Pharmaceutical composition comprising isolated mitochondria and use thereof in 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 through the use of mitochondria isolated from normal gastric epithelial cells. [Background technology]

[0002] Gastric cancer, which arises from the gastric mucosa, is a cancer with a worldwide prevalence. In Asia, gastric cancer ranks third on the list of common cancers after breast and lung cancer, and remains the second leading cause of cancer-related death after lung cancer. Despite gradual declines in incidence and mortality due to changes in dietary habits and proper food storage and handling techniques, gastric cancer continues to pose a significant public health challenge in many Asian countries.

[0003] While advanced-stage gastric cancer requires treatments such as chemotherapy, radiation therapy, surgery, or a combination thereof, treatment of early-stage gastric cancer involves minimally invasive techniques such as endoscopic therapy and laparoscopic surgery. However, frequent recurrence, metastasis, and drug resistance of gastric cancer contribute to the poor prognosis of diagnosed patients. Over the past few decades, rapid advances in cancer stem cell (CSC) research have provided increasing evidence supporting their important role in the progression and treatment of gastric cancer. These cells are the underlying cause of tumor initiation, recurrence, metastasis, and drug resistance. Because cancer stem cells often remain in a dormant or quiescent state, they can also evade antitumor treatments, ultimately leading to tumor recurrence and metastasis after treatment. Therefore, significant advances have been made in targeting the characteristics of gastric cancer stem cells.

[0004] In recent years, many studies have revealed that mitochondria can move between cells, regardless of whether they are normal or cancerous. It has also been observed that the transfer of mitochondria to injured tissues and cells can increase survival and reduce damage. Meanwhile, the functional role of mitochondria varies between cells depending on the 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 unknown.

[0005] In view of the above, there is a need in the related art for new treatments and pharmaceutical compositions for treating gastric cancer. Summary of the Invention

[0006] The following presents a simplified summary of the disclosure in order to provide the reader with a basic understanding. This summary is not an exhaustive overview of the disclosure, and it does not identify key / critical elements of the invention or delineate the scope of the invention. Its sole purpose is to present some concepts disclosed herein in a simplified form as a prelude to the more detailed description that is presented later.

[0007] As embodied and generally described herein, one aspect of the present disclosure is directed to a pharmaceutical composition for treating gastric cancer, the pharmaceutical composition comprising mitochondria isolated from normal gastric epithelial cells of a subject with gastric cancer or a healthy subject.

[0008] According to an alternative or optional embodiment of the present disclosure, the gastric cancer is resistant to an anticancer drug. Examples of anticancer drugs to which gastric cancer may 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 another aspect, the present disclosure is directed to a pharmaceutical kit for treating gastric cancer comprising a first container for housing the pharmaceutical composition described above and a second container for housing an anti-cancer agent.

[0010] According to embodiments of the present disclosure, examples of anti-cancer 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] Yet another aspect of the present disclosure is directed to the use of mitochondria for the manufacture of a medicament in the treatment of gastric cancer in a subject in need thereof, wherein the mitochondria are isolated from the subject or normal gastric epithelial cells from a healthy subject.

[0012] According to embodiments of the present disclosure, the medicament is administered in an amount of 0.005 mg / kg to 2 mg / kg every other day. In one effective embodiment, the medicament is administered in an amount of 0.01 mg / kg every other day. In another preferred effective embodiment, the medicament is administered in an amount of 0.1 mg / kg every other day. In yet another effective embodiment, the medicament is administered in an amount of 1 mg / kg every other day.

[0013] According to embodiments of the present disclosure, gastric cancer may be resistant to anticancer drugs. Examples of anticancer drugs to which gastric cancer may 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 the present disclosure, the subject is preferably a human.

[0015] Due to the above characteristics, the present pharmaceutical composition containing mitochondria isolated from normal gastric epithelial cells provides an effective strategy for treating gastric cancer. Furthermore, administration of mitochondria also reduces the resistance of gastric cancer cells to chemotherapy drugs. Overall, the pharmaceutical composition of the present invention can effectively cure gastric cancer.

[0016] Many of the attendant features and advantages of the present disclosure will become better understood by reference to the following detailed description considered in connection with the accompanying drawings.

[0017] The present description is better understood from the following detailed description read in light of the accompanying drawings. [Brief explanation of the drawings]

[0018] [Figure 1] FIG. 1 is a flow cytometry histogram showing the fluorescence intensity emitted from (A) unstained gastric cancer AGS cells, (B) AGS cells stained with a red fluorescent dye, (C) GES-1 cells stained with a 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] FIG. 2 is a bar graph showing Western blot quantification results of (A) SOX2, (B) NANOG, (C) GRP78, and (D) NOTCH proteins, and (E) phosphorylated JNK proteins, in gastric cancer MKN-45 cells after co-culture with mitochondria, according to one embodiment of the present disclosure. [Figure 3]Figure 3 shows line graphs showing (A) body weight and (B) tumor volume of mice after AGS cell inoculation. Mean ± SEM, n≧4, two-tailed 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. Mean ± SEM, n≧4, two-tailed Student's t-test: *, p<0.05, **, p<0.01, ***, p<0.005. [Figure 5] Figure 5 consists of bar graphs showing 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, respectively. Mean ± SEM, n≧4, two-tailed Student's t-test: *, p<0.05, **, p<0.01, ***, p<0.005. [Figure 6] Figure 6 is a bar graph showing cell viability of (A) AGS cells and (B) MKN-45 cells, respectively, with or without co-culture with mitochondria under low-dose chemotherapy drug 5-FU. 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 shows bar graphs showing Western blot quantification of Bax protein expression in (A) AGS cells and (B) MKN-45 cells, 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 8] Figure 8 is a bar graph showing the results of Western blot quantification of the expression ratio between phosphorylated and non-phosphorylated AKT in MNK-45 cells, mean ± SEM, n≧3, two-tailed Student's t-test: *, p<0.05, **, p<0.01, ***, p<0.005. DETAILED DESCRIPTION OF THE INVENTION

[0019] The detailed description provided below in connection with the accompanying drawings is intended as a description of the present embodiment and is not intended to represent the only manner in which the present embodiment may be constructed or utilized. The description sets forth the functions of the embodiment and the sequence of steps for constructing and operating the embodiment, although the same or equivalent functions and sequences may be accomplished by different embodiments.

[0020] 1.Definition For convenience, certain terms employed in the specification, examples, and appended claims are collected here. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

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

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

[0023] As used herein, the term "isolated mitochondria" refers to mitochondria isolated from specific cells of eukaryotic organisms by a variety of known methods, for example, using commercially available isolation kits that isolate intact mitochondria from gastric epithelial cells while maintaining biological mitochondrial activity.

[0024] The terms "administering," "administered," or "administration" are used interchangeably herein and refer to the application of isolated mitochondria, pharmaceutical compositions containing same, and / or pharmaceutical products made with isolated mitochondria to a subject via a medically acceptable route, such as oral ingestion, injection, topical application, inhalation, and rectal administration. The process of administration also includes determining the appropriate dosage and frequency to effectively achieve its intended purpose, e.g., inhibiting tumor growth.

[0025] As used herein, the term "amount" refers to an amount of isolated mitochondria, at a dosage and for a period of time necessary, effective to provide a therapeutic effect in treating a condition or to delay or minimize one or more symptoms associated with the condition, in order to achieve a desired therapeutic outcome for the treatment of gastric cancer. An effective amount of isolated mitochondria, as described herein, refers to an amount of isolated mitochondria that, alone or in combination with other therapeutic agents, provides a therapeutic effect in treating a condition. In some effective embodiments of the present disclosure, the amount of isolated mitochondria is effective to inhibit tumor growth in a subject with gastric cancer.

[0026] As used herein, the term "treating" or "treatment" is intended to mean obtaining a desired pharmacological and / or physiological effect in a subject, e.g., reducing tumor or cancer cell growth and / or inhibiting the progression of metastatic and / or drug-resistant cancer. The effect may be prophylactic, in that the symptoms are completely or partially prevented, and / or therapeutic, in that the disease and / or side effects resulting from the disease are partially or completely cured. "Treatment," as used herein, includes curative or palliative treatment of disease in mammals, particularly humans, and includes (1) inhibiting the condition (e.g., by halting the progression of cancer in a subject), or (2) alleviating the condition (e.g., inhibiting tumor growth in a subject).

[0027] The terms "subject" or "patient" are used interchangeably herein to refer to mammals, including the human species, treatable by the methods of the present disclosure. The term "mammal" refers to all members of the class Mammalia, including humans, primates, domestic and farm animals such as rabbits, pigs, sheep, and cattle, other zoo, sport, or pet animals, and rodents such as mice and rats. Furthermore, the terms "subject" or "patient" are intended to refer to both male and female genders, unless one gender is specifically indicated.

[0028] 2. Description of the Invention The present disclosure is based, at least in part, on the discovery that when mitochondria isolated from normal gastric epithelial cells are transplanted into gastric cancer cells, significant inhibition of cancer cell growth is observed. Accordingly, the present disclosure aims to provide a pharmaceutical composition comprising mitochondria isolated from normal gastric epithelial cells. Furthermore, the present disclosure aims to provide the use of mitochondria for the manufacture of a medicament for the treatment of gastric cancer in a subject in need thereof.

[0029] 2.1 Pharmaceutical Compositions Comprising Isolated Mitochondria One aspect of the present 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 an embodiment of the present disclosure, the normal gastric epithelial cells are derived from a subject with 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 and homogenizing a tissue or cell sample, at least one of the following common methods can be used to isolate and purify mitochondria: repetitive and differential centrifugation, differential sedimentation, density gradient fractionation, immunomagnetic separation, and / or filtration. Generally, reagents and consumables used for mitochondrial isolation are commercially available in kit form. In one effective embodiment, mitochondria are isolated from normal gastric epithelial cells (i.e., GES-1 cell line) using a mitochondrial isolation kit. Optionally, the isolated mitochondria can then be maintained in vitro by any means known to those skilled in the art. In one effective embodiment, the isolated mitochondria are suspended in phosphate-buffered saline (PBS) solution and stored at 4°C while retaining their original biological activity, just as they were in cells.

[0031] According to an embodiment of the present disclosure, the pharmaceutical composition is prepared in a form suitable for injection (e.g., subcutaneous injection) at a target site. 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, saline, dextran solution, amino acid solution, Ringer's solution, lactated Ringer's solution, phosphate buffer, phosphate-buffered saline, and combinations thereof. According to an effective embodiment of the present disclosure, the pharmaceutical composition is prepared by suspending isolated mitochondria in saline or PBS solution. Optionally, the injectable formulation can be sterilized by filtration through a bacteria-retaining filter before use.

[0032] 2.2 Pharmaceutical Kits Another aspect of the present disclosure is directed to a pharmaceutical kit (e.g., a pharmaceutical pack). The pharmaceutical kit provided herein primarily comprises a first container and a second container. Specifically, the first container contains a pharmaceutical composition described herein, and the second container contains an anti-cancer agent for co-administration with isolated mitochondria or the pharmaceutical composition comprising the same. Examples of first and second containers suitable for use in the pharmaceutical kit include vials, ampoules, bottles, syringes, dispenser packages, and / or other suitable containers. In some embodiments of the present disclosure, the pharmaceutical kit is useful for treating cancer and / or eliminating the progression of cancer in a subject in need thereof.

[0033] Alternatively or optionally, the pharmaceutical kit of the present disclosure may further include instructions providing directions for directing a user on how to use the pharmaceutical kit or the pharmaceutical composition contained therein. Preferably, the pharmaceutical kit may also include information required by regulatory agencies, such as the Pharmaceuticals and Medical Devices Agency (PMDA) of Japan. In certain embodiments of the present disclosure, the instructions in the pharmaceutical kit are prescribing 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 developing cancer in a subject in need thereof. In effective embodiments of the present disclosure, the dosage regimen directs the sequential or combined administration of the pharmaceutical composition and an anticancer agent described in Section 2.1 at a specific frequency and dosage to treat gastric cancer in the subject.

[0034] Examples of anti-cancer drugs suitable for co-administration with isolated mitochondria in the 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 The present disclosure is further directed to the use of isolated mitochondria in treating or delaying the onset of cancer in a subject in need thereof. According to embodiments of the present disclosure, the mitochondria and / or pharmaceutical compositions comprising same are used for the manufacture of a medicament in the treatment of gastric cancer in a subject, wherein the mitochondria are isolated from a subject with cancer or from normal gastric epithelial cells from a healthy subject, as described in Section 2.1.

[0036] According to effective embodiments, the medicament is administered to a subject having gastric cancer with or without anti-cancer drug resistance in an amount sufficient to inhibit tumor growth. According to some embodiments of the present disclosure, the medicament is administered to a subject in an amount of 0.005 mg / kg to 2 mg / kg of 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 pharmaceutical agent is administered to the subject in an amount between 0.01 mg / kg and 1 mg / kg. In one effective embodiment of the present disclosure, the pharmaceutical agent is administered to the subject with gastric cancer in an amount of about 0.01 mg / kg. In an alternative embodiment of the present disclosure, the pharmaceutical agent is administered to the cancerous subject in an amount of about 0.1 mg / kg. In another embodiment of the present disclosure, the pharmaceutical agent is administered to the subject with gastric cancer in an amount of about 0.3 mg / kg. In yet another embodiment of the present disclosure, the pharmaceutical agent is administered to the subject in an amount of 1 mg / kg.

[0037] As disclosed herein, the pharmaceutical agent may be formulated in a single dose or multiple doses (e.g., single-use or multi-dose vials) in an amount sufficient to provide a therapeutic effect in the treatment of gastric cancer. According to embodiments of the present disclosure, when multiple doses are administered to a subject, each of the multiple doses contains the same amount of the pharmaceutical agent described herein. In effective examples, the pharmaceutical agent is formulated in a three-dose vial that can be used for at least three injections. According to embodiments of the present disclosure, when multiple doses of the pharmaceutical agent are administered to a subject, each dose of the pharmaceutical agent is administered four times per day to once every three months. Specifically, the pharmaceutical agent described herein may be administered four times per day, three times per day, twice per day, once per day, once every two days, once every three days, once per week, once every two weeks, once per month, once every two months, or once per three months. In some effective embodiments, the pharmaceutical agent is administered to a subject every two days. According to embodiments of the present disclosure, when multiple doses of a pharmaceutical agent are administered to a subject, the period between the first and last dose of the multiple doses is 1 day, 2 days, 4 days, 6 days, or 1 week. In an effective embodiment, the period between the first and last dose of the multiple doses is 6 days.

[0038] The isolated mitochondria and / or pharmaceuticals produced therefrom 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 injection) to a suitable or desired site. Exemplary suitable parenteral routes include, but are not limited to, intraarticular, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracerebroventricular, intradermal, subcutaneous, and intraperitoneal injection. Specifically, contemplated routes are intravenous administration (e.g., systemic intravenous injection) and / or local administration to the affected site (e.g., direct subcutaneous injection). The appropriate route will vary depending on the particular condition being treated, its severity, individual patient parameters such as age, physical condition, size, sex, and weight, duration of treatment, the nature of concomitant therapy (if any), dosage and active ingredients, genetic factors, and other factors within the knowledge and expertise of the medical practitioner recognized by those skilled in the art. These factors are well known to those skilled in the art and can be addressed with no more than routine experimentation. Generally, the most suitable administration route 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 the present disclosure, the isolated mitochondria and / or pharmaceutical agent are administered via subcutaneous injection. In some effective embodiments, the pharmaceutical agent is administered to the tumor lesion via subcutaneous injection.

[0039] According to an alternative or optional embodiment of the present disclosure, the pharmaceutical agent can be used to treat drug-resistant gastric cancer, i.e., gastric cancer resistant to anticancer drugs. Examples of anticancer drugs traditionally 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 an effective embodiment of the present disclosure, gastric cancer develops resistance to 5-FU drugs.

[0040] In accordance with the present disclosure, the term "subject" refers to an animal that can be administered and benefit from the pharmaceutical agents disclosed herein. Examples of animals include, but are not limited to, humans, rats, mice, guinea pigs, monkeys, pigs, goats, cows, 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 above characteristics, the 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 the pharmaceutical composition / medicine to improve the therapeutic effect of gastric cancer. [Example]

[0042] Example Materials and Methods cell culture The human gastric cancer cell lines AGS and MKN45 were cultured separately in Roswell Park Memorial Institute (RPMI 1640) medium (Gibco, Waltham, MA, USA), and the human normal gastric epithelial cell line GES-1 was cultured in Dulbecco's Modified Eagle Medium (DMEM, Thermo Fisher Scientific, Waltham, MA, 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 with 5% CO2 (v / v) at 37°C.

[0043] Cells were passaged every three days. After rinsing the cells with 1x PBS, a recombinant enzyme (e.g., TrypLE, Thermo Fisher Scientific, Waltham, MA, USA) was used for cell detachment for approximately 5 minutes. The reaction was terminated by adding three volumes of culture medium (i.e., RPMI 1640 or DMEM). Subsequently, the culture medium containing the recombinant enzyme was removed by centrifugation and resuspended in the same medium (i.e., RPMI 1640 or DMEM) used to culture the cells. Cell counts were performed using trypan blue, and the cells were seeded into 75T flasks for continued culture. For experimental purposes, cells were seeded into various sized culture plates, including 96-well plates, 6-well plates, and 10 cm culture dishes, at 6 x 10 cells per well, respectively. 3 pieces, 1×10 5 pcs and 1 x 10 6 The cells were seeded at different cell densities.

[0044] Isolation of mitochondria from normal gastric epithelial cells GES-1 cells were trypsinized with TryPLE for 5 minutes, after which the reaction was stopped and the culture medium containing TryPLE was removed by centrifugation, followed by one wash with PBS. The cells were then centrifuged at 1000 rpm to remove the supernatant and 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 cultured at 1 × 10 7Each sample was suspended in 400 μL of Reagent A containing 1x EDTA-free protease inhibitors. After vortexing for 5 minutes, the reaction was stopped by placing it on ice. Within 2 minutes, 10 μL of Reagent B was added, followed by high-speed vortexing for 10 seconds every minute for a total of 5 minutes. Next, 400 μL of Reagent C containing 1x EDTA-free protease inhibitors was added, and the mixture was centrifuged at 700 g for another 10 minutes at 4°C. The supernatant was collected and centrifuged again at 3000 g for 15 minutes at 4°C. The resulting pellet was collected and resuspended in 100 μL of Reagent C. The mixture was then centrifuged at 12000 g for 5 minutes at 4°C to wash the mitochondria. After removing the supernatant, the mitochondria were suspended in 100 μL of PBS for this study.

[0046] Quantification of isolated mitochondria Mitochondria harvested from cells were sonicated in 100 μL of lysis buffer (M-PER™ Mammalian Protein Extraction Reagent, Thermo Fisher Scientific, Waltham, MA, USA). After centrifugation, the supernatant was transferred to a new tube, and the mitochondrial protein concentration was determined using a sulforhodamine B Assay Kit (Abcam).

[0047] Flow cytometry Confluent cells were trypsinized and harvested by centrifugation. Cells were resuspended in culture medium and incubated with fluorescently conjugated antibodies against CD24, CD44, and Lgr5 (BD Biosciences) for 45 minutes, then washed twice with PBS. Fluorescence was detected by flow cytometry (BD FACSCalibur, BD Biosciences) and analyzed using Flow Software 2 software.

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

[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, after which the reaction was stopped with three volumes of culture medium. The TryPLE-containing culture medium was then removed by centrifugation. The cells were washed with PBS to remove any remaining FBS and resuspended in 100 μL of serum-free culture medium mixed with matrix gel (1:1 ratio) to form a cell suspension. Finally, the cell suspension was placed on ice for further use.

[0050] II. Cancer cell transplantation Before being subcutaneously implanted into the back of the mice, the cell suspension was mixed homogeneously and aspirated with an appropriate hypodermic syringe, and the whole process was carried out on ice.

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

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

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

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

[0055] Example 1 Isolation of GES-1 mitochondria and determination of effective dose The goal of this experiment was to quantify isolated mitochondria and determine an effective dosage for subsequent experiments. To this end, mitochondria were first isolated from normal gastric epithelial cells (GES-1) according to the procedure outlined in the "Materials and Methods" section, at a concentration of 1 x 10 cells. 7 The total weight of mitochondria extracted per cell was calculated. Subsequently, various concentrations of mitochondria (μg / ml) in PBS solution were co-cultured with gastric cancer MKN-45 cells for 24 hours to find the effective dose.

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

[0057] Example 2 Transplantation of GES-1 mitochondria into gastric cancer cells In this experiment, we investigated whether mitochondria isolated from GES-1 cells could be transplanted into gastric cancer cells. Specifically, AGS cell lines stained with a 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 pre-stained with a green fluorescent dye (MitoTracker Green FM, Invitrogen). Meanwhile, the control group cells were treated with a phagocytosis inhibitor (i.e., methyl-β-cyclodextrin), a nanotube formation inhibitor, and an actin-dependent endocytosis inhibitor (i.e., cytochalasin D) for 1 hour before co-culture with isolated mitochondria. Treatment with the phagocytosis and endocytosis inhibitors allowed for observation of mitochondrial movement. AGS cells from both groups were then 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 coculture with GES-1 mitochondria implied the intercellular transfer of these organelles. Furthermore, the decreased green fluorescence intensity in AGS cells treated with the inhibitor compared with their untreated counterparts (i.e., AGS cells in the experimental group) suggested a potential regulatory role for phagocytosis and actin-dependent endocytosis pathways in promoting the internalization and endocytosis of GES-1 mitochondria into 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 stemness of gastric cancer cells. To this end, we measured the expression levels of stemness-associated proteins SOX2, NANOG, GRP78, NOTCH, and phosphorylated JNK in MKN-45 cells cocultured with mitochondria using Western blotting according to the procedures 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 stemness-related proteins in gastric cancer cells after co-culture with GES-1 cell mitochondria, indicating that mitochondria from GES-1 cells can attenuate the stemness and potential of gastric cancer cells.

[0061] Example 4 GES-1 mitochondria affected the tumor-initiating ability of gastric cancer cells In this study, we investigated whether mitochondria isolated from GES-1 cells could inhibit tumor formation in vivo. To this end, mitochondria isolated from GES-1 cells were cocultured with AGS cells for 24 hours, after which cancer cells were subcutaneously implanted into 6-week-old mice. Mouse weights and tumor sizes 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 with the control group of mice without mitochondrial treatment.

[0063] Example 5 GES-1 mitochondria inhibit tumor growth and progression in vivo In this example, we investigated the ability of mitochondria isolated from GES-1 cells to inhibit tumor growth and progression in vivo. To this end, mice were randomly divided into three groups: Groups I and II received direct subcutaneous implantation of MKN-45 cells; Group II mice received an additional subcutaneous injection of mitochondria beneath tumor-forming sites; and Group III mice were co-cultured with MKN-45 cells and GES-1 mitochondria for 24 hours, followed by subcutaneous implantation of cancer cells. Mouse weights and tumor sizes were recorded every two days. Tumors were removed from the mice one week after mitochondria injection for further protein expression analysis via Western blotting. The results are shown in Figures 4 and 5.

[0064] As evidenced by 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 tissues, the quantitative results shown in Figure 5 reveal that mice treated with mitochondria showed significantly reduced protein expression, including stemness-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 with the group without mitochondria treatment. Collectively, the data indicate that administration of isolated mitochondria from normal gastric epithelial cells can effectively inhibit tumor growth, progression, and stemness.

[0066] Example 6 GES-1 mitochondria reduced gastric cancer cell resistance to anticancer drugs In this experiment, we investigated whether mitochondria isolated from GES-1 cells could increase the sensitivity of gastric cancer cells to anticancer drugs. To this end, the gastric cancer cell lines AGS and MKN-45 were cocultured with GES-1 mitochondria for 24 hours, and then the culture medium was replaced with medium containing 5-FU (0.5 μM) and 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 in Figure 6 show 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 confirm whether mitochondria directly affect cell 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 revealed a consistent trend in the two gastric cancer cell lines: Bax protein expression was reduced in cancer cells after treatment with low-dose 5-FU, while mitochondria restored the reduced Bax expression induced by low-dose 5-FU treatment in both AGS and MKN-45 cells. Furthermore, the data in Figure 8 show that the ratio of phosphorylated AKT in MKN-45 cells decreased over time after co-culture with mitochondria. These data collectively demonstrate that mitochondria from GES-1 cells effectively reduce drug resistance in gastric cancer cells.

[0070] Taking the results of Examples 1 to 6 together, 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 drugs and enhance the cytotoxicity of these drugs to cancer cells. Therefore, pharmaceutical compositions containing isolated mitochondria provided by the present disclosure can be effectively used to treat gastric cancer, including drug-resistant gastric cancer.

[0071] It should be understood that the above description of the embodiments is given by way of example only, and that various modifications may be made by those skilled in the art. The above specification, examples, and data provide a complete description of the structure and use of exemplary embodiments of the invention. While various embodiments of the invention have been described above with a certain degree of particularity or with reference to one or more specific embodiments, those skilled in the art could 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.

2. The pharmaceutical composition of claim 1 , wherein the gastric cancer is resistant to an anticancer drug.

3. 3. The pharmaceutical composition of claim 2, wherein the anticancer drug 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.

4. A pharmaceutical kit for treating gastric cancer, comprising a first container for housing the pharmaceutical composition of claim 1 and a second container for housing an anti-cancer agent.

5. 5. The pharmaceutical kit of claim 4, wherein the anticancer drug 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. 1. Use of mitochondria for the manufacture of a medicament in the treatment of gastric cancer in a subject in need thereof, wherein the mitochondria are isolated from normal gastric epithelial cells from said subject or a healthy subject.

7. 7. The use of claim 6, wherein the medicament is administered to the subject once every two days in an amount of 0.005 mg / kg to 2 mg / kg.

8. The use according to claim 6, wherein the gastric cancer is resistant to an anticancer drug.

9. The use according to claim 8, wherein the anticancer drug 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.

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

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