Use of dengue virus for preparing Anti-hepatoma drug

Abstract

A method to treat hepatoma with dengue viruses which infect liver tumor stem cells for annihilation of hepatocellular carcinoma tissues. The liver tumor stem cells expressing the biomarker of CD133 in a tumor part are the objects infected by dengue viruses preferentially and killed due to specific protein expressions for suppression of hepatoma.

Description

[Technical Field]

[0001] This invention relates to the use of dengue virus in the preparation of anti-liver cancer drugs, specifically the use of dengue virus to induce the death of liver cancer tissue by infecting liver tumor stem cells. [Previous Technology]

[0002] Liver cancer is currently the second most common cancer in Taiwan, second only to lung cancer, and it also ranks among the top ten in mortality. Even after surgical resection, radiation therapy, or chemotherapy, liver cancer patients still have a relatively high recurrence rate (70%) compared to other cancers. There are many reasons for recurrence, the most important being cancer stem cells. Cancer stem cells are derived from mutations of normal stem cells, thus possessing self-renewal and differentiation capabilities. While they constitute a very small proportion of the cancer cell population (approximately 0.1%), they can still form tumors again. Furthermore, cancer stem cells have developed resistance to some drugs, so chemotherapy cannot completely eliminate them, leading to cancer recurrence.

[0003] Therefore, the problem to be solved by this invention is to provide a cancer stem cell (hereinafter also referred to as "liver tumor stem cell") that can preferentially infect liver cancer tissue and remove liver tumor stem cells with low impact on normal liver cells, thereby promoting the death of liver cancer tissue and preventing the recurrence of liver cancer.

[0004] Past research has found that dengue virus (DENV) can infect hematopoietic stem cells in the bone marrow, thereby affecting the hematopoietic system and impairing stem cell differentiation capacity. Current literature shows that dengue virus antigens have been detected in liver cells and Kupffer cells of patients with severe dengue fever, and the virus can be recultured from liver biopsy. Furthermore, numerous clinical observations and mouse experiments have confirmed that the liver is one of the target organs for dengue virus infection. Studies have also found that dengue virus infection of liver cancer cell lines can induce cytopathic effects; however, currently, no literature indicates that dengue virus can infect liver tumor stem cells and induce their death. [Summary of the Invention]

[0005] In view of this, the inventor of this case has a deep understanding of the shortcomings and defects of the previous case, and has been eager to improve and innovate. After years of research, the inventor has successfully developed the use of dengue virus in the preparation of anti-liver cancer drugs and the method of dengue virus killing liver cancer tissue.

[0006] To achieve the above objective, this invention provides a use of dengue virus in the preparation of anti-liver cancer drugs, which induces the death of liver cancer tissue by infecting liver tumor stem cells with dengue virus.

[0007] In one embodiment of the invention, the liver cancer tissue includes fully cancerous tumor tissue and non-tumor tissue between normal liver and cancerous tissue; liver tumor stem cells are located in the tumor tissue.

[0008] In one embodiment of the invention, after dengue virus infects liver cancer tissue, it can produce infectious dengue virus.

[0009] In one embodiment of the invention, the concentration of dengue virus produced by dengue virus infection of tumor tissue is higher than the concentration of dengue virus produced by dengue virus infection of non-tumor tissue.

[0010] In one embodiment of the invention, the concentration of dengue virus produced by dengue virus infection of liver tumor stem cells is higher than the concentration of dengue virus produced by infection of cells other than liver tumor stem cells.

[0011] In one embodiment of the invention, the biomarkers expressed by liver tumor stem cells include at least one selected from the group consisting of CD133, CD117 and CD34.

[0012] In one embodiment of the invention, the biomarker expressed by liver tumor stem cells includes CD133.

[0013] In one embodiment of the invention, compared to cells that express CD133 biomarkers in non-tumor tissues, liver tumor stem cells that express CD133 biomarkers in tumor tissues become targets of dengue virus infection due to the affected expression level of specific proteins, which further leads to the death of liver tumor stem cells.

[0014] In one embodiment of the invention, the amount of dengue virus used is 0.5 MOI to 1.5 MOI relative to the number of cells in the liver cancer tissue to be infected.

[0015] In one embodiment of the invention, the amount of dengue virus used is 1 MOI relative to the number of cells in the liver cancer tissue to be infected.

[0016] This invention also provides a method for killing liver cancer tissue with dengue virus, which is to induce the death of liver cancer tissue by infecting liver tumor stem cells in liver cancer tissue with dengue virus.

Implementation Method

[0025] [Terminology Definitions] Many technical and scientific terms commonly used in the field of biotechnology are used extensively in this specification. In the following description, the following definitions are provided to provide a clear and consistent understanding of this specification and the scope of the claims, as well as the scope to which such terms are given. Other terms not specifically defined below have meanings that are commonly understood by those skilled in the art.

[0026] The terms "individual," "patient," and similar terms are used herein to refer to a mammal that is in the treatment evaluation stage and / or is being treated. In one embodiment, the mammal is a human. Thus, the term "patient" encompasses an individual with liver cancer, including individuals who have undergone resection (surgery) to remove cancerous tissue or who are candidates for such resection (surgery). The individual may be human, but also includes other mammals, particularly those used as laboratory models of human diseases, such as mice, rats, etc.

[0027] The term "treatment" and similar terms are used herein to refer to the administration of a drug to achieve a certain effect. This effect is a therapeutic, partial, or complete cure of a disease and / or the symptoms of that disease. As used herein, "treatment" covers any treatment of liver cancer in mammals (particularly humans) and includes: (a) suppressing the disease, i.e., halting its development; and (b) alleviating the disease, i.e., resolving the disease. In the treatment of tumors (e.g., liver cancer), therapeutic agents can directly reduce the growth and metastasis of tumor cells.

[0028] The term "cell culture" and similar terms are used herein to refer to the maintenance of cells in an artificial, living in vitro environment. However, it should be understood that the term "cell culture" is a general term and can be used not only to cover the cultivation of individual cells but also to cover the cultivation of tissues or organs.

[0029] The term “tumor” is used herein to refer to the growth and proliferation of all proliferative cells (malignant or benign), as well as all precancerous and cancerous cells and tissues.

[0030] The term "liver cancer" is used herein to refer to cells exhibiting spontaneous, unregulated growth to the point of exhibiting an abnormal growth phenotype characterized by markedly uncontrolled cell proliferation. In this invention, the relevant cells include precancerous (e.g., benign), malignant, pre-metastatic, metastatic, and non-metastatic cells in the detection, analysis, classification, or treatment.

[0031] The term "liver cancer tissue specimen" and similar terms are used herein to refer to any cells obtained from liver cancer tissue, including fully cancerous tumor tissue and non-tumor tissue intermediate between normal liver and cancerous tissue. In the case of solid tumors that have not metastasized, tissue specimens are usually obtained from surgically removed tumors, and test specimens are prepared using known techniques. Similarly, in the case of metastatic cancer, cells may be collected from bodily fluids such as lymph, blood, serum, or exudates from distant infected organs or their distal sources.

[0032] The term “biomarker” is used herein to refer to a specific biomolecule (e.g., CD133) that is highly expressed in a particular cell population (e.g., liver tumor stem cells).

[0033] The term "hepatocellular carcinoma recurrence" is used in this article to refer to the further growth of neoplastic or cancerous cells after a diagnosis of hepatocellular carcinoma. Specifically, hepatocellular carcinoma recurrence refers to the situation where liver tumor stem cells are not completely eliminated during treatment, leading to further growth of liver cancer tissue.

[0034] The terms "related to" or "associated with" and similar terms are used herein to refer to a statistical correlation between two events, including numbers, data sets, and the like. For example, when the events involve numbers, a positive correlation means that as one increases, the other also increases. A negative correlation means that as one increases, the other decreases.

[0035] The term "multiplicity of infection (MOI)" in this paper refers to the ratio of the infectious agent to the target of infection, and its calculation formula is: Multiplicity of infection = Number of viral particles / Number of host cells.

[0036] The term "plaque forming unit (PFU)" in this document refers to the number of viruses that form a single viral plaque (phage plaque) on a monolayer cultured animal cell, and is used as a unit for quantifying viral content.

[0037] The term “plaque viral titer” in this article refers to the number of viruses contained in one milliliter of culture medium. The calculation formula is: viral titer (PFU / ml) = (number of viral plaques) * 1000 / 400 * (dilution factor).

[0038] [Dengue Virus] The dengue virus of the present invention preferentially infects liver tumor stem cells. In this invention, as long as it can preferentially infect liver tumor stem cells and induce liver cancer tissue death with minimal impact on normal liver cells, the serotype of the dengue virus is not particularly limited. As an example, the following can be listed: dengue virus type 1 (DENV-1), dengue virus type 2 (DENV-2), dengue virus type 3 (DENV-3), dengue virus type 4 (DENV-4), dengue virus type 5 (DENV-5), or derivative viruses of the above. One of the above dengue viruses can be used, or two or more can be used in combination. As an example, dengue virus type 2 is preferred, and more preferably, dengue virus strain 16681 of dengue virus type 2 is used. The source of the dengue virus in this invention is not particularly limited; it can be obtained through conventional commercial purchases or isolated in a laboratory or clinical setting.

[0039] [Liver Stem Cells and Liver Tumor Stem Cells] Liver stem cells have the potential to differentiate into hepatocytes, bile duct epithelial cells, and other cell types. Their biomarkers include CD133, CD117, and CD34. Liver tumor stem cells are derived from mutations of liver stem cells and also have self-renewal and differentiation capabilities. They are the main cell population infected by dengue virus in the early stages. Liver tumor stem cells account for a very small proportion (about 0.1%) of liver cancer cells and have the ability to promote the growth of liver cancer cells. As liver tumor stem cells that are the target of dengue virus infection, it is preferable that they express at least one of the biomarkers in the group composed of CD133, CD117, and CD34, and more preferably that they express the biomarker CD133. Liver tumor stem cells have developed resistance to some drugs, which is one of the main causes of liver cancer recurrence.

[0040] [Method for Treating Liver Cancer] This invention discloses a method for using dengue virus to treat liver cancer. The method involves administering a therapeutically effective dose of dengue virus to a patient in need. The administered dengue virus preferentially infects and kills liver tumor stem cells to treat liver cancer.

[0041] The treatment method of this invention can be used in the treatment of liver cancer in mammalian individuals (especially humans). Individuals who have or are at risk of developing tumors are covered by the treatment method described herein.

[0042] The method of this invention relates to administering dengue virus to an individual (e.g., a human patient) to inhibit the growth of liver cancer cells. The treatment method according to this invention can also be applied to reduce tumor size, decrease tumor burden, and / or improve clinical outcomes in patients.

[0043] [Dengue Virus Administration] Targeting liver cancer tissue with dengue virus can be achieved through various methods, including intratumoral, intravenous, intradermal, subcutaneous, oral (e.g., inhalation), percutaneous (i.e., local), mucosal, intraperitoneal, intraarterial, and rectal administration. Other suitable routes include administration of the composition via: oral, buccal, nasal, nasopharyngeal, non-enteric, enteric, gastric, local, percutaneous, subcutaneous, intramuscular, in tablet, solid, powder, liquid, or aerosol form, intralesional injection into the tumor, intralesional injection near the tumor, intravenous infusion, and intraarterial infusion. Local or systemic administration can be performed with or without excipients. Slow-release administration can also be performed at or around the tumor site in an individual.

[0044] [Dosage] In this treatment method, an effective amount of dengue virus is administered to the individual in need. Specifically, the dosage varies depending on the purpose of administration, the health and physical condition of the individual to be treated, age, the individual's taxonomic group (e.g., human, non-human primate, primate, etc.), the dengue virus formulation, the treating clinician's assessment of the medical situation, and other relevant factors. This dosage is expected to be within a relatively wide range, which can be determined through routine testing. For example, to adequately induce the death of liver tumor stem cells, it is preferable to use 0.5 MOI to 1.5 MOI of dengue virus relative to the number of cells in the liver cancer tissue to be infected, and more preferably, 1 MOI.

[0045] [Pharmaceutical Compositions] Dengue virus can be mixed with pharmaceutically acceptable carriers (e.g., in the form of pharmaceutically acceptable salts) to prepare pharmaceutical compositions; it can also be packaged in containers and prepared as kits or products together with packaging inserts containing information on the use and method of use of dengue virus.

[0046] The invention is illustrated by the following embodiments. However, the following embodiments are merely illustrative of the invention and should not be regarded as a limitation of the scope of the invention in any way. In addition, the dengue virus and materials used below are readily available from the market.

[0047] Figure 1 is a flowchart of the relevant experiment for dengue virus infection of liver cancer tissue according to one embodiment of the invention. First, 41 groups of liver cancer specimens were collected from liver cancer patients. Each group of specimens contained tumor tissue and non-tumor tissue. After obtaining the specimens, they were processed by cutting and grinding. Red blood cell lysis buffer was added to remove red blood cells, leaving nucleated cells. Then, dengue virus was used to infect tumor tissue and non-tumor tissue cells at a 1 MOI infection condition (virus to cell ratio of 1:1). The infection supernatant and cells were collected at specific time points (1, 2, 3, 5, 7, 10 and 14 days after infection). The supernatant was also used to quantify the virus concentration through a plaque assay. The cells were analyzed by multicolor flow cytometry (FACS) to analyze the expression of cell surface markers.

[0048] [Hepatocellular Carcinoma Specimens Processing] Hepatocellular carcinoma specimens were obtained from 41 patients (National Cheng Kung University Hospital Human Research Ethics Review Committee Project No.: B-ER-103-187). The gender and age of the patients from whom the specimens were obtained are shown in Table 1 below. Each specimen was paired and divided into tumor part and non-tumor part. Freshly obtained specimens were first cut into small pieces with scissors, soaked in PBS, and then 2-3 ml of collagenase was added and cultured at 37°C for 5 minutes to remove connective tissue. The enzyme reaction was then neutralized with RPMI medium containing 10% fetal bovine serum, and the primary cells were separated by centrifugation (300 rcf, 8 minutes). Next, red blood cell lysis buffer was added and incubated on a vortex mixer for 8 minutes, followed by centrifugation (300 rcf, 8 minutes) to remove red blood cells. This step was repeated twice to ensure complete removal of red blood cells. Cells were rehydrated with RPMI medium containing 10% fetal bovine serum, and the cells were filtered through a 100 μm filter to remove cell clumps. Finally, the cell count was calculated for subsequent experiments.

[0049] [Table 1]

[0050] [Plaque Assay] 7 x 10⁵ mouse kidney epithelial cells (BHK cells) per well were dissolved in 1.5 mL of DMEM medium containing 5% fetal bovine serum and seeded on a 6-well plate. The virus solution to be tested was first serially diluted by adding 100 μL of unknown virus solution to 900 μL of DMEM medium containing 2% fetal bovine serum to achieve a 10-fold dilution. After the seeded cells adhered to the plate, the culture medium was removed, 400 μL of the virus solution to be tested was added, and the plate was incubated at 37°C for 2 hours, shaking the plate every 15 minutes to ensure that the infection solution evenly covered the cells. After two hours, the infection solution was removed, methylcellulose was added to limit the range of virus movement, and the plate was returned to the 37°C incubator for 7 days. Seven days later, the six-well plates were removed, and each well was washed with PBS. Then, crystal violet was added for staining for 1 hour (cells will be stained; no cells or dead cells will not be stained). Finally, the stain was washed off with water, and the number of viral plaques was counted to calculate the plaque viral titer.

[0051] [Ex vivo infection of DENV] 8 x 10⁶ tumor tissues and non-tumor tissues were dissolved in RPMI medium containing 10% fetal bovine serum for in vitro dengue virus infection. The infection conditions were 1 MOI, and the total volume of virus and cell solution was 2 mL. Type II dengue virus (strain 16681) was used. The infection mixture was incubated at 37°C for 2 hours, with the tubes shaken every 30 minutes to increase the opportunity for virus-cell contact. Two hours later, the virus that had not come into contact with the cells was removed by centrifugation (300 rcf, 8 minutes). The cells were then rehydrated with 700 μL of RPMI medium containing 10% fetal bovine serum (FBS), and then aliquoted into seven centrifuge tubes containing 2 mL of RPMI medium containing 10% FBS. The centrifuge tubes were then placed in a 37°C incubator, and the virus solution and cells were collected at specific time points (1, 2, 3, 5, 7, 10, and 14 days post-infection). At the specific time points, the infection mixture was removed and centrifuged (300 rcf, 8 minutes) to separate the infection supernatant from the cells. The infection supernatant was used to detect viral titer using viral plaque assay. The cells were washed with PBS and used for subsequent experiments.

[0052] [Magnetic Bead Isolation] Wash 5 x 10⁷ tumor tissues and non-tumor tissues separately with MACS buffer to remove residual cell culture medium. Rehydrate the cells with an appropriate amount of MACS buffer and add antibody-linked magnetic beads. For every 10⁷ cells, add 20 μL of antibody-linked magnetic beads and 80 μL of MACS buffer. Place the cell and magnetic bead mixture on a vortex mixer and incubate at 4°C for 40 minutes. After incubation, add 2-3 mL of MACS buffer to the mixture and centrifuge (1000 rpm, 5 minutes) to separate the cells from the unlinked magnetic beads. Rehydrate the cells with 500 μL of MACS buffer and then add them to an LSCollumn mounted on a magnetic base (pre-washed with 1 mL of MACS buffer). In this step, cells attached to the magnetic beads will remain in the column and are considered positive cells, while cells not attached to the beads will flow out of the column into the centrifuge tube and are considered negative cells. The LS column is then rinsed with MACS buffer to ensure that all cells remaining in the column are truly positive. Finally, the magnetic base of the LS column is removed, and the cells are transferred to new centrifuge tubes. Both tubes are centrifuged (300 rpm, 8 minutes), the MACS buffer is removed, and the cells are rehydrated with RPMI medium containing 10% fetal bovine serum. The cell count is then determined.

[0053] [Multicolor FACS Analysis] 2 x 10⁶ cells were resuspended in 200 μL of staining buffer and divided into a staining group and an isotype group. Immunofluorescence antibodies targeting specific cell surface markers were added to the staining group, while isotype immunofluorescence antibodies were added to the isotype group for staining. Staining was performed in the dark, and the cells were incubated at 4°C for 30 minutes. After staining, the cells were washed with 1 mL of staining buffer and centrifuged (300 rcf, 8 minutes) to remove antibodies not attached to the cells. Finally, the cells were resuspended in 200 μL of staining buffer, and the expression of cell surface markers was analyzed using a flow cytometer (LSR Fortessa, BD).

[0054] [Example 1] Please refer to Figure 2. Figure 2 shows the experimental results of the virus concentration produced by dengue virus infection of liver cancer tissue according to one embodiment of the present invention. Figure 2a is the infection curve of dengue virus infection of tumor tissue and non-tumor tissue (n=41). From the results of virus concentration, it can be seen that liver cancer tissue can be infected by dengue virus and can produce infectious virus. The circle is the infection curve of non-tumor tissue and the square is the infection curve of tumor tissue. There is a statistical difference between the two curves (*, p value=0.0469). That is, dengue virus prefers to infect tumor tissue and produces a larger amount of dengue virus.

[0055] In addition, the peak viral load concentrations of tumor tissue and non-tumor tissue after dengue virus infection were extracted and analyzed in each group of specimens. Figure 2b is a comparison of the peak viral load concentrations of tumor tissue and non-tumor tissue after dengue virus infection (n=41). The results of viral load concentration show that the viral load concentration produced by tumor tissue after dengue virus infection is significantly higher than that produced by non-tumor tissue after dengue virus infection, which is statistically significant (****, p value < 0.0001); that is, dengue virus prefers to infect tumor tissue and produces a larger amount of dengue virus.

[0056] Furthermore, the correlation between the viral concentrations produced after dengue virus infection was analyzed between each group of specimens, namely between tumor tissue and non-tumor tissue. Figure 2c shows the relationship between the highest viral concentrations produced by tumor tissue and non-tumor tissue in each group of liver cancer specimens (n=41). From the results of viral concentration, it can be seen that dengue virus infection of tumor tissue can produce a higher concentration of dengue virus; that is, dengue virus prefers to infect tumor tissue and produces a larger amount of dengue virus.

[0057] [Example 2] Please refer to Figure 3. Figure 3 is a comparison of the highest viral concentration produced by dengue virus infection of liver cancer tissue and the number of cells in the tumor tissue in one embodiment of the present invention. Figure 3a is a scatter plot of the highest viral concentration produced by dengue virus infection of tumor tissue and the number of liver tumor stem cells (n=23). From the cell staining results, it can be seen that the highest viral concentration (viral titer) produced by dengue virus infection of tumor tissue is significantly positively correlated with the number of liver tumor stem cells (CD133 + / - and / or CD117 + / - and / or CD34 + / -) in the tumor tissue (r>0), which has a statistical difference (*, p value=0.0461); that is, dengue virus prefers to infect liver tumor stem cells and produces a larger amount of dengue virus.

[0058] Conversely, Figure 3b is a scatter plot of the highest viral concentration produced by dengue virus infection of tumor tissue and the percentage of cells other than liver tumor stem cells (n=23). The cell staining results show that the highest viral concentration produced by dengue virus infection of tumor tissue cells is significantly negatively correlated with the proportion of cells other than liver tumor stem cells in the tumor tissue (r<0), which is statistically significant (*, p=0.0487). That is, dengue virus is less inclined to infect cells other than liver tumor stem cells and produces a smaller amount of dengue virus when infecting cells other than liver tumor stem cells.

[0059] Next, the biomarker performance of liver tumor stem cells in tumor tissue and non-tumor tissue was analyzed. Figure 3c is a comparison of the percentage of gated cells expressing the CD133 biomarker in tumor tissue and non-tumor tissue. The results show that there is a significant difference in the CD133 biomarker between tumor tissue and non-tumor tissue; that is, there are more liver tumor stem cells (cells expressing CD133) in tumor tissue.

[0060] The results above show that the concentration of infectious virus produced in tumor tissues infected by dengue virus is significantly correlated with the number of liver tumor stem cells in liver cancer tissues, and the CD133+ cell population shows great differences in expression between tumor tissues and non-tumor tissues.

[0061] [Example 3] Please refer to Figure 4. Figure 4 shows the experimental results of CD133 biomarker expression in tumor tissue infected with dengue virus according to one embodiment of the present invention. CD133+ cells and CD133- cells in the tumor tissue were separated by magnetic bead sorting, and the cells were infected with dengue virus. The infection supernatant was collected at a specific time point, and the virus was quantified by viral plaque detection method. In Figure 4, the blue line represents the viral infection curve generated by infecting tumor tissue (intact cells before sorting) with dengue virus, the red line represents the viral infection curve generated by infecting tumor tissue with cells expressing the CD133 biomarker (CD133+) with dengue virus, and the green line represents the viral infection curve generated by infecting tumor tissue with cells not expressing the CD133 biomarker (CD133-) with dengue virus. The results show that in the early stage of dengue virus infection (1-3 days), CD133+ cells mainly dominate dengue virus infection and produce infectious viruses; while in the middle and late stages after infection (after 3 days), CD133- cells take over to support viral infection and virus production.

[0062] [Example 4] Please refer to Figure 5. Figure 5 shows the cell survival status of non-tumor tissue and tumor tissue cells that have not been infected with dengue virus after long-term culture in one embodiment of the present invention. The tumor tissue and non-tumor tissue were cultured at 37°C with an initial cell count of 6 x 10⁴. During the 1-14 days of the culture process, the cell count of the tumor tissue and non-tumor tissue was calculated every day. The red line is the cell survival curve of the tumor tissue after long-term culture (N=1), and the blue line is the cell survival curve of the non-tumor tissue after long-term culture (N=1). From this result, it can be seen that the survival rate of the cells of the tumor tissue and non-tumor tissue is not significantly different when they have not been infected with dengue virus, and both can survive for at least 14 days. In summary, when there was no significant difference in cell count between tumor and non-tumor tissues within 14 days, non-tumor tissues were less susceptible to dengue virus infection compared to tumor tissues. The specific reasons for this are still unclear, but it is inferred that dengue virus primarily infects liver tumor stem cells, mainly CD133+ liver tumor stem cells. Since non-tumor tissues have fewer liver tumor stem cells, they are less susceptible to dengue virus infection. In fact, since non-tumor tissues are between normal liver and cancerous tissue, they should not contain CD133+ liver tumor stem cells. However, CD133+ liver tumor stem cells were detected in non-tumor tissues, suggesting that they may have migrated from tumor tissues, thus making these non-tumor tissues susceptible to dengue virus infection.

[0063] [Example 5] Figure 6 shows proteins with significantly different expression levels in CD133+ cells in tumor tissues and non-tumor tissues according to one embodiment of the present invention; CD133+ cells were isolated from tumor tissues and non-tumor tissues respectively, and their protein identity was analyzed to identify the differences in the expression levels of various proteins in the two types of cells. The raw results were analyzed using DEanalysisApp to identify proteins with significantly different expression levels between CD133+ cells in tumor tissues and non-tumor tissues. The results are shown in Figure 6a, which is a comparison chart of volcano expression levels in CD133+ cells in tumor tissues and non-tumor tissues. In the comparison of the expression levels of various proteins in cells, the proteins with higher expression levels in tumor tissue are selected by the blue box on the right and arranged in Figure 6b according to the degree of difference in expression levels. They are listed in descending order of significant difference in expression levels, as follows: Protein IDs: Q9Y305, Q9UBV2, Q9HCS2, Q9BUB7, Q99541, Q5BJF2, P55145, P40925, P05556, P04114, P01861, P00390, O95573, O15228, O15254, P01903, P62 Protein 937; similarly, the proteins with higher expression levels in non-tumor tissues are selected by the red box on the left and arranged in Figure 6c according to the degree of difference in their expression levels. They are arranged in descending order of significant difference in expression levels, and are proteins with the following IDs: O15229, O75477, O96000, P01008, P02743, P11509, P11712, P16190, P34810, P46782, P48449, P50225, P62269, Q5VT66, Q9BYV1, and Q9NTJ5.

[0064] From the above results, it can be seen that the expression levels of proteins with the following IDs in liver tumor stem cells expressing CD133 biomarkers in tumor tissue are increased: Q9Y305, Q9UBV2, Q9HCS2, Q9BUB7, Q99541, Q5BJF2, P55145, P40925, P05556, P04114, P01861, P00390, O95573, O15228, O15254, P01903, and P62937, while protein I... The decreased expression levels of proteins D for the following genes—O15229, O75477, O96000, P01008, P02743, P11509, P11712, P16190, P34810, P46782, P48449, P50225, P62269, Q5VT66, Q9BYV1, and Q9NTJ5—make liver tumor stem cells expressing the CD133 biomarker in tumor tissues a preferred target for dengue virus infection, further leading to the death of liver tumor stem cells.

[0065] [Example 6] Please refer to Figures 7A-7D. Figures 7A-7D are a list of biological pathways involving proteins with significant differences in expression, analyzed using the PANTHER database according to one embodiment of the invention. The proteins with significant differences in expression obtained in Example 4 were analyzed using the PANTHER database to determine their physiological functions and pathways of action. The results classified these proteins into molecular functions, cellular components, biological processes, and protein classes. Figure 7A shows the results of classifying these proteins with different expression levels by molecular function. As shown in Figure 7A, in CD133+ cells between tumor and non-tumor tissues, their transporter activity, structural molecular activity, molecular function regulator, catalytic activity, and binding functions are affected. Figure 7B shows the results of classifying these proteins with different expression levels by cellular component. As shown in Figure 7B, in CD133+ cells between tumor and non-tumor tissues... +The intercellular structure, including the cell, extracellular region, organelles, protein-containing complexes, membranes, and cell junctions, is affected;Figure 7C shows the classification of these differentially expressed proteins according to biological processes. As shown in Figure 7C, between CD133+ cells in tumor and non-tumor tissues, biological processes such as response to stimulus, multicellular organismal process, metabolic process, localization, immune system process, cellular process, cellular component organization or biogene, biological regulation, and biological adhesion are affected. Figure 7D shows the classification of these differentially expressed proteins according to protein class. As shown in the figure, between CD133+ cells in tumor and non-tumor tissues, transferases, transfer carrier proteins, receptors, oxidoreductases, nucleic acid binding, and membrane transport proteins are affected. The classification of proteins such as proteins, ligases, hydrolases, enzyme modulators, defense immunity proteins, and cell adhesion molecules is affected.

[0066] As can be seen from the above results, the molecular functions, cellular composition, biological processes and protein classification of liver tumor stem cells expressing CD133 biomarkers in tumor tissue are affected. As a result, liver tumor stem cells expressing CD133 biomarkers in tumor tissue become the preferred targets of dengue virus infection, which further leads to the death of liver tumor stem cells.

[0067] [Example 7] Please refer to Figures 8A and 8B. Figures 8A and 8B show the proportion of each pathway involved in the biological pathways in which proteins with significant differences are mainly involved, analyzed using the KEGG database in one embodiment of the invention. A -log P value greater than 2 represents P < 0.05, indicating a statistically significant difference. Figure 8A shows the biological pathways involved by proteins with high expression levels in CD133+ cells of tumor tissue. Among the biological pathways with significant differences, they are arranged in descending order of significance: detoxification of reactive oxygen species pathway, fatty acid metabolism pathway, metabolism pathway, intracellular metabolism of fatty acids regulating insulin secretion pathway, and the metabolism of ingested H₂SeO₄ and H₂SeO₃ into H₂Se. The pathways involved include: 2Se), localization of the PINCH-ILK-PARVIN complex to focal adhesions, MET interacts with TNS proteins, lipid metabolism, fibronectin matrix formation, pristanoyl-CoA beta-oxidation, CHL1 interactions, and lipophagy. Figure 8B shows the biological pathways involved by proteins with high expression levels in CD133+ cells of non-tumor tissues. Among the biological pathways with significant differences, they are arranged in descending order of significance: CYP2E1 reactions, xenobiotics, formation of a ternary complex, and subsequently, the 43S complex.The following pathways are involved: ribosome scanning and start codon recognition, translation initiation complex formation, activation of mRNA upon binding of the cap-binding complex and elFs, and subsequent binding to 43S, cytochrome P450-arranged by substrate type, peptide chain elongation, selenocysteine ​​synthesis, eukaryotic translation termination, eukaryotic translation elongation, and nonsense-mediated decay (NMD) independent of the exon junction complex (EJC). The following pathways are involved: the Complex (EJC) pathway, the viral mRNA translation pathway, the ElF2AK4 (GCN2) response to amino acid deficiency pathway, the formation of a pool of free 40S subunits pathway, the Phase I functionalization of compounds pathway, the L13a-mediated translational silencing of ceruloplasmin expression pathway, and GTP hydrolysis and joining of the 60S ribosomal subunits.The following pathways are involved in the translation of mRNA: subunit, SRP-dependent cotranslational protein targeting to membrane, exon junction complex (EJC) enhanced nonsense-mediated degradation (NMD), nonsense-mediated degradation (NMD), selenoamino acid metabolism, eukaryotic translation initiation, cap-dependent translation initiation, biosynthesis of maresin-like SPMs, influenza viral RNA transcription and replication, influenza life cycle, and biosynthesis of maresins. The pathways of rRNA processing include: the maresins pathway, the synthesis of epoxy (EET) and dihydroxyeicosatrienoic acids (DHET), the influenza infection pathway, the regulation of expression of SLITs and ROBOs pathway, the synthesis of (16-20)-hydroxyeicosatetraenoic acids (HETE) pathway, the major pathway of rRNA processing in the nucleolus and cytosol pathway, and rRNA processing in the nucleus and cytosol pathway.The pathways include cytosol, metabolism, rRNA processing, ROBO receptor signaling, and biological oxidation.

[0068] As can be seen from the above results, the above-mentioned biological pathways in liver tumor stem cells that express CD133 biomarkers in tumor tissue are affected. As a result, liver tumor stem cells that express CD133 biomarkers in tumor tissue become the preferred targets of dengue virus infection, which further leads to the death of liver tumor stem cells.

[0069] In summary, after dengue virus infection of liver cancer tissue, a greater amount of infectious dengue virus can be produced. Furthermore, the higher the proportion of liver tumor stem cells (CD133+) in the tumor tissue, the higher the concentration of dengue virus produced after infection; there is a significant positive correlation between the two. Therefore, dengue virus can infect liver tumor stem cells and cause their death.

[0070] The dengue virus used in this invention has a preference for infecting liver tumor stem cells (CD133+) in tumor tissue. The reason for this is that there are more liver tumor stem cells in tumor tissue that express CD133 biomarkers than cells that express CD133 biomarkers in non-tumor tissues. Furthermore, liver tumor stem cells that express CD133 biomarkers in tumor tissues are affected by the expression level of a specific protein (see Figure 6) and thus become the preferred targets of dengue virus infection, which further leads to the death of liver tumor stem cells.

[0071] The above-described embodiments are only for illustrating the technical ideas and features of the present invention. Their purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be used to limit the patent scope of the present invention. All equivalent changes or modifications made in accordance with the spirit disclosed in the present invention should still be covered within the patent scope of the present invention. [Simplified Explanation of the Diagram]

[0017] Figure 1 is a flowchart of an experiment related to dengue virus infection of liver cancer tissue according to one embodiment of the invention.

[0018] Figure 2 shows the experimental results of the viral concentration produced by dengue virus infection of liver cancer tissue according to one embodiment of the present invention. Figure 2a is the infection curve of dengue virus infection of tumor tissue and non-tumor tissue (n=41), Figure 2b is a comparison of the highest viral concentration produced by dengue virus infection of tumor tissue and non-tumor tissue (n=41), and Figure 2c is a relationship between the highest viral concentration produced by tumor tissue and non-tumor tissue in each group of liver cancer tissue samples (n=41).

[0019] Figure 3 is a comparison of the highest viral concentration produced by dengue virus infection of liver cancer tissue and the number of cells in the tumor tissue according to one embodiment of the invention. Figure 3a is a scatter plot of the highest viral concentration produced by dengue virus infection of tumor tissue and the number of liver tumor stem cells (n=23). Figure 3b is a scatter plot of the highest viral concentration produced by dengue virus infection of tumor tissue and the percentage of cells other than liver tumor stem cells (n=23). Figure 3c is a comparison plot of the percentage of gated cells expressing the CD133 biomarker in tumor tissue and non-tumor tissue.

[0020] Figure 4 shows the experimental results of the expression of CD133 biomarker in tumor tissue infected with dengue virus according to one embodiment of the invention. The blue line is the virus infection curve generated by infecting tumor tissue (intact cells before sorting) with dengue virus, the red line is the virus infection curve generated by the cell population expressing CD133 biomarker (CD133+) in the tumor tissue infected with dengue virus, and the green line is the virus infection curve generated by the cell population not expressing CD133 biomarker (CD133-) in the tumor tissue infected with dengue virus.

[0021] Figure 5 shows the cell survival status of non-tumor tissue and tumor tissue cells that have not been infected with dengue virus in one embodiment of the present invention after long-term culture. The red line is the cell survival curve of tumor tissue cultured for a long time (N=1), and the blue line is the cell survival curve of non-tumor tissue cultured for a long time (N=1).

[0022] Figure 6 shows the proteins expressed in CD133+ cells in tumor tissue and non-tumor tissue with significantly different expression levels according to one embodiment of the invention. Figure 6a is a comparison chart of volcano expression. In the comparison of the expression levels of various proteins in CD133+ cells in tumor tissue and non-tumor tissue, the proteins with higher expression levels in tumor tissue are selected by the blue box on the right and arranged in Figure 6b according to the degree of difference in expression levels. Similarly, the proteins with higher expression levels in non-tumor tissue are selected by the red box on the left and arranged in Figure 6c according to the degree of difference in expression levels.

[0023] Figures 7A to 7D are a list of biological pathways involving proteins with significant differences in expression analyzed using the PANTHER database according to one embodiment of the invention. Figure 7A shows the results of classifying these proteins with significant differences in expression by molecular function; Figure 7B shows the results of classifying these proteins with significant differences in expression by cellular component; Figure 7C shows the results of classifying these proteins with significant differences in expression by biological process; and Figure 7D shows the results of classifying these proteins with significant differences in expression by protein class.

[0024] Figures 8A and 8B show the proportion of each pathway in the biological pathways mainly involved by proteins with significant differences in expression, analyzed using the KEGG database according to one embodiment of the invention. Figure 8A shows the biological pathways involved by proteins with higher expression levels in CD133+ cells of tumor tissue; Figure 8B shows the biological pathways involved by proteins with higher expression levels in CD133+ cells of non-tumor tissue. The -log P value greater than 2 represents P < 0.05, which is statistically significant.

Claims

1. The use of dengue virus in the preparation of an anti-hepatocellular carcinoma tissue drug, wherein the dengue virus is used to infect liver tumor stem cells to induce the death of the liver cancer tissue.

2. The use of dengue virus as claimed in claim 1 for the preparation of an anti-hepatocellular carcinoma drug, wherein the hepatocellular carcinoma tissue comprises fully cancerous tumor tissue and non-tumor tissue intermediate between normal liver and cancerous tissue; and the hepatocellular carcinoma stem cells are located in the tumor tissue.

3. The use of dengue virus as described in claim 2 for the preparation of an anti-hepatocellular carcinoma drug, wherein the dengue virus, after infecting the hepatocellular carcinoma tissue, can produce an infectious dengue virus.

4. The use of dengue virus as claimed in claim 3 for the preparation of an anti-hepatocellular carcinoma drug, wherein the concentration of dengue virus produced by infecting the tumor tissue is higher than the concentration of dengue virus produced by infecting the non-tumor tissue.

5. The use of dengue virus as claimed in claim 3 for the preparation of an anti-hepatocellular carcinoma drug, wherein the dengue virus concentration produced by infecting the liver tumor stem cells is higher than the dengue virus concentration produced by infecting cells other than liver tumor stem cells.

6. Use of dengue virus as claimed in any one of claims 1 to 5 for the preparation of an anti-hepatocellular carcinoma drug, wherein the biomarker expressed by the hepatocellular carcinoma stem cells comprises at least one selected from the group consisting of CD133, CD117 and CD34.

7. Use of dengue virus as claimed in claim 6 for the preparation of an anti-hepatocellular carcinoma drug, wherein the biomarker expressed by the hepatocellular carcinoma stem cells comprises CD133.

8. The use of dengue virus as described in claim 6 for the preparation of an anti-hepatocellular carcinoma drug, wherein the liver tumor stem cells expressing the CD133 biomarker in tumor tissue become the preferred targets of the dengue virus due to the affected expression level of a specific protein compared to cells expressing the CD133 biomarker in non-tumor tissues, further leading to the death of the liver tumor stem cells.

9. The use of dengue virus as claimed in claim 1 for the preparation of an anti-hepatocellular carcinoma drug, wherein the amount of dengue virus used is 0.5 MOI to 1.5 MOI relative to the number of cells in the hepatocellular carcinoma tissue to which the infection is directed.

10. The use of dengue virus as claimed in claim 1 for the preparation of an anti-hepatocellular carcinoma drug, wherein the amount of dengue virus used is 1 MOI relative to the number of cells in the hepatocellular carcinoma tissue to which the infection is directed.

11. A method for killing liver cancer tissue with dengue virus, wherein the dengue virus infects liver tumor stem cells in the liver cancer tissue, thereby causing the liver cancer tissue to die.

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