Pharmaceutical compositions containing isolated mitochondria and their use in the treatment of gastric cancer

Abstract

To provide a pharmaceutical composition for the treatment of gastric cancer in a subject.SOLUTION: A pharmaceutical composition comprises mitochondria isolated from normal gastric epithelial cells of a subject having gastric cancer or a healthy subject. Also disclosed herein is the use of the mitochondria and / or the pharmaceutical composition containing the same for the manufacture of a medicament for treating gastric cancer in a subject in need thereof. According to some embodiments of the present disclosure, the medicament is administered once every two days in an amount of 0.005 mg / kg to 2 mg / kg.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present disclosure relates to the treatment of gastric cancer. More specifically, the disclosed invention relates to the treatment of gastric cancer by using mitochondria isolated from normal gastric epithelial cells.

Background Art

[0002] Gastric cancer arising from the gastric mucosa is a globally spreading cancer. In Asia, gastric cancer is ranked third in the list of cancers commonly seen after breast cancer and lung cancer, and is still the second leading cause of cancer-related deaths after lung cancer. Despite the gradual decline in the morbidity and mortality rates due to changes in eating habits and proper food preservation and handling methods, gastric cancer continues to pose a major public health problem in many Asian countries.

[0003] While advanced gastric cancer requires treatments such as chemotherapy, radiotherapy, surgery or combinations thereof, the treatment of early gastric cancer involves minimally invasive techniques such as endoscopic treatment and laparoscopic surgery. On the other hand, the frequent recurrence, metastasis and drug resistance of gastric cancer contribute to the poor prognosis of diagnosed patients. Over the past few decades, the rapid progress of cancer stem cell (CSC) research has led to an increasing amount of evidence supporting their important role in the progression and treatment of gastric cancer. These cells are the root cause of tumorigenesis, recurrence, metastasis and drug resistance. Since cancer stem cells are often in a quiescent or dormant state, they may avoid anti-tumor treatments and ultimately lead to tumor recurrence and metastasis after treatment. Therefore, targeting the characteristics of gastric cancer stem cells has greatly developed.

[0004] In recent years, numerous studies have revealed that mitochondria are mobile between cells, regardless of whether they are normal or cancerous. Furthermore, the movement of mitochondria to damaged tissues and cells has been observed to enhance survival rates and mitigate damage. On the other hand, the functional roles of mitochondria vary between cells depending on their relative demands for energy distribution, metabolite biosynthesis, and signal transduction. How and to what extent mitochondria are involved in the treatment of gastric cancer remains largely unknown.

[0005] In light of the above, there is a need for novel treatments and pharmaceutical compositions for treating gastric cancer in the related technologies. [Overview of the project]

[0006] Below is a simplified summary of this disclosure to give the reader a basic understanding. This summary is not a comprehensive overview of the disclosure, nor does it identify key / important elements of the invention or define its scope. Its sole purpose is to present some of the concepts disclosed herein in a simplified form as an introduction to the more detailed explanation below.

[0007] As embodied and described herein in general terms, one aspect of the present disclosure is directed toward a pharmaceutical composition for treating gastric cancer. The pharmaceutical composition comprises mitochondria isolated from normal gastric epithelial cells of a subject with gastric cancer or a healthy subject.

[0008] According to alternative or optional embodiments of this disclosure, gastric cancer is resistant to anticancer drugs. Examples of anticancer drugs that can cause gastric cancer to develop drug resistance include, but are not limited to, trastuzumab, ramucirumab, pembrolizumab, nivolumab, sunitinib, regorafenib, oxaliplatin, capecitabine, irinotecan, docetaxel, folinic acid, fluorouracil (5-FU), cisplatin, paclitaxel, epirubicin, doxorubicin, mitomycin, lenvatinib, apatinib, and irinotecan.

[0009] In other embodiments, the disclosure is directed to a pharmaceutical kit for treating gastric cancer, comprising a first container for containing the above-mentioned pharmaceutical composition and a second container for containing an anticancer agent.

[0010] According to embodiments of this disclosure, examples of anticancer agents suitable for use in the pharmaceutical kit include, but are not limited to, trastuzumab, ramucirumab, pembrolizumab, nivolumab, sunitinib, regorafenib, oxaliplatin, capecitabine, irinotecan, docetaxel, folinic acid, 5-fluorouracil (5-FU), cisplatin, paclitaxel, epirubicin, doxorubicin, mitomycin, lenvatinib, apatinib, and irinotecan.

[0011] Further aspects of this disclosure are directed toward the use of mitochondria for the manufacture of pharmaceuticals in the treatment of gastric cancer in subjects requiring such treatment, wherein mitochondria are isolated from normal gastric epithelial cells from subjects or healthy subjects.

[0012] According to embodiments of this disclosure, the drug is administered every two days at a dose of 0.005 mg / kg to 2 mg / kg. In one effective embodiment, the drug is administered every two days at a dose of 0.01 mg / kg. In another preferred effective embodiment, the drug is administered every two days at a dose of 0.1 mg / kg. In yet another effective embodiment, the drug is administered every two days at a dose of 1 mg / kg.

[0013] According to embodiments of the present disclosure, gastric cancer may be resistant to anticancer drugs. Examples of anticancer drugs that can cause gastric cancer to develop drug resistance include, but are not limited to, trastuzumab, ramucirumab, pembrolizumab, nivolumab, sunitinib, regorafenib, oxaliplatin, capecitabine, irinotecan, docetaxel, folinic acid, 5-fluorouracil (5-FU), cisplatin, paclitaxel, epirubicin, doxorubicin, mitomycin, lenvatinib, apatinib, and irinotecan.

[0014] According to embodiments of this disclosure, the subject is preferably a human.

[0015] Due to the characteristics described above, this pharmaceutical composition containing mitochondria isolated from normal gastric epithelial cells provides an effective strategy in the treatment of gastric cancer. Furthermore, administration of mitochondria also reduces the resistance of gastric cancer cells to chemotherapeutic agents. Overall, the pharmaceutical composition of this invention can effectively cure gastric cancer.

[0016] Many of the accompanying features and advantages of this disclosure will be better understood by referring to the following detailed description, which will be considered in conjunction with the attached drawings.

[0017] This explanation will be better understood by referring to the following detailed explanation, which should be read in conjunction with the attached drawings. [Brief explanation of the drawing]

[0018] [Figure 1] Figure 1 is a flow cytometry histogram showing the fluorescence intensity emitted from (A) unstained gastric cancer AGS cells, (B) AGS cells stained with red fluorescent dye, (C) GES-1 cells stained with green fluorescent dye, (D) AGS cells co-cultured with GES-1 mitochondria, (E) AGS cells treated with methyl-β-cyclodextrin (MβCD) and co-cultured with GES-1 mitochondria, and (F) AGS cells treated with cytochalasin D and co-cultured with GES-1 mitochondria, according to one embodiment of the present disclosure. [Figure 2] Figure 2 consists of bar graphs showing the results of Western blot quantification of (A) SOX2, (B) NANOG, (C) GRP78, and (D) NOTCH proteins, and (E) phosphorylated JNK protein, in gastric cancer MKN-45 cells after co-culture with mitochondria, according to one embodiment of the present disclosure. [Figure 3]Figure 3 is a line graph showing (A) body weight and (B) tumor volume of mice after inoculation with AGS cells, respectively. Mean ± SEM, n≧4, two-sided Student's t-test: * = p<0.05, ** = p<0.01, *** = p<0.005. [Figure 4] Figure 4 is a line graph showing (A) body weight and (B) tumor volume of mice after inoculation with MKN-45 cells, respectively. Mean ± SEM, n≧4, two-sided Student's t-test: * = p<0.05, ** = p<0.01, *** = p<0.005. [Figure 5] Figure 5 consists of bar graphs showing the results of Western blot quantification of (A) YY-1, (B) SOX2, (C) NOTCH-1, (D) PGC-1α, (E) NANOG, (F) MCP-1, and (G) GRP78 protein expression in mice with and without mitochondrial injection. Mean ± SEM, n≧4, two-sided Student's t-test: * = p<0.05, ** = p<0.01, *** = p<0.005. [Figure 6] Figure 6 is a bar graph showing the cell viability of (A) AGS cells and (B) MKN-45 cells with and without co-culture with mitochondria under low-dose chemotherapeutic drug 5-FU, respectively. Mean ± SEM, n≧3, two-tailed Student's t-test: * = p<0.05, ** = p<0.01, *** = p<0.005, # = p<0.05, ## = p<0.01, ### = p<0.005. [Figure 7] Figure 7 is a bar graph showing the results of Western blot quantification of Bax protein expression in (A) AGS cells and (B) MKN-45 cells, respectively. Mean ± SEM, n≧3, two-sided Student's t-test: * = p<0.05, ** = p<0.01, *** = p<0.005, # = p<0.05, ## = p<0.01, ### = p<0.005. [Figure 8] Figure 8 is a bar graph showing the results of Western blot quantification of the expression ratio between phosphorylated and unphosphorylated AKT in MNK-45 cells, mean ± SEM, n≧3, two-sided Student's t-test: * = p<0.05, ** = p<0.01, *** = p<0.005.

Mode for Carrying Out the Invention

[0019] The detailed description given below in connection with the accompanying drawings is for the purpose of explaining this embodiment and does not represent the only form in which this embodiment can be configured or utilized. The description explains the functions of the embodiment and the sequence of steps for configuring and operating the embodiment. However, the same or equivalent functions and sequences can also be realized by different embodiments.

[0020] 1. Definitions For convenience, the specific terms used in the specification, the embodiments, and the appended claims are summarized here. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present invention pertains.

[0021] The singular forms "a", "an", and "the" are used herein to include plural references unless the context clearly indicates otherwise.

[0022] Values expressed in a range format include only the numerical values clearly indicated as the limits of the range, but should be interpreted in a flexible manner so as to also include all individual numerical values or sub-ranges subsumed within that range as if each numerical value and sub-range were clearly indicated. For example, an amount of "0.1 to 3 mg / kg" should be interpreted to include not only the explicitly stated delivery range of 0.1 mg / kg to 3 mg / kg, but also the individual amounts (e.g., 1 mg / kg, 2 mg / kg, and 3 mg / kg) and sub-ranges (e.g., 0.1 to 0.5 mg / kg, 1.1 to 2.2 mg / kg, etc.) within the indicated range.

[0023] The term "isolated mitochondria" used herein refers to mitochondria isolated from a specific cell of a eukaryote by various known methods, for example, using a commercially available isolation kit that separates intact mitochondria from gastric epithelial cells while maintaining biological mitochondrial activity.

[0024] The terms “administering,” “being administered,” or “administering” are used interchangeably here and refer to the application of isolated mitochondria, pharmaceutical compositions containing them, and / or pharmaceuticals manufactured with isolated mitochondria to a subject through medically acceptable routes such as oral ingestion, infusion, topical application, inhalation, and transrectal administration. The administration process also includes the step of determining an appropriate dosage and frequency to effectively achieve its intended purpose, e.g., inhibition of tumor growth.

[0025] The term "amount" as used herein refers to the amount of isolated mitochondria administered and over the required period of time that is effective in providing a therapeutic effect in the treatment of a condition or in delaying or minimizing one or more symptoms associated with the condition, in order to achieve a desired therapeutic outcome with respect to the treatment of gastric cancer. The effective amount of isolated mitochondria described herein means the amount of isolated mitochondria that provides a therapeutic effect in the treatment of a condition, either alone or in combination with other therapeutic agents. In some effective embodiments of this disclosure, the amount of isolated mitochondria is effective in inhibiting tumor growth in subjects with gastric cancer.

[0026] As used herein, the terms “treating” or “treatment” are intended to mean obtaining a desired pharmacological and / or physiological effect in a subject, for example, reducing the growth of tumors or cancer cells and / or inhibiting the progression of metastatic and / or drug-resistant cancer. The effect may be prophylactic in that it completely or partially prevents the symptoms and / or therapeutic in that it partially or completely cures the disease and / or side effects caused by the disease. As used herein, “treatment” includes curative or temporary palliative treatment of a disease in mammals, in particular humans, and includes (1) inhibiting a condition (for example, by stopping the progression of cancer in a subject) or (2) mitigating a condition (for example, by inhibiting tumor growth in a subject).

[0027] The terms “subject” or “patient” are used interchangeably here to mean mammals, including the human species, that can be treated by the methods of this disclosure. The term “mammal” means all members of the class Mammalia, including humans, primates, domesticated and livestock such as rabbits, pigs, sheep and cattle, other animals kept in zoos, for sport or as pets, and rodents such as mice and rats. Furthermore, the terms “subject” or “patient” are intended to mean both male and female unless one sex is specifically indicated.

[0028] 2. Description of the present invention This disclosure is based, at least to some extent, on the finding that transplantation of mitochondria isolated from normal gastric epithelial cells into gastric cancer cells results in significant inhibition of cancer cell growth. Therefore, this disclosure aims to provide a pharmaceutical composition comprising mitochondria isolated from normal gastric epithelial cells. Furthermore, this disclosure also aims to provide the use of mitochondria for the manufacture of pharmaceuticals in the treatment of gastric cancer in subjects requiring it.

[0029] 2.1 Pharmaceutical compositions containing isolated mitochondria One aspect of this disclosure is directed to a pharmaceutical composition for treating gastric cancer in a subject. The pharmaceutical composition comprises mitochondria isolated from normal gastric epithelial cells. According to embodiments of this disclosure, the normal gastric epithelial cells are derived from a subject having gastric cancer. Alternatively, or optionally, the normal gastric epithelial cells are derived from a healthy subject.

[0030] Mitochondria can be isolated from normal gastric epithelial cells using any tools and means known to those skilled in the art. After collecting a tissue or cell sample and homogenizing it, at least one of the usual methods, including repetitive and differential centrifugation, differential precipitation, density gradient fractionation, immunomagnetic separation, and / or filtration, can be applied to isolate and purify mitochondria. Generally, reagents and consumables used for mitochondrial isolation are commercially available in kit form. In one effective example, mitochondria are isolated from normal gastric epithelial cells (i.e., GES-1 cell line) using a mitochondrial isolation kit. Optionally, isolated mitochondria can then be maintained in vitro by any means known to those skilled in the art. In one effective example, isolated mitochondria are suspended in phosphate-buffered saline (PBS) solution and stored at 4°C while retaining their intrinsic biological activity as they were in the cells.

[0031] According to embodiments of this disclosure, the pharmaceutical composition is prepared in a form suitable for injection at a target site (e.g., subcutaneous injection). Preferably, the pharmaceutical composition is formulated in an aqueous solution to form an injectable formulation. Examples of aqueous solutions suitable for preparing the pharmaceutical composition include, but are not limited to, distilled water, glucose solution, xylitol solution, D-mannitol solution, fructose solution, physiological saline, dextran solution, amino acid solution, Ringer's solution, lactated Ringer's solution, phosphate buffer, phosphate-buffered physiological saline, and combinations thereof. According to effective embodiments of this disclosure, the pharmaceutical composition is prepared by suspending isolated mitochondria in physiological saline or PBS solution. Optionally, the injectable formulation can be sterilized by filtration through a bacterial-retaining filter before use.

[0032] 2.2 Pharmaceutical Kits Other aspects of this disclosure are directed toward pharmaceutical kits (e.g., pharmaceutical packs). The pharmaceutical kits provided herein mainly comprise a first container and a second container. Specifically, the first container contains the pharmaceutical composition described herein, and the second container contains an anticancer agent for co-administration with isolated mitochondria or the pharmaceutical composition containing them. Examples of first and second containers suitable for use in the pharmaceutical kit include vials, ampoules, bottles, syringes, dispenser packages and / or other suitable ones. In some embodiments of this disclosure, the pharmaceutical kit is useful for treating cancer and / or eliminating cancer progression in subjects requiring it.

[0033] Alternatively or optionally, the pharmaceutical kits of the present disclosure may further include instructions that provide directions to the user regarding the use of the pharmaceutical kit or the pharmaceutical composition contained herein. Preferably, the pharmaceutical kits may also include information required by regulatory bodies such as the Pharmaceuticals and Medical Devices Agency (PMDA) in Japan. In certain embodiments of the present disclosure, the instructions of the pharmaceutical kit are prescription information. According to embodiments of the present disclosure, the instructions in the pharmaceutical kit provide a dosage regimen for treating and / or reducing the risk of cancer development in a subject requiring it. In effective embodiments of the present disclosure, the dosage regimen directs sequential or combined administration of the pharmaceutical composition and anticancer agents described in Chapter 2.1 at a specific frequency and dosage for treating gastric cancer in a subject.

[0034] Examples of anticancer agents suitable for co-administration with isolated mitochondria in this kit include, but are not limited to, trastuzumab, ramucirumab, pembrolizumab, nivolumab, sunitinib, regorafenib, oxaliplatin, capecitabine, irinotecan, docetaxel, folinic acid, 5-fluorouracil (5-FU), cisplatin, paclitaxel, epirubicin, doxorubicin, mitomycin, lenvatinib, apatinib, and irinotecan.

[0035] 2.3 Use of isolated mitochondria in cancer treatment This disclosure is further directed toward the use of isolated mitochondria in the treatment of cancer development or delay of disease onset in subjects requiring it. According to embodiments of this disclosure, the mitochondria and / or a pharmaceutical composition containing them are used for the manufacture of a drug in the treatment of gastric cancer in a subject, and the mitochondria are isolated from normal gastric epithelial cells from a subject with cancer or a healthy subject, as described in Chapter 2.1.

[0036] According to an effective embodiment, the drug is administered to a subject having gastric cancer, with or without resistance to anticancer drugs, in an amount sufficient to inhibit tumor growth. According to some embodiments of the present disclosure, the drug is administered to the subject in an amount of 0.005 mg / kg to 2 mg / kg per the subject's body weight, for example, about 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 mg / kg. Preferably, the drug is administered to the subject in an amount of 0.01 mg / kg to 1 mg / kg. In one effective embodiment of this disclosure, the drug is administered to a subject with gastric cancer in an amount of about 0.01 mg / kg. In an alternative embodiment of this disclosure, the drug is administered to a cancerous subject in an amount of about 0.1 mg / kg. In another embodiment of this disclosure, the drug is administered to a subject with gastric cancer in an amount of about 0.3 mg / kg. In yet another embodiment of this disclosure, the drug is administered to the subject in an amount of 1 mg / kg.

[0037] As disclosed herein, a sufficient amount of the drug to provide a therapeutic effect in the treatment of gastric cancer may be prescribed in single doses or multi-dose doses (e.g., single-use vials or multi-dose vials). According to embodiments of the disclosure, when multi-dose doses are administered to a subject, each multi-dose dose contains the same amount of the drug described herein. In effective embodiments, the drug is prescribed in three-dose vials available for at least three infusions. According to embodiments of the disclosure, when multi-dose doses of the drug are administered to a subject, each dose of the drug is administered at a frequency of four times a day to once every three months. Specifically, the drug described herein is administered at a frequency of four times a day, three times a day, twice a day, once a day, once every two days, once every three days, once a week, once every two weeks, once a month, once every two months, or once every three months. In some effective embodiments, the drug is administered to a subject at a frequency of once every two days. According to embodiments of this disclosure, when a multi-dose drug is administered to a subject, the interval between the first and last doses of the multi-dose drug is 1 day, 2 days, 4 days, 6 days, or 1 week. In effective embodiments, the interval between the first and last doses of the multi-dose drug is 6 days.

[0038] The isolated mitochondria and / or pharmaceuticals produced thereby can be administered by any route that effectively transports the isolated mitochondria to a suitable or desired site of action. In some embodiments, the isolated mitochondria are administered parenterally (e.g., via infusion) to a suitable or desired site. Exemplary suitable parenteral routes include, but are not limited to, joint, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, intradermal, subcutaneous, and intraperitoneal infusions. Specifically, routes considered are intravenous administration (e.g., systemic intravenous infusion) and / or local administration to the site of effect (e.g., direct subcutaneous infusion). The suitable route varies depending on the specific condition being treated, its severity, individual patient parameters such as age, physical condition, build, sex, and weight, duration of treatment, (if any) nature of combination therapy, dosage and active ingredients, genetic factors, and other factors within the knowledge and expertise of healthcare professionals recognized by those skilled in the art. These factors are well known to those skilled in the art and can be addressed through routine experimentation alone. Generally, the most suitable route of administration depends on various factors, including the stability of the drug in the circulatory environment and / or the condition of the subject (e.g., whether the subject can tolerate subcutaneous injection). According to some embodiments of this disclosure, isolated mitochondria and / or pharmaceuticals are administered via subcutaneous injection. In some effective embodiments, pharmaceuticals are administered to tumor lesions via subcutaneous injection.

[0039] According to alternative or optional embodiments of the present disclosure, the drug may be used in the treatment of drug-resistant gastric cancer, i.e., gastric cancer resistant to anticancer agents. Examples of anticancer agents conventionally used in the treatment of gastric cancer include, but are not limited to, trastuzumab, ramucirumab, pembrolizumab, nivolumab, sunitinib, regorafenib, oxaliplatin, capecitabine, irinotecan, docetaxel, folinic acid, 5-fluorouracil (5-FU), cisplatin, paclitaxel, epirubicin, doxorubicin, mitomycin, lenvatinib, apatinib, irinotecan, and combinations thereof. In effective embodiments of the present disclosure, gastric cancer develops resistance to 5-FU drugs.

[0040] According to this disclosure, the term “subject” means an animal that is administered with and can benefit from the medicinal products disclosed herein. Examples of animals include, but are not limited to, humans, rats, mice, guinea pigs, monkeys, pigs, goats, cattle, horses, dogs, cats, birds, and poultry. In one exemplary embodiment, the subject is a rat. In another exemplary embodiment, the subject is a human.

[0041] Due to the characteristics described above, this pharmaceutical composition / medicine and its use can effectively inhibit the growth of gastric cancer. Furthermore, the administration of intact mitochondria also stimulates the cytotoxic effect of chemotherapeutic agents on cancer cells, thereby enabling this pharmaceutical composition / medicine to improve the therapeutic effect of gastric cancer. [Examples]

[0042] Examples Materials and methods cell culture Human gastric cancer cell lines AGS and MKN45 were cultured individually in Roswell Park Memorial Institute (RPMI1640) medium (Gibco, Waltham, Massachusetts, USA), and human normal gastric epithelial cell line GES-1 was cultured in Dulbecco's Modified Eagle Medium (DMEM, Thermo Fisher Scientific, Waltham, Massachusetts, USA). All media were supplemented with 10% fetal bovine serum (FBS) and antibiotics (100 U / ml penicillin / 100 μg / ml streptomycin). Cells were incubated in a growth chamber at 37°C with CO2 (5%, v / v).

[0043] The cells were subculturified every three days. After rinsing the cells with 1×PBS, recombinant enzyme (e.g., TrypLE, Thermo Fisher Scientific, Waltham, Massachusetts, USA) was used for cell detachment for approximately 5 minutes. The reaction was terminated by adding three times the volume of culture medium (i.e., RPMI1640 or DMEM). Subsequently, the culture medium containing the recombinant enzyme was removed by centrifugation and resuspended in the same medium used to culture the cells (i.e., RPMI1640 or DMEM). Cell counting was performed using trypan blue, and cells for continued culture were seeded in 75T flasks. For experimental purposes, the cells were placed in various sizes of culture plates, including 96-well plates, 6-well plates, and 10cm culture dishes, with 6×10 cells per well. 3 pieces, 1×10 5 pieces and 1 × 10 6 The seeds were seeded at different cell densities.

[0044] Isolation of mitochondria from normal gastric epithelial cells GES-1 cells were trypsinized using TryPLE for 5 minutes, then the reaction was stopped, the culture medium containing TryPLE was removed by centrifugation, and followed by a single wash with PBS. Subsequently, the cells were centrifuged at 1000 rpm to remove the supernatant, and the cells were placed on ice for further use.

[0045] Mitochondria were extracted from GES-1 cells using a commercially available mitochondrial isolation kit (Mitochondria Isolation Kit for Cultured Cells, Thermo Fisher Scientific, Waltham, Massachusetts, USA). The mitochondrial isolation kit included reagents A, B, and C. Specifically, GES-1 cells were isolated (1 × 10⁻⁶). 7Each mitochondrion was suspended in 400 μL of reagent A containing 1× EDTA-free protease inhibitor. After vortexing for 5 minutes, the reaction was paused by placing it on ice. Within 2 minutes, 10 μL of reagent B was added, and the reaction mixture was rapidly vortexed for 10 seconds every minute for a total of 5 minutes. Subsequently, 400 μL of reagent C containing 1× EDTA-free protease inhibitor was added, and the mixture was centrifuged at 700 g at 4°C for a further 10 minutes. The supernatant was collected and centrifuged again at 3000 g at 4°C for 15 minutes. The resulting pellet was collected and resuspended in 100 μL of reagent C. The mixture was then centrifuged at 12000 g at 4°C for 5 minutes to wash the mitochondria. After removing the supernatant, the mitochondria were suspended in 100 μL of PBS for this study.

[0046] Quantitative analysis of isolated mitochondria Mitochondria harvested from cells were dissolved by sonication in 100 μL of lysis buffer (M-PER® Mammalian Protein Extraction Reagent, Thermo Fisher Scientific, Waltham, Massachusetts, USA). After centrifugation, the supernatant was transferred to a new tube, and the concentration of mitochondrial proteins was determined using a sulforhodamine (SRB) assay kit (Sulforhodamine B Assay Kit, Abcam).

[0047] Flow cytometry Confluent cells were trypsin-treated and collected by centrifugation. The cells were resuspended in culture medium and incubated with fluorescently conjugated antibodies to CD24, CD44, and Lgr5 (BD Biosciences) for 45 minutes, followed by two washes with PBS. Fluorescence was detected by flow cytometry (BD FACSCalibur, BD Biosciences) and analyzed using analytical software (Flowing Software 2).

[0048] Animals and housing conditions Male BALB / c nude mice (5 weeks old, weighing approximately 20g) were housed in a room maintained at a temperature of 22±2℃ with a 12 / 12-hour light-dark cycle, with free access to food and water.

[0049] Establishment of xenograft tumor models I. Cell culture Human gastric cancer cell lines (including MKN45 and AGS) were trypsinized with TryPLE for 5 minutes, and the reaction was stopped using 3x the volume of culture medium. The culture medium containing TryPLE was then removed by centrifugation. The cells were washed with PBS to remove any remaining FBS, and a cell suspension was prepared by resuspending them in 100 μL of serum-free culture medium mixed with matrix gel (in a 1:1 ratio). Finally, the cell suspension was placed on ice for further use.

[0050] II. Transplantation of cancer cells Before subcutaneous transplantation into the backs of mice, the cell suspension was homogenized, aspirated using a suitable subcutaneous syringe, and the entire process was performed on ice.

[0051] BALB / c nude mice were randomly assigned to one of two groups that underwent the treatments listed below. Group I: Human gastric cancer cell lines (e.g., MKN45 or AGS) were co-cultured for 24 hours with or without mitochondria before subcutaneous transplantation into mice. Tumor growth was monitored every two days. Group II: Human gastric cancer cell lines (i.e., MKN45) were subcutaneously transplanted into mice, followed by injection of saline solution beneath the tumors. Tumor growth was monitored after one week and recorded every two days.

[0052] Western blotting Proteins from gastric cancer cells co-cultured with GES-1 mitochondria were extracted using a lysis buffer containing a protease inhibitor (1%) and a phosphatase inhibitor (1%) (M-PER® Mammalian Protein Extraction Reagent, Thermo Fisher Scientific, Waltham, Massachusetts, USA). 20 micrograms of protein were mixed in a 3:1 ratio with a sample buffer containing SDS (4%), glycerol (20%), 2-mercaptoethanol (10%), bromophenol blue (0.004%), and TrisHCl (0.125M, pH 6.8), and separated by SDS-PAGE. The gel was blotted onto a PVDF membrane (Millipore, Billerica, Massachusetts, USA) and incubated with the first and second antibodies. The membranes were developed with Enhanced Chemiluminescence (ECL) substrate (Sigma-Aldrich) and observed using a luminescence imaging system (Syngene® G:BOX Mini9, Gel Documentation System). Protein expression was calculated using image software (ImageJ, Rasband, WS, ImageJ, US National Institutes of Health, Bethesda, Maryland, USA).

[0053] Sulfolodamine B (SRB) assay A quarter volume of fixation solution was added to the sample cell culture, and the cells were fixed by incubation at 4°C for 1 hour. The fixed cell culture medium was gently aspirated, and the cells were washed three times with 200 μl of sterile water to remove excess fluid. SRB solution (45 μl) was added for staining, and the cells were incubated at room temperature, away from light, for 15 minutes. After removing the dye, the cells were washed four times with 200 μl of 1× wash buffer. Then, 200 μl of 1× solubilizer was added, and the cell sample was thoroughly mixed using a plate shaker at 700 rpm for 10 minutes. The absorbance of the mixed sample (at 565 nm) was measured using a microplate spectrophotometer (Synergy® HTX Multi-Mode Microplate Reader, Bio-Tek Epoch), and the cell viability (%) was calculated using the following formula: Cell viability (%)=((ODSample-ODBlank)) / ((ODControl-ODBlank))×100% The calculation was performed using [this method].

[0054] statistical analysis All results are presented as mean ± standard deviation. Statistical analysis was performed using one-way analysis of variance (ANOVA) or a two-tailed Student's t-test for multiple comparisons, and statistically significant differences were indicated with a p-value < 0.05.

[0055] Example 1: Isolation of GES-1 mitochondria and determination of effective dose. The objective of this experiment was to quantify isolated mitochondria and determine the effective dose for subsequent experiments. To this end, mitochondria were first isolated from normal gastric epithelial cells (GES-1) following the procedure outlined in the "Materials and Methods" section, with 1 × 10⁶ cells. 7 The total weight of mitochondria extracted per cell was calculated. Subsequently, mitochondria at various concentrations (μg / ml) in PBS solution were co-cultured with gastric cancer MKN-45 cells for 24 hours to determine the effective dose.

[0056] Doses in the range of 2.5–250 μg / ml were found to effectively reduce the viability of MKN-45 cells. Therefore, a dose of 25 μg / ml was selected as the representative concentration for subsequent in vitro and in vivo experiments.

[0057] Example 2: Transplantation of GES-1 mitochondria into gastric cancer cells This experiment investigated whether mitochondria isolated from GES-1 cells could be transplanted into gastric cancer cells. Specifically, AGS cell lines stained with red fluorescent dye (MitoTracker Deep Red FM, Invitrogen) were divided into an experimental group and a control group. The experimental group was cultured for 24 hours with mitochondria isolated from GES-1 cells that had been pre-stained with green fluorescent dye (MitoTracker Green FM, Invitrogen). On the other hand, the cells of the control group were treated for 1 hour with a phagocytosis inhibitor (i.e., methyl-β-cyclodextrin), a nanotube formation inhibitor, and an actin-dependent endocytosis inhibitor (i.e., cytochalasin D) before co-culturing with the isolated mitochondria. The treatment with the phagocytosis inhibitor and endocytosis inhibitor allowed for observation of mitochondrial movement. Then, AGS cells from both groups were analyzed by flow cytometry. The results are shown in Figure 1.

[0058] The results shown in Figure 1 revealed the uptake of exogenous GES-1 mitochondria by AGS cells. As shown in Figure 1(D), the presence of green fluorescence in AGS cells following co-culture with GES-1 mitochondria indicated intercellular transfer of these organelles. Furthermore, the decrease in green fluorescence intensity in AGS cells treated with the inhibitor compared to untreated counterparts (i.e., AGS cells from the experimental group) suggested a potential regulatory role of phagocytosis and actin-dependent endocytosis pathways in promoting the internalization and endocytosis of GES-1 mitochondria in gastric cancer cells (see panels (E) and (F) in Figure 1).

[0059] Example 3: GES-1 mitochondria reduced the expression of gastric cancer stem cell (GCSC) markers. In this example, we investigated whether mitochondria from GES-1 cells attenuate the stem cell properties of gastric cancer cells. For this purpose, the expression levels of stem cell-related proteins SOX2, NANOG, GRP78, NOTCH, and phosphorylated JNK in MKN-45 cells co-cultured with mitochondria were measured using Western blotting according to the procedure described in the "Materials and Methods" section. The quantitative results of the Western blot analysis are shown in Figure 2.

[0060] The results shown in Figure 2 clearly demonstrate a significant decrease in the expression levels of stem cell-related proteins in gastric cancer cells after co-culture with GES-1 cell mitochondria, indicating that mitochondria from GES-1 cells can attenuate the stem cell properties and capabilities of gastric cancer cells.

[0061] Example 4: GES-1 mitochondria influenced the tumorigenicity of gastric cancer cells. In this study, we investigated whether mitochondria isolated from GES-1 cells could inhibit tumor formation in vivo. For this purpose, mitochondria isolated from GES-1 cells were co-cultured with AGS cells for 24 hours, and then the cancer cells were subcutaneously transplanted into 6-week-old mice. The body weight and tumor size of the mice were recorded every two days. The results are summarized in Figure 3.

[0062] The results shown in Figure 3 clearly demonstrate that isolated mitochondria from GES-1 cells significantly inhibited the development of AGS tumors in mice compared to a control group of mice without mitochondrial treatment.

[0063] Example 5: GES-1 mitochondria inhibit tumor growth and progression in vivo. In this study, we investigated the ability of mitochondria isolated from GES-1 cells to inhibit tumor growth and progression in vivo. For this purpose, mice were randomly divided into three groups to receive the following treatments: Groups I and II received direct subcutaneous transplantation of MKN-45 cells; Group II mice received further subcutaneous injection of mitochondria beneath the tumor once it developed; and Group III co-cultured MKN-45 cells with GES-1 mitochondria for 24 hours, after which the cancer cells were subcutaneously transplanted into the mice. Mouse body weight and tumor size were recorded every two days. Tumors were removed from mice one week after mitochondrial injection for further protein expression analysis via Western blotting. The results are shown in Figures 4 and 5.

[0064] As is evident from the significant reduction in tumor volume shown in panel B of Figure 4, tumor growth was found to be suppressed after the test mice received mitochondrial injection, compared to group I (i.e., the group without mitochondrial treatment).

[0065] Regarding protein analysis in tumor tissue, the quantitative results shown in Figure 5 reveal that mice treated with mitochondria showed a significant decrease in protein expression, including stem cell-related proteins (i.e., SOX2, NANOG, GRP78, and NOTCH-1), glycolysis-related protein PGC-1α, and tumor progression-related proteins YY-1 and MCP-1, compared to the untreated group. The data comprehensively demonstrate that administration of isolated mitochondria from normal gastric epithelial cells can effectively inhibit tumor growth, progression, and stem cell characteristics.

[0066] Example 6: GES-1 mitochondria reduced the resistance of gastric cancer cells to anticancer drugs. This experiment investigated whether mitochondria isolated from GES-1 cells could increase the sensitivity of gastric cancer cells to anticancer drugs. For this purpose, gastric cancer cell lines AGS and MKN-45 were co-cultured with GES-1 mitochondria for 24 hours, then the culture medium was replaced with a medium containing 5-FU (0.5 μM), and the cells were incubated again for another 24 hours. The cancer cell viability of each cell line was analyzed using a sulforhodamine B (SRB) assay according to the procedure described in the "Materials and Methods" section. The results are shown in Figure 6.

[0067] The results shown in Figure 6 demonstrate that mitochondria isolated from GES-1 cells enhanced the cytotoxicity of 5-FU against gastric cancer cell lines AGS and MKN-45.

[0068] To further investigate whether mitochondria directly influence cellular apoptosis and drug resistance in cancer cells, Western blot analysis was performed to quantify the protein expression of the apoptosis-related gene Bax and the drug resistance-related protein phosphorylated AKT. The results are shown in Figures 7 and 8, respectively.

[0069] The quantified data in Figure 7 reveals a consistent trend in two gastric cancer cell lines: Bax protein expression decreased in cancer cells after treatment with low-dose 5-FU, while mitochondria restored the decrease in Bax expression induced by low-dose 5-FU treatment in both AGS and MKN-45 cells. Furthermore, the data in Figure 8 shows that the ratio of phosphorylated AKT in MKN-45 cells decreased over time after co-culture with mitochondria. These data collectively demonstrate that mitochondria in GES-1 cells effectively reduce drug resistance in gastric cancer cells.

[0070] Based on the combined results of Examples 1-6, it is clear that mitochondria obtained from normal gastric epithelial cells have the ability to prevent tumor formation and inhibit tumor growth. Furthermore, isolated mitochondria can effectively reduce the resistance of gastric cancer cells to chemotherapeutic agents and enhance the cytotoxicity of these drugs against cancer cells. Therefore, the pharmaceutical compositions comprising isolated mitochondria provided herein can be effectively used in the treatment of gastric cancer, including drug-resistant gastric cancer.

[0071] It should be understood that the above description of embodiments is given for illustrative purposes only and that various modifications can be made by those skilled in the art. The above specifications, examples and data provide a complete description of the structure and use of exemplary embodiments of the invention. Although various embodiments of the invention have been described above with some degree of specificity or by reference to one or more individual embodiments, those skilled in the art should be able to make numerous modifications to the disclosed embodiments without departing from the spirit or scope of the invention.

Claims

1. A pharmaceutical composition for treating gastric cancer, comprising mitochondria isolated from normal gastric epithelial cells of a subject with gastric cancer or a healthy subject, The aforementioned gastric cancer is resistant to anticancer drugs and exhibits cancer stem cell properties; this is a pharmaceutical composition.

2. The pharmaceutical composition according to claim 1, wherein the anticancer agent is selected from the group consisting of trastuzumab, ramucirumab, pembrolizumab, nivolumab, sunitinib, regorafenib, oxaliplatin, capecitabine, irinotecan, docetaxel, folinic acid, 5-fluorouracil (5-FU), cisplatin, paclitaxel, epirubicin, doxorubicin, mitomycin, lenvatinib, apatinib, irinotecan, and combinations thereof.

3. The use of mitochondria for the manufacture of a pharmaceutical for the treatment of gastric cancer in a subject requiring such use, wherein the mitochondria are isolated from normal gastric epithelial cells from the subject or a healthy subject, and the gastric cancer is resistant to anticancer drugs and exhibits cancer stem cell characteristics.

4. The use according to claim 3, wherein the pharmaceutical is administered to the subject once every two days in an amount of 0.005 mg / kg to 2 mg / kg.

5. The use according to claim 3, wherein the anticancer agent is selected from the group consisting of trastuzumab, ramucirumab, pembrolizumab, nivolumab, sunitinib, regorafenib, oxaliplatin, capecitabine, irinotecan, docetaxel, folinic acid, 5-fluorouracil (5-FU), cisplatin, paclitaxel, epirubicin, doxorubicin, mitomycin, lenvatinib, apatinib, irinotecan, and combinations thereof.

6. The use according to claim 3, wherein the subject is a human.

Images