.Cell metabolism 7,11-20(2008).

[0238] https: / / doi.org / 10.1016 / j.cmet.2007.10.002

[0239] 10 Hitosugi,T.et al.Phosphoglycerate mutase 1 coordinates

[0240] glycolysis and biosynthesis to promote tumor growth.Cancer Cell

[0241] 22,585-600(2012). https: / / doi.org / 10.1016 / j.ccr.2012.09.020 11Chaneton,B.&Gottlieb,E.PGAMgnam style:a glycolytic switchcontrolsbiosynthesis.Cancer Cell 22,565-566(2012).

[0242] https: / / doi.org / 10.1016 / j.ccr.2012.10.014

[0243] 12 Schwarze,S.R.,Hruska,K.A.&Dowdy,S.F.Protein

[0244] transduction:unrestricted delivery into all cells?Trends in cell

[0245] biology 10,290-295(2000).

[0246] https: / / doi.org / 10.1016 / s0962-8924(00)01771-2

[0247] 13 Ramsey,J.D.&Flynn,N.H.Cell-penetrating peptides transport

[0248] therapeutics into cells.Pharmacology&therapeutics 154,78-86

[0249] (2015). https: / / doi.org / 10.1016 / j.pharmthera.2015.07.003

[0250] 14 Wadia,J.S.,Stan,R.V.&Dowdy,S.F.Transducible TAT-HA

[0251] fusogenic peptide enhances escape ofTAT-fusion proteins after

[0252] lipid raft macropinocytosis.Nature medicine 10,310-315(2004).

[0253] https: / / doi.org / 10.1038 / nm996。

Claims

1. A cell-penetrating peptide Y119E, characterized in that: Using the cell-penetrating peptide TAT as a carrier, a cell-penetrating peptide containing phosphorylated PGAM1Y119 is delivered into the cell. The cell-penetrating peptide Y119E containing phosphorylated Y119 has the sequence YGRKKRRQRRR-KIWRRSEDVP.

2. The use of the cell-penetrating peptide Y119E as described in claim 1 in the preparation of a drug to inhibit cisplatin-resistant non-small cell lung cancer.

Images