CN114920802B - Polypeptide probe for regulating movement of telomerase speed-limiting protein, and complex and method thereof - Google Patents
Polypeptide probe for regulating movement of telomerase speed-limiting protein, and complex and method thereof Download PDFInfo
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Abstract
The invention provides a polypeptide probe for regulating telomerase speed-limiting protein movement, a complex thereof and an application method thereof. The invention designs a PKK-TTP polypeptide probe and a PKK-TTP/hs polypeptide probe compound, wherein PKK-TTP can promote telomerase speed-limiting protein in cell nucleus to transfer into cytoplasm under the stimulation of white light (PKK-TTP+L); PKK-TTP/hs complex can carry hs into cancer cells to inhibit the expression of hTERT protein in cytoplasm, and cut off the source of hTERT protein in nucleus; in addition, under white light stimulation, the PKK-TTP/hs complex (PKK-TTP/hs+L) can not only successfully deliver hs into cells, reduce hTERT protein in cell nuclei, but also promote the migration of hTERT protein from the cell nuclei into cytoplasm; the two compounds provided by the invention can effectively reduce the content of hTERT protein in the cell nucleus in different application scenes, thereby achieving the technical effect of inhibiting the proliferation of tumor cells.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a polypeptide probe for regulating movement of telomerase speed-limiting protein, a compound and a method thereof.
Background
The nucleus is the center of control of cytogenetic and metabolic activities. The nuclear membrane is a bilayer membrane that completely encapsulates the nucleus, providing an intracellular compartmentalized environment in which many biological processes occur. Nuclear shuttling is an important biological process that can transfer signals from the cytoplasm to the nucleus, regulating cell behavior. Wherein, telomerase is a ribonucleoprotein complex that plays an active role in the nucleus, and the 3' end of telomeres is catalyzed and synthesized by taking self RNA as a template to maintain the length of the telomeres. Human telomerase is composed of ribonucleic acid templates, reverse transcriptase catalytic components (hTERT proteins) and other related proteins. Among them, the reverse transcriptase is the main rate-limiting protein of telomerase activity, catalyzing the reverse transcriptase activity of telomerase.
In normal human tissue, telomerase activity is inhibited. However, in about 85% of cancer cells, telomerase is reactivated, enabling tumor cells to maintain telomere length in cell division, effecting immortalization. The research finds that: the reverse transcriptase has a positive correlation with telomerase activity in cancer cells. Human telomerase reverse transcriptase (hTERT) down-regulation in telomerase positive cancer cells can lead to tumor cell growth arrest. Thus, reverse transcriptase may be a further target of research for targeting cancer cells with telomerase.
Disclosure of Invention
Based on the above, it is necessary to provide a polypeptide probe complex capable of regulating the movement of telomerase rate-limiting protein and an application method thereof, which can reduce the content of hTERT protein in the cell nucleus, inhibit the activity of telomerase in the cell nucleus and achieve the purpose of preventing cancer cell proliferation.
The invention adopts the following technical scheme:
the invention provides a polypeptide probe for regulating telomerase speed-limiting protein movement in cytoplasm, which is mainly prepared by reacting a substance capable of generating ROS through light induction with PKKKRKVKAARRRRRRRR n-alkynyl Peptide (PKK).
Specifically, the invention provides a polypeptide probe PKK-TTP for regulating telomerase speed-limiting protein movement, which has the structure shown in the following formula:
the preparation method of the PKK-TTP comprises the following steps: adopting TTP and polypeptide PKK with a sequence of PKKKKVKAARRRRRRRRRRRRra, placing in a mixed solvent system containing DMSO and water, adding sodium ascorbate and cuprous bromide, reacting under inert atmosphere, and purifying to obtain the final product. In some of these embodiments, preferably, the reaction molar ratio of TTP to polypeptide PKK is 2:1.
The invention also provides a polypeptide probe complex for regulating movement of telomerase speed-limiting protein, which is formed by synthesizing a polypeptide probe after coupling a substance capable of generating ROS by light induction and PKKKRKVKAARRRRRRRR orthoalkynyl Peptide (PKK) and then loading small interfering RNA of hTERT protein.
Preferably, PKK-TTP/hs is a PKK-TTP/hs complex prepared by reacting PKK-TTP with hTERT siRNA. The preparation method comprises the following steps: preparing PKK-TTP according to the method, and then incubating the PKK-TTP with hTERT siRNA to react, thus obtaining the product. In some of these embodiments, the molar ratio of PKK-TTP to hTERT siRNA is 40:1.
The invention also provides application of the polypeptide probe complex for regulating telomerase speed-limiting protein movement, PKK-TTP and PKK-TTP/hs in preparation of products for reducing the content of telomerase speed-limiting protein in cell nucleus. White light stimulation is required to reduce the telomerase rate-limiting protein content in the nucleus by PKK-TTP and/or PKK-TTP/hs.
Compared with the prior art, the invention has the beneficial effects that:
three sets of corresponding polypeptide/nucleic acid probes were developed using strategies to cut/reduce the "turn in" of the telomerase rate-limiting protein in the nucleus and the "turn out" of the telomerase-promoting protein, depending on the nature of the telomerase protein itself: (1) the PKK-TTP polypeptide probe can generate Reactive Oxygen Species (ROS) under the stimulation of white light, and promote the transfer of proteins in the cell nucleus into cytoplasm; (2) PKK-TTP polypeptide loaded nucleic acid human telomerase reverse transcriptase small interfering RNA (hTERT siRNA, hs) is constructed to form PKK-TTP/hs complex nano-probes, and the PKK-TTP/hs can carry hs into cancer cells to silence mRNA in cytoplasm, inhibit expression of cytoplasmic hTERT protein and cut off the source of nuclear protein, so that the protein content in the cell nucleus is reduced; (3) the PKK-TTP/hs+L complex can successfully deliver hs into cells on one hand, and active oxygen generated by PKK-TPP can promote the removal of hTERT protein from the nucleus into cytoplasm under illumination on the other hand. The combination of the two can reduce the hTERT protein located in the cell nucleus to a greater extent.
The invention discloses three methods for regulating nuclear telomerase reverse transcription rate-limiting protein (hTERT) movement in cytoplasm for the first time, wherein the methods comprise two polypeptide probe complexes with monotonous hTERT control function, namely PKK-TTP+L and PKK-TTP/hs, and a multifunctional polypeptide probe complex with double regulation modes, namely PKK-TTP/hs+L, and the content of hTERT protein in the nucleus is reduced through relevant passages of the polypeptide probe complex in cytoplasm. The three methods can effectively reduce the content of hTERT protein in the nucleus in vitro and in vivo, wherein the third combination strategy has the best effect, and can achieve the purpose of effectively inhibiting the proliferation of tumor cells.
Drawings
FIG. 1 is a synthetic scheme of PKK-TTP polypeptide probes.
FIG. 2 is a diagram showing LC-MS characterization of PKK-TTP polypeptide probes.
FIG. 3 is a representation of the optical properties of PKK-TTP polypeptide probes in solution and ROS-producing properties; wherein (A) is an optical characterization of the PKK-TTP polypeptide probe; (B) Characterization of ROS production by PKK-TTP polypeptide probes in solution systems.
FIG. 4 is a characterization of the intracellular ROS-producing properties of a PKK-TTP polypeptide probe. Wherein (A) is cytotoxicity study of PKK-TTP polypeptide probes with different concentrations; (B) Relative absorbance of DCFH-DA after different times of illumination after 6. Mu.M of PKK-TTP polypeptide probe and cell co-incubation; (C) Fluorescent response of intracellular ROS indicators after various times of illumination following incubation of 6. Mu.M PKK-TTP polypeptide probes with cells.
FIG. 5 is a graph showing the results of a test for PKK-TTP+L in vitro modulation of intracellular movement of the cell telomerase TERT protein; wherein (A) shows an immunofluorescence image of HeLa cells incubated with 6. Mu.M PKK-TTP under white light (50 mW/cm 2 2 min), translocation of hTERT protein from the cytoplasm to the nucleus; (B) counting the relative quantification of the fluorescence intensity; (C) After incubation of 6. Mu.M PKK-TTP with HeLa cells, white light L (50 mW/cm 2 2 min) total proteins of nuclei and cytoplasm were extracted after stimulation, compared to the amount of hTERT protein measured by ELISA in the absence of light (expressed in ng/mL, n=3).
FIG. 6 shows the results of a nuclear telomerase activity assay following PKK-TTP+L action; wherein (A-B) is 6 mu M PWhite light L (50 mW/cm after KK-TTP co-incubation with HeLa cells 2 ) Extracting telomerase of cell nucleus after 2min of stimulation, and comparing with TRAP determination and corresponding quantitative result of cell nucleus telomerase activity without illumination; (C) After incubation of 6. Mu.M PKK-TTP with HeLa cells, white light L (50 mW/cm 2 ) Cell viability after 2min stimulation compared to that without light.
FIG. 7 is a graph showing statistics of test results of PKK-TTP probe load hs; wherein (A) is a polyacrylamide gel electrophoresis diagram of PKK-TTP/hs complexes in different proportions; (B) particle size diagram of PKK-TTP/hs complex with different proportion; (C) Potential characterization graphs for different ratios of PKK-TTP polypeptides and siRNA complexes.
FIG. 8 is a graph showing PKK-TTP/hs modulation of intracellular telomerase reverse transcriptase; wherein (A) is an IF image of PKK-TTP/hs (6. Mu.M PKK-TTP,150nM hs) after 24 hours of HeLa cell treatment; (B) quantitatively counting the expression of hTERT protein in the nucleus thereof; (C) Determination of the content of hTERT protein (expressed as ng/mL, n=3) in HeLa nuclei after incubation for 24h with PKK-TTP/hs (6 μm PKK-TTP,150nM hs) for ELISA; (D-E) TRAP assay and corresponding quantification of telomerase activity in HeLa nuclear extract after 24 hours incubation with PKK-TTP/hs; (F) Cytotoxicity to HeLa cells after incubation for 24h for PKK-TTP/hs (6. Mu.M PKK-TTP,150nM hs).
FIG. 9 is an in vitro modulation of telomerase reverse transcriptase in the nucleus of PKK-TTP/hs+L; wherein (A-B) is PKK-TTP/hs (40:1, 6. Mu.M PKK-TTP,150nM hs) with or without white light laser irradiation (L, 2min,50 mW/cm) 2 ) Post HeLa cell confocal images and fluorescence intensity statistics, green fluorescence (DCFH-DA, ex: 48nm, em:500-530 nm), yellow fluorescence (hs-Cy 5, ex:633nm, em:700-750 nm); (C) Quantitative PCR test results of TERT mRNA in cells after PKK-TTP/hs+L treatment; (D-E) quantitative statistics of expression of hTERT protein in the IF image and nuclei of PKK-TTP/hs+l treated cells; (F) Determination of the content of hTERT protein in HeLa nuclei of PKK-TTP/hs+l for ELISA (expressed as ng/mL, n=3)
FIG. 10 is a graph showing the comparative test effects of PKK-TTP and PKK-TTP/hs+L, wherein (A-B) is 6. Mu.M PKK-TTP and PKK-TTP/hs+L (40:1, 6. Mu.M PKK-TTP,150nM hs,2min,50mW/cm) 2 ) HeLa cells after incubationMeasuring telomerase activity in the nuclear extract and corresponding quantitative results; (C) is PKK-TTP/hs+L cytotoxicity after treatment.
FIG. 11 is a graph showing the effect of protein modulation in tumors of mice, wherein (A) is the anti-tumor effect of the therapeutic effect of PKK-TTP/hs+L combination treatment, and the tumor-bearing mice are treated schematically; (B) The tumor sections were stained for nuclear and cytoplasmic hTERT immunofluorescence and (C) corresponding nuclear fluorescence intensities after PBS, PKK-TTP, PKK-TTP+ L, PKK-TTP/hs and PKK-TTP/hs+L treatment.
FIG. 12 is a graph showing the results of various examples on a mouse tumor model, wherein (A) is the anti-tumor effect of the PKK-TTP/hs+L combined treatment effect, and the tumor-bearing mouse treatment is schematically shown; when the transplanted tumor grows to about 200mm 3 Injecting PBS, PKK-TTP/hs and PKK-TTP/hs into five groups of mice by intratumoral injection, and irradiating the tumor part of the mice with white light laser after 24 hours; (B) To change the relative volume of each group of tumors during treatment (V 1 /V 0 ) (n=4); (C) Representative tumor images of different groups after 15 days of intratumoral injection treatment; (D) Weight change (n=4) throughout the treatment period for mice in PBS, PKK-TTP/hs and PKK-TTP/hs; (E) Ki67 immunofluorescence staining of tumor sections after treatment with PBS, PKK-TTP, PKK-TTP+ L, PKK-TTP/hs and PKK-TTP/hs+L and (F) corresponding fluorescence intensities; (G) HE staining of tumor sections following treatment with PBS, PKK-TTP, PKK-TTP+ L, PKK-TTP/hs and PKK-TTP/hs+L.
FIG. 13 is an explanatory diagram of a technical route and an action mechanism of PKK-TTP/hs+L of the present invention.
Detailed Description
The present invention will be described in further detail with reference to specific examples so as to more clearly understand the present invention by those skilled in the art.
The following examples are given for illustration of the invention only and are not intended to limit the scope of the invention. All other embodiments obtained by those skilled in the art without creative efforts are within the protection scope of the present invention based on the specific embodiments of the present invention.
In the examples of the present invention, all raw material components are commercially available products well known to those skilled in the art unless specified otherwise; in the embodiments of the present invention, unless specifically indicated, all technical means used are conventional means well known to those skilled in the art.
Test example 1
As shown in FIG. 1, the test example provides a PKK-TTP polypeptide probe, and the synthesis method comprises the following steps: 2 equivalents of TTP molecules and one equivalent of polypeptide molecules (sequence PKKKKVKAARRRRRRRRRRRRra) were dissolved in DMSO/H2O (v/v=1:1), 2 equivalents of sodium ascorbate and 2 equivalents of cuprous bromide were added and reacted under nitrogen at 40℃for 24H. After the reaction is finished, the solvent is dried by spin, and the synthetic product can be obtained after HPLC purification. The synthesized product was freeze-dried to give a red solid (yield 50%). The mass spectrum of the product is shown in FIG. 2, and the high-resolution mass spectrum shows mass-to-charge ratio [ M+9H ]] 9+ calcd,353.5487；found,353.5504.[M+8H] 8+ calcd,397.6164；found,397.6187.[M+7H] 7+ calcd,454.2748；found,454.2767.[M+6H] 6+ calcd,529.8194；found,529.8217.[M+5H] 5+ calcd,635.5819; found,635.5839, demonstrates successful synthesis of the product PKK-TTP polypeptide probe.
The PKK-TTP polypeptide probe consists of 2 modules: 1) TPETP-N 3 (TTP), azide-modified tetraphenylethylene thiophene with red emission, designed for bioimaging and ROS generation; 2) PKKKKVKAARRRRRRRRra-alkynyl Polypeptide (PKK) utilizing cationic polypeptides to increase the efficiency of cell internalization.
Further explores the ROS and the efficiency of the PKK-TTP polypeptide probe, and the method comprises the following steps:
the PKK-TTP polypeptide probe was added to water and 10. Mu. MABDA was added to investigate the ability of the PKK-TTP polypeptide probe to generate ROS under white light irradiation in solution. The absorbance values of ABDA at 378nm at different illumination times were recorded to obtain the decay rate of the photosensitizing process. In addition, the singlet oxygen test in the cells can be carried out in a 96-well plate and a confocal dish, after PKK-TTP polypeptide probes and the cells are incubated in the dark for a designated time, 10 mu M DCFH-DA is added into each well/dish, after incubation for 30 minutes, the cells are washed by PBS to remove redundant DCFH-DA, illumination stimulation is carried out for a corresponding time, and after illumination is finished, the cells are placed under a reader of an enzyme-labeling instrument or photographed under a laser confocal microscope, so that the effect of generating ROS in the cells can be obtained.
The results are shown in FIGS. 3 and 4. The results show that: the PKK-TTP polypeptide probe has satisfactory ROS production efficiency in both solution and in cells, but as the illumination time increases, ROS produced by the PKK-TTP polypeptide probe increases, which promotes apoptosis. To further ensure the safety of the stimulated cell illumination in the experiment, the screening test demonstrated that the white light illumination (50 mW/cm 2 ) The transfer of hTERT protein can be observed in the treated cells.
Further research on the effect of PKK-TTP polypeptide probes in inhibiting nuclear telomerase activity under the effect of white light (PKK-TTP+L for short), the method comprises the following steps:
after 24 hours incubation of PKK-TTP polypeptide probes with cells, 50mW/m was re-used 2 The cells were irradiated with the white light source of intensity for 2 minutes, and the results of the treatment were tested for localization and content of the proteins by immunofluorescence imaging technique and enzyme-linked immunosorbent assay, and as shown in fig. 5, the enhanced distribution of nuclear hTERT protein in the cytoplasm was observed from the image, and the corresponding fluorescence intensity statistics showed that nuclear hTERT protein was transferred from the nucleus to the cytoplasm, and the amount of hTERT protein in the cytoplasm was increased. The hTERT protein in the components is tested and calculated by ELISA protein quantitative analysis method, and the calculated result of ELISA is consistent with the result of the observed immunofluorescence imaging, which proves that ROS (50 mW/cm 2 2 min) did allow transfer of the hTERT protein in the nucleus into the cytoplasm, resulting in a reduced content of hTERT protein in the nucleus, demonstrating that PKK-ttp+l can mediate the transfer of hTERT protein out of the nucleus into the cytoplasm.
The effect on cell viability after translocation of the nuclear hTERT protein following PKK-ttp+l stimulation was further examined by enzyme activity assay and MTT method and the results are shown in figure 6.
Compared with PKK-TTP polypeptide probes, PKK-TTP+L can mediate that telomerase activity in cell nucleus is obviously reduced, and the inhibition rate is 18.8%. Subsequently, PKK-TTP+L showed higher cytotoxicity than PKK-TTP polypeptide probes, and cell viability was reduced by 13.8% after ROS production.
These results demonstrate that PKK-ttp+l can promote translocation of nuclear hTERT protein into the cytoplasm through the nuclear pores, thereby inhibiting nuclear telomerase activity, affecting cell viability.
Test example 2
The test example further provides a preparation method for forming a PKK-TTP/hs complex by loading hTERT siRNA with a PKK-TTP polypeptide probe, which comprises the following steps:
preparing hTERT siRNA (hs) solution by using DEPC water, mixing PKK-TTP polypeptide probe solutions with different concentrations and hs solutions with equal volumes and equal concentrations according to molar concentration ratios of (0-1000): 1 (1:0, 1:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 1000:1), diluting to required volumes by using the DEPC water, and incubating for 30min at normal temperature to form PKK-TTP/hs complexes with different reaction ratios.
The formation of PKK-TTP/hs complexes after electrostatic binding of PKK-TTP polypeptides to hs was analyzed by polyacrylamide gel electrophoresis and the results are shown as A in FIG. 3. Particle size and potential analyses were further performed on PKK-TTP/hs complexes of different reaction ratios, and the results are shown as B and C in FIG. 3. To avoid too much cation to produce strong toxicity to cells, the PKK-TTP polypeptide probe and hs are reacted in a molar concentration of 40:1 to select the PKK-TTP/hs complex with the optimal proportion of carrying hs in combination with polyacrylamide gel electrophoresis result and particle size result.
The PKK-TTP/hs complex aims to regulate the content of hTERT protein in the cell nucleus through gene silencing after entering cells with high efficiency, thereby reducing the telomerase activity related to the hTERT protein in the cell nucleus.
Further explores the gene silencing effect of PKK-TTP/hs complex, the test method comprises the following steps:
after 24 hours incubation of PKK-TTP/hs complex (40:1) with cells, intracellular RNA was extracted, and the expression of hTERT mRNA in cells was detected by quantitative reverse transcription polymerase chain reaction (RT-PCR) experiments, the results of which are shown in FIG. 7, the expression level of hTERT mRNA in cells was significantly reduced, which suggests that PKK-TTP/hs complex successfully exerted gene silencing effect in cells.
Further observations were made that hs promoted downregulation of hTERT protein in the nucleus after 24h incubation of HeLa cells with PKK-TTP/hs complexes. The results of immunofluorescence Imaging (IF) and enzyme-linked immunosorbent assay (ELISA) analysis of the anti-hTERT protein showed localization and content of hTERT protein in the nucleus and cytoplasm, and the results are shown in fig. 8. The results indicate that PKK-TTP/hs complexes can successfully reduce the content of nuclear hTERT protein in HeLa cells.
Test example 3
Further explores the action effect of PKK-TTP/hs complex under white light treatment (PKK-TTP/hs+L for short), the method comprises the following steps:
after incubation of PKK-TTP/hs complexes with cells for 24 hours, 50mW/m was re-used 2 The white light source with the intensity irradiates the cells for 2 minutes, and the processing result can be used for testing the positioning and the content of the protein through immunofluorescence imaging technology and enzyme-linked immunosorbent assay.
The results are shown in FIGS. 9 and 10, and the laser confocal image results indicate PKK-TTP/hs+L (2 min,50 mW/cm) 2 ) After incubation, immunofluorescence imaging experiments (IF) and corresponding average fluorescence intensity quantitative analysis results for anti-hTERT protein showed: PKK-TTP/hs+L can sufficiently reduce the content of hTERT protein in cell nucleus (the inhibition rate is 44.3%).
In addition, the results of hTERT protein ELISA of the cell nucleus and the corresponding cell parts also prove that compared with PKK-TTP (without gene silencing and ROS effect), PKK-TTP/hs+L can effectively reduce the content of hTERT protein in the cell nucleus (the inhibition rate is 40.6%).
In general, the principle of the nucleoprotein inhibition strategy of the invention to modulate nucleoprotein-related pathways is shown in figure 11. The invention designs a multifunctional compound consisting of three modules:
1) TTP, an azide-functionalized tetraphenyl thiophene, for use in bioimaging and ROS production.
2) PKKKRKVKAARRRRRRRR n-alkynyl Peptide (PKK), a cationic cell-penetrating peptide, can improve cell internalization efficiency and gene loading capacity by electrostatic interactions.
3) The specific small interfering RNA of hs, telomerase reverse transcriptase will be used to induce gene silencing and subsequent low expression of hTERT protein.
Specifically, the PKK-TTP/hs complex can carry hs into cancer cells, silence mRNA in cytoplasm thereof, and inhibit expression of cytoplasmic hTERT protein, thereby cutting off protein source in hTERT cell nucleus. At the same time, PKK-TPP produces modest ROS under light conditions that can promote the removal of hTERT protein from the nucleus into the cytoplasm through the nuclear pores.
Test example 4
Further explored the effect of PKK-TTP+ L, PKK-TTP/hs complex and PKK-TTP/hs complex on mouse model under white light treatment (called PKK-TTP/hs+L for short), the method comprises the following steps:
HeLa cells are planted at the armpit of a mouse to form a transplantation tumor, after the tumor grows to a certain volume, the mouse is randomly divided into 5 groups, and 4 mice in each group are named PBS, PKK-TTP, PKK-TTP+ L, PKK-TTP/hs and PKK-TTP/hs+L respectively. The PKK-TTP polypeptide probe and the PKK-TTP/hs complex are injected into tumor, after incubation for 24 hours, the tumor is irradiated by a white light source, and the treatment result can be tested by observing the tumor volume and tumor slice immunofluorescence imaging technology.
As a result, as shown in FIGS. 11 and 12, fewer nuclear hTERT protein red signals (selected circles) could be detected in the PKK-TTP/hs group compared to the PBS and PKK-TTP groups, indicating that hs was able to effectively inhibit hTERT protein expression by PKK-TTP/hs. In addition, a significant increase in the signal of hTERT protein in the cytoplasm was observed after white light irradiation (as indicated by the arrow), demonstrating that the PKK-ttp+l system has the effect of regulating nuclear protein transfer to the cytoplasm. More effectively, the mean fluorescence intensity of hTERT protein immunofluorescence showed that nuclear hTERT protein was drastically reduced (inhibition rate 42.3%) after PKK-TTP/hs+L treatment (FIG. 11C), indicating that PKK-TTP/hs+L could minimize the amount of nuclear hTERT protein by combining the two methods.
Furthermore, the inhibitory activity of the different groups on mouse tumor growth was judged by continuously monitoring the volume change of the tumor over a 15 day treatment period. Relative tumor volumes showed no significant tumor growth inhibiting effect of PBS and PKK-TTP. In sharp contrast, PKK-TTP/hs+L has a significant tumor-inhibiting effect (FIG. 12). This dual modulation system significantly inhibited tumor growth in a PKK-TTP/hs+L dependent manner, as demonstrated by a tumor growth curve similar to the PKK-TTP control when treatment was stopped on day 11. Finally, synergistic therapeutic results were further carefully confirmed using ki67 immunofluorescence staining and H & E staining, widely used in oncology to predict tumor proliferation tendencies, and significantly enhanced tumor suppression effect was observed with PKK-TTP/hs+l compared to other treatment groups (fig. 12).
Taken together, these results indicate that PKK-TTP/hs can deliver hs into tumor tissue to interfere with hTERT protein, thereby effecting antiproliferative therapy. On the other hand, PKK-TTP+L can generate ROS, promote the transportation of hTERT protein from the nucleus to cytoplasm, and realize antiproliferative treatment. The inhibition of tumor growth by the PKK-TTP/hs+L combination was significantly improved compared to other treatments alone. There was little difference in mouse body weight throughout the treatment, indicating negligible systemic cytotoxicity of PKK-TTP/hs, PKK-TTP+L and PKK-TTP/hs+L.
The invention regulates the low expression of hTERT protein in cytoplasm from two aspects, and can have certain influence on the telomerase activity and cell activity in the cell nucleus after cutting off the protein source of the cell nucleus and promoting the protein transfer in the cell nucleus. PKK-TPP+L successfully promotes the removal of hTERT protein from the nucleus into the cytoplasm through the nuclear pores, thereby reducing the content of nuclear protein, PKK-TTP/hs successfully mediates gene silencing and leads to low expression of nuclear hTERT protein in the cytoplasm of HeLa cells, so that the source of nuclear hTERT protein can be cut off. The result shows that PKK-TTP+L is effectively reduced by 18.8% compared with the telomerase activity in the cell nucleus of the single polypeptide probe test group; PKK-TTP/hs was 26.3% less potent than telomerase activity in the nuclei of the polypeptide probe test group alone; PKK-TTP/hs+L is effectively reduced by 40.1% compared with the activity of telomerase in the cell nucleus of the single polypeptide probe test group, and the strategy can effectively inhibit the activity of nuclear telomerase, so that in vivo and in vitro experiments prove that the strategy can effectively reduce the proliferation rate of tumor HeLa cells.
It should be noted that the above examples are only for further illustrating and describing the technical solution of the present invention, and are not intended to limit the technical solution of the present invention, and the method of the present invention is only a preferred embodiment and is not intended to limit the scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (9)
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