Application of mitochondrial transplantation preparation in reversing lung cancer EGFR targeted drug resistance

Abstract

The invention relates to an application of a mitochondrial transplantation preparation in reversing drug resistance after lung cancer EGFR targeted therapy, namely an application of the mitochondrial transplantation preparation in preparing a drug for reversing EGFR-TKIs drug resistance, and discovers that the mitochondrial transplantation preparation can easily enter an EGFR-TKIs drug-resistant NSCLC cell, and the EGFR protein expression level of the NSCLC cell is reduced, so that the drug resistance of the EGFR-TKIs drug-resistant NSCLC cell is reduced, and the drug resistance of the EGFR-TKIs drug-resistant NSCLC cell is reduced. The acquired drug resistance of the NSCLC cells to the EGFR-TKIs is reversed, and the treatment effect of the EGFR-TKIs on the NSCLC is improved.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to the application of a mitochondrial transplantation preparation in reversing the drug resistance of lung cancer to EGFR targeted drug treatment. Background Art

[0002] Lung cancer is the most common malignant tumor worldwide, with over 2 million patients diagnosed with the disease and approximately 1.8 million deaths each year. Non-small cell lung cancer (NSCLC), the predominant histological type of lung cancer, accounts for nearly 85% of all lung cancer cases.

[0003] Conventional treatments for lung cancer include surgery, radiotherapy, and neoadjuvant or adjuvant chemotherapy. The vast majority of patients are diagnosed with lung cancer in the advanced stages, ineligible for radical surgery and requiring only comprehensive treatment based on traditional chemotherapy. Traditional chemotherapy for NSCLC includes paclitaxels, pemetrexed, platinum analogs, and gemcitabine. The overall survival rate for patients treated solely with traditional chemotherapy is only 26%, resulting in a very high mortality rate.

[0004] With the rapid development of precision medicine, epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) have become an effective targeted therapy for advanced NSCLC. Studies have shown that 40% to 50% of lung adenocarcinoma patients in China harbor EGFR mutations, making them suitable candidates for EGFR-TKIs. EGFR mutations can lead to abnormal and persistent activation of tyrosine kinase (TK), increasing phosphorylation levels and causing uncontrolled cell growth and cancer. Compared with traditional chemotherapy, EGFR-TKIs can significantly prolong patients' progression-free survival, improve objective response rates, and enhance quality of life. EGFR-TKIs have transformed the chemotherapy paradigm for cancer and have become a first-line clinical treatment for this population.

[0005] The first-generation EGFR-TKIs include gefitinib, icotinib, and erlotinib, and the second-generation EGFR-TKIs include afatinib and dacomitinib. Clinically, it has been found that even with effective treatment with first- and second-generation EGFR-TKIs, 50-80% of patients will develop acquired resistance after 11 months of treatment (Acquired resistance to TKIs in solid tumors: learning from lung cancer. Nat Rev Clin Oncol. 2014 Aug; 11(8): 473-81.; Systemic treatment in EGFR-ALK NSCLC patients: second line therapy and beyond. Cancer Biol Med. 2014 Sep; 11(3): 173-81.). Third-generation EGFR-TKIs, represented by osimertinib, will also develop acquired resistance after 9-13 months of treatment (AZD9291, an irreversible EGFR TKI, overcomes T790M-mediated resistance to EGFR inhibitors in lung cancer. Cancer Discov. 2014 Sep; 4(9): 1046-61.). This greatly limits the survival benefit of lung cancer patients after using EGFR-TKIs.

[0006] Almost all EGFR-TKIs develop acquired resistance after a period of use. First- and second-generation EGFR-TKIs target exon 19 deletion mutations or the L858R mutation, while the third-generation osimertinib targets the T790M mutation. The mechanisms of resistance include protein conformational changes that conceal the drug's active site and new gene mutations that create new activation sites on the target protein. Ultimately, clinical manifestations include elevated EGFR phosphorylation (p-EGFR) levels in tumor tissue, overactivation of EGFR and its downstream signaling pathways, and continued abnormal tumor cell proliferation.

[0007] Currently, there is no effective clinical treatment for patients with EGFR-TKI resistance, and the only option is chemotherapy. Furthermore, once a patient develops resistance to a full line of EGFR-TKIs, more complex resistance mechanisms may emerge, and even a shift in tumor pathology may occur, making treatment more difficult clinically. This is a global dilemma and a clinical bottleneck, leading to an urgent need to develop an effective method to reverse EGFR-TKI resistance. By reversing EGFR-TKI resistance, especially the clinical bottleneck of first-generation EGFR-TKI resistance, we can extend the time lung cancer patients can effectively use EGFR-targeted therapy and improve their survival and quality of life.

[0008] The main functions of mitochondria include energy metabolism, calcium homeostasis, redox, and apoptosis. Mitochondria are dynamic organelles whose dynamic nature is not only reflected in the regulation of energy production, but also in their biogenesis and fusion / fission, which are processes of mitochondrial stress adaptation.

[0009] The increasing understanding of these key processes involved in neoplastic transformation makes mitochondria an attractive therapeutic target. Mitochondrial transplantation is an emerging approach with anti-tumor potential by restoring mitochondrial function. Therapeutic applications of mitochondrial transplantation Transplanting mitochondria from healthy cells to abnormal cells has emerged as a novel and attractive therapeutic strategy for treating diseases caused by mitochondrial damage or dysfunction in somatic cells.

[0010] Previously, Zhang et al. transplanted mitochondria into breast cancer cell lines. Their results showed that mitochondrial transplantation-induced apoptosis inhibited cell growth and reduced oxidative stress, thereby increasing the sensitivity of MCF-7 and MDA-MB-231 breast cancer cells to the chemotherapy drugs doxorubicin and paclitaxel (Mitochondrial transplantation regulates antitumouractivity, chemoresistance and mitochondrial dynamics in breast cancer. J ExpClin Cancer Res. 2019; 38: 30.). Mitochondrial transplantation research is still in its infancy, and there are no reports of mitochondrial transplantation in targeted therapy or reversing resistance to targeted therapy. Summary of the Invention

[0011] The purpose of the present invention is to provide an application of a mitochondrial transplantation preparation to reverse the EGFR targeted drug resistance of lung cancer, and to solve the clinical medication problem of drug resistance after EGFR targeted treatment of lung cancer by using the mitochondrial transplantation preparation.

[0012] To achieve the above-mentioned purpose of the invention, the present invention first provides a use of a mitochondrial transplantation preparation in the preparation of a drug for reversing EGFR-TKIs resistance.

[0013] In particular, the present invention provides a use of a mitochondrial transplant preparation in the preparation of a drug for reversing first-generation EGFR-TKIs resistance.

[0014] More specifically, the first-generation EGFR-TKIs are gefitinib, icotinib or erlotinib.

[0015] The experiments of the present invention found that the mitochondrial transplant preparation can easily enter the interior of EGFR-TKIs-resistant non-small cell lung cancer cells, and by reducing the EGFR protein expression level of non-small cell lung cancer cells, reverse the acquired resistance of non-small cell lung cancer cells to first-generation EGFR-TKIs, thereby improving the therapeutic effect of first-generation EGFR-TKIs on non-small cell lung cancer.

[0016] Therefore, the present invention further provides the use of a mitochondrial transplant preparation combined with EGFR-TKIs in the preparation of a drug for treating lung cancer.

[0017] Specifically, the lung cancer is EGFR-TKIs-resistant lung cancer.

[0018] More specifically, the lung cancer is first-generation EGFR-TKIs-resistant lung cancer.

[0019] Furthermore, the present invention further provides a combination drug for treating EGFR-TKIs-resistant lung cancer, wherein the active ingredients of the drug include a mitochondrial transplant preparation and first-generation EGFR-TKIs, and the mitochondrial transplant preparation and first-generation EGFR-TKIs respectively become independent dosing units.

[0020] The present invention provides a method for effectively improving the acquired resistance of non-small cell lung cancer to EGFR-TKIs, especially first-generation EGFR-TKIs, through mitochondrial transplantation preparations. Cell experiments and animal experiments on two first-generation EGFR-TKIs-resistant cell lines (PC9 / GR and H1975 lung cancer cell lines) have demonstrated that mitochondrial transplantation preparations can effectively improve the resistance to EGFR-TKIs. By exploring the mechanism, it is revealed that improving mitochondrial function can effectively reduce the resistance to EGFR-TKIs in non-small cell lung cancer. This provides a new strategy and theoretical basis for improving the resistance to EGFR-TKIs in non-small cell lung cancer, and is the first study in the field of EGFR targeted therapy resistance in lung cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1This figure shows the entry of mitochondria with GFP fluorescent labeling into PC9 / GR tumor cells.

[0022] Figure 2 is the IC of gefitinib against PC9 / GR tumor cells 50 concentration.

[0023] Figure 3 is the IC of erlotinib against PC9 / GR tumor cells 50 concentration.

[0024] Figure 4 is the IC of gefitinib against H1975 tumor cells 50 concentration.

[0025] Figure 5 is the IC of erlotinib against H1975 tumor cells 50 concentration.

[0026] Figure 6 This is the inhibitory effect of mitochondria on the growth of recipient cells PC9 / GR.

[0027] Figure 7 Flow cytometry analysis was used to analyze whether mitochondria enhanced the killing effect of gefitinib and erlotinib on PC9 / GR cells.

[0028] Figure 8 Flow cytometry analysis was used to analyze whether mitochondria enhanced the killing effect of gefitinib and erlotinib on H1975 cells.

[0029] Figure 9 Western Blot was used to detect the expression level of EGFR protein in PC9 / GR cells co-incubated with mitochondria.

[0030] Figure 10 is the tumor growth curve of tumor-bearing animals under different intervention measures.

[0031] Figure 11 are the final tumor volume and weight of tumor-bearing animals under different intervention measures.

[0032] Figure 12 Western Blot was used to detect the phosphorylation (p-EGFR) level of EGFR protein extracted from the tumor of each group of mice.

[0033] Figure 13 Elisa was used to detect the expression level of EGFR protein in the serum of each group of mice.

[0034] Figure 14 These are the results of pathological staining of the organ tissues of mice in each group. Implementation Method

[0035] The following embodiments are only used to more clearly illustrate the technical solutions of the present invention so that those skilled in the art can better understand and utilize the present invention, but are not intended to limit the scope of protection of the present invention.

[0036] Unless otherwise specified, the production processes, experimental methods or detection methods involved in the embodiments of the present invention are all conventional methods in the prior art, and their names and / or abbreviations are conventional names in the field and are very clear and unambiguous in the relevant fields of use. Those skilled in the art can understand the conventional process steps based on the names and apply the corresponding equipment to implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0037] The various instruments, equipment, raw materials or reagents used in the embodiments of the present invention are not particularly limited in their sources and are all conventional products that can be purchased through regular commercial channels or prepared according to conventional methods well known to those skilled in the art. Example

[0038] Example 1

[0039] 293T human embryonic kidney cells were transfected with a mitochondrial fluorescent protein plasmid, GFP, resulting in green fluorescence in the mitochondria. 48 hours after transfection, the plasmid transfection efficiency exceeded 80%.

[0040] 293T cells grown at 80-90% density in a 10 cm cell culture dish were collected. The original culture medium was discarded, and 1 ml of trypsin was added to each dish to digest the cells. The cells were then placed in a 37°C incubator. After 3 minutes, the digestion was terminated with complete culture medium containing 10% fetal bovine serum.

[0041] Transfer the cell suspension to a 15 ml centrifuge tube and centrifuge at 1000 rpm for 3 minutes. Discard the supernatant and retain the pellet. Resuspend the pellet in 3 ml of PBS buffer and count the cells.

[0042] Take 10 million cells and centrifuge at 1000 rpm for 3 minutes. Retain the pellet and discard the supernatant. Add 2 ml of Mitochondrial Isolation Buffer (Biyuntian, C3601-1) to suspend the cells and place in an ice bath for 10 minutes.

[0043] Transfer the cell suspension to a glass homogenizer and homogenize 25-35 times. Take 2µl of the cell suspension and add 50µl of trypan blue staining solution. Mix thoroughly and observe the proportion of cells that stain positive (blue) under a microscope. Stop homogenization when the proportion of positive cells exceeds 50%.

[0044] After homogenization, the suspension was aliquoted and transferred to 1.5 ml sterile, enzyme-free centrifuge tubes. Centrifuge at 1000 g for 10 min in a pre-cooled 4°C centrifuge, and the pellet was discarded. The supernatant was transferred to a new 1.5 ml sterile, enzyme-free centrifuge tube and centrifuged again at 3500 g for 10 min in a pre-cooled 4°C centrifuge. The supernatant was discarded, and the pellet was retained to obtain purified mitochondria tagged with GFP.

[0045] The mitochondria with GFP green fluorescence label obtained above were added to the culture medium of gefitinib-resistant non-small cell lung cancer cell line PC9 / GR. After incubation for 6 hours, the recipient cells were observed under a fluorescence microscope.

[0046] Observation results such as Figure 1 The left side shows a green fluorescence field fluorescence image, and the right side shows a bright field cell image under the same field of view. In the image, a large amount of fluorescence is visible in the cell region, indicating mitochondria labeled with GFP green fluorescence, while there is no obvious punctate fluorescence in the intercellular space, indicating that no mitochondria are present. These results indicate that mitochondria labeled with GFP green fluorescence can be internalized by the gefitinib-resistant non-small cell lung cancer cell line PC9 / GR, and mitochondria can enter PC9 / GR cells.

[0047] Example 2

[0048] In this example, first-generation targeted drug-resistant lung cancer cells PC9 / GR and H1975 were used as test cells, gefitinib and erlotinib were used as test drugs, and the CCK8 assay was used to assess the half-lethal concentration of the drugs for the cells.

[0049] In a 6-well plate, mitochondria purified from AC16 cells were added to recipient cells PC9 / GR or H1975 and co-incubated. After 24 hours, PC9 / GR or H1975 cells in the logarithmic growth phase were digested with trypsin, resuspended in culture medium and counted, and the cells were seeded in a 96-well plate at a density of 5000 cells per well.

[0050] A continuous concentration gradient of gefitinib or erlotinib-containing culture medium was prepared using the serial dilution method and added to PC9 / GR or H1975 cells at the corresponding drug concentrations. Six replicate wells were set up for each drug concentration. After 24 hours of continuous cell culture, the culture medium in the 96-well plate was discarded, and 100 μl of culture medium and 10 μl of CCK8 were added to each well. After incubation at 37°C for 2-4 hours, the absorbance of each well at OD492 nm was measured using a microplate reader.

[0051] The growth inhibition rate of each concentration of gefitinib or erlotinib on PC9 / GR or H1975 cells was calculated based on the absorbance values of the control wells without CCK8 and the drug-added wells with gefitinib or erlotinib. The half-lethal concentration IC of gefitinib or erlotinib on PC9 / GR or H1975 cells was calculated based on the inhibition rate. 50 .

[0052] The same method was used to calculate the half-lethal concentration (IC) of gefitinib or erlotinib for PC9 / GR or H1975 cells co-incubated without mitochondria. 50 .

[0053] The final result is as follows Figures 2 to 5 As shown, compared with PC9 / GR and H1975 cells co-incubated without mitochondria, the inhibition curves of two first-generation targeted drugs, gefitinib and erlotinib, shifted to the left, and the fitted IC 50 The IC values of gefitinib or erlotinib were reduced in PC9 / GR and H1975 cells, two first-generation targeted drug-resistant lung cancer cells co-incubated with mitochondria. 50 concentration, reversing gefitinib or erlotinib resistance.

[0054] Among them, the IC of gefitinib on PC9 / GR cells 50 The concentration decreased from 24.5 μM to 19.6 μM ( Figure 2 IC of erlotinib on PC9 / GR cells 50 The concentration decreased from 6.23 μM to 1.53 μM ( Figure 3 IC of gefitinib on H1975 cells 50 The concentration decreased from 51.5 μM to 37.3 μM ( Figure 4 IC of erlotinib on H1975 cells 50 The concentration decreased from 62.3 μM to 24.57 μM ( Figure 5 ).

[0055] Example 3

[0056] To evaluate whether mitochondria can have an independent growth inhibitory effect on tumor cells, in this example, mitochondria purified from AC16 cells were added to recipient cells PC9 / GR and co-incubated without adding drugs. Cell growth was assessed using the CCK8 assay.

[0057] After the recipient cells were incubated with mitochondria for 24 h, the culture medium in the 96-well plate was discarded, and 100 μl of culture medium and 10 μl of CCK8 were added to each well. After incubation in a 37°C incubator for 2-4 h, the absorbance of each well at OD492 nm was detected using a microplate reader.

[0058] According to the absorbance values of the control wells without CCK8 and the control group cell wells without mitochondria, the cell growth inhibition rates of low, medium and high concentrations of mitochondria were calculated. Among them, the high dose of mitochondria was defined as the number of mitochondria: the number of recipient cells = 100:1; the medium dose of mitochondria was defined as the number of mitochondria: the number of recipient cells = 10:1; and the low dose of mitochondria was defined as the number of mitochondria: the number of recipient cells = 1:1.

[0059] The results are as follows Figure 6 As shown, it shows that no matter low dose, medium dose or high dose of mitochondria, they cannot produce growth inhibitory effect on recipient tumor cells.

[0060] Example 4

[0061] Mitochondria extracted from AC16 cells were co-incubated with gefitinib-resistant PC9 / GR cells for 24 hours, and then gefitinib (Ge) or erlotinib (Erlo) was added for intervention for 48 hours. Subsequently, cell apoptosis was detected by flow cytometry.

[0062] Among them, the high-dose mitochondria is stipulated as the number of mitochondria: the number of recipient cells = 100:1; the low-dose mitochondria is stipulated as the number of mitochondria: the number of recipient cells = 10:1.

[0063] Figure 7 The results showed that transplantation of AC16 cell line mitochondria into PC9 / GR cells enhanced the killing effect of two first-generation targeted drugs against first-generation targeted drug-resistant cells in a concentration-dependent manner. Compared with gefitinib alone, high-dose mitochondria combined with gefitinib increased the apoptosis rate of gefitinib in PC9 / GR cells from 4.6% to 8.7%, an 89.1% improvement in killing efficiency. Compared with erlotinib alone, high-dose mitochondria combined with erlotinib increased the apoptosis rate of erlotinib in PC9 / GR cells from 11.5% to 19.7%, a 71.3% improvement in killing efficiency.

[0064] Mitochondria extracted from AC16 cells were incubated with gefitinib-resistant H1975 cells for 24 hours before being treated with gefitinib (Ge) or erlotinib (Erlotinib) for 48 hours. Flow cytometry was then used to analyze cell apoptosis. Similarly, the high-dose mitochondria ratio was defined as 100:1 for the number of mitochondria to the number of recipient cells, and the low-dose mitochondria ratio was defined as 10:1 for the number of mitochondria to the number of recipient cells.

[0065] Figure 8Results also showed that transplantation of AC16 cell line mitochondria into H1975 cells enhanced the killing effect of two first-generation targeted drugs against first-generation targeted drug-resistant cells, also in a concentration-dependent manner. Compared with gefitinib alone, high-dose mitochondria combined with gefitinib increased the apoptosis rate of gefitinib in H1975 cells from 15.8% to 24.0%, a 51.9% increase in killing efficiency. Compared with erlotinib alone, high-dose mitochondria combined with erlotinib increased the apoptosis rate of erlotinib in H1975 cells from 3.22% to 10.9%, a two-fold increase in killing efficiency.

[0066] Example 5

[0067] Mitochondria extracted from AC16 cells were co-incubated with gefitinib-resistant PC9 / GR cells for 24 h, and then whole protein lysates of gefitinib-resistant PC9 / GR cells were extracted and Western Blot assay was performed to detect the expression level of EGFR protein in the cells.

[0068] Figure 9 The test results showed that after the mitochondria of the AC16 cell line were transplanted into PC9 / GR cells, the expression level of EGFR protein in the latter cells decreased in a concentration gradient-dependent manner.

[0069] Example 6

[0070] Nude mice were subcutaneously implanted with PC9 / GR cells to establish a drug-resistant tumor animal model, and the mice were randomly divided into two groups.

[0071] One week after tumor implantation (day 0), the baseline tumor volume of the two groups of mice was measured. On days 1 to 5, 8 to 12, and 15 to 16, both groups of mice were treated with gefitinib at a dose of 10 mg / kg via oral gavage. On days 1, 4, 8, 11, and 15, mice in the mitochondria + gefitinib group received mitochondrial transplantation at a dose of 1×10 7 The mice in the gefitinib group were injected with 200 μl of mitochondrial suspension through the tail vein. The mice in the gefitinib group were given an equal amount of normal saline in the same way.

[0072] Tumor measurements were performed on days 2, 5, 7, 9, 12, 14, and 16. The results showed that mitochondrial transplantation helped improve the efficacy of gefitinib and inhibited tumor growth ( Figure 10 ), the sensitivity of tumor cells to gefitinib was significantly increased.

[0073] On the 16th day, the mice were killed, the tumors were dissected out, and the tumor sizes were measured, photographed, and counted. Figure 11The tumor weight and volume of mice in the mitochondria+gefitinib group were significantly smaller than those in the gefitinib group, with statistically significant differences, indicating that mitochondria can enhance the sensitivity of gefitinib-resistant cells to gefitinib and reverse resistance.

[0074] Example 7

[0075] The whole protein lysate was extracted from the mouse tumor after dissection and used for Western Blot experiment. Figure 12 The results showed that the phosphorylation level of EGFR protein in the tumor protein of mice in the mitochondria + gefitinib group was lower than that in the tumor protein of the gefitinib group, proving that mitochondria can inhibit the phosphorylation level of EGFR protein in gefitinib-resistant cells, thereby reversing drug resistance and improving therapeutic efficacy.

[0076] Furthermore, serum was extracted from the blood of mice in each group for ELISA experiments to detect the expression level of EGFR protein in the mouse blood. Figure 13 The results showed that the expression of EGFR protein in the serum of mice in the mitochondria + gefitinib group was lower than that in the gefitinib group, further indicating that mitochondria combined with gefitinib can reduce the expression of EGFR protein in mice and enhance the efficacy of gefitinib.

[0077] Example 8

[0078] Liver, spleen, lung and kidney tissues were extracted from each group of mice and stained with HE to observe whether mitochondrial transplantation affects the organ structure of the animals.

[0079] Figure 14 Results showed that alveolar structure in lung tissue was stable, epithelial cells were normal, and there was no inflammatory infiltration or pulmonary fibrosis. In kidney tissue, epithelial cells were densely packed, without congestion or edema, and no inflammatory cell infiltration or fibrosis was observed. In liver tissue, hepatocytes were densely packed and structurally stable, with normal nuclei and no inflammatory cell infiltration or fibrosis was observed. In spleen tissue, there were no significant changes in spleen structure, with normal size and distribution of red and white pulp areas, and no dilation or inflammatory cell infiltration within the sinusoids. These results indicate that mitochondria did not affect changes in animal tissues, did not induce an inflammatory response, and had a good safety profile.

[0080] The above embodiments of the present invention do not describe all details in detail, nor do they limit the present invention to the above embodiments. Various changes, modifications, substitutions, and variations made by those skilled in the art without departing from the principles and purpose of the present invention are intended to be included within the scope of protection of the present invention.

Claims

1. Application of mitochondrial transplantation preparations in the preparation of drugs to reverse EGFR-TKIs resistance.

2. Application of mitochondrial transplant preparations in the preparation of drugs to reverse resistance to first-generation EGFR-TKIs.

3. The use according to claim 2, wherein the first-generation EGFR-TKIs is gefitinib, icotinib or erlotinib.

4. Application of mitochondrial transplant preparations combined with EGFR-TKIs in the preparation of drugs for the treatment of lung cancer.

5. The use according to claim 4, wherein the lung cancer is EGFR-TKIs-resistant lung cancer.

6. The use according to claim 4, wherein the lung cancer is first-generation EGFR-TKIs-resistant lung cancer.

7. A combination drug for treating EGFR-TKIs-resistant lung cancer, wherein the active ingredients of the drug include a mitochondrial transplant preparation and first-generation EGFR-TKIs, and the mitochondrial transplant preparation and the first-generation EGFR-TKIs are independent dosing units.

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