TW201347767A - Application of LZ-8 protein derived from the extract of Ganoderma for inhibiting cancer cell growth - Google Patents

Abstract

The present invention provides an application of Ling Zhi-8 (LZ-8) protein, derived from an extract of Ganoderma, for inhibiting cancer cell growth by inducing a ribosomal stress in the cancer cell and preventing the association of murine double minute 2 (MDM2) protein and p53 protein, so as to activate the p53 protein and inhibit the cancer cell growth.

Description

Application of a Ganoderma lucidum extract component LZ-8 protein to inhibit cancer cell growth

The invention provides an application of LZ-8 protein in Ganoderma lucidum extract to inhibit cancer cell growth

Ganoderma lucidum ( G. lucidum for short) is a kind of medical fungus that has been used for a long time in traditional Chinese medicine. Its efficacy is to prolong life and fight various diseases. Studies have confirmed that G. lucidum contains immunoregulatory factors and tumor antagonists. For example, studies have shown that Ganoderma lucidum inhibits cell differentiation, causes apoptosis, halts growth, and inhibits various cancer cell migration. G. lucidum also inhibits tumorigenesis and metastasis of human hepatoma cells. However, the detailed mechanism for inhibiting tumor differentiation has not been fully revealed.

Ling Zhi-8 (LZ-8 for short) is an immunomodulatory protein isolated from the mycelium of G. lucidum . The gene responsible for the production of the LZ-8 protein and its cDNA have been transferred. This LZ-8 protein is considered to be the main biologically active substance in G. lucidum . LZ-8 protein has excellent heat and acid resistance and is resistant to alkali and dehydration. LZ-8 is composed of 110 amino acids with a molecular weight of 12.4 kDa, which is similar to the variable region of the immunoglobulin heavy chain. In vitro studies have shown that the proliferative activity of the LZ-8 protein (mitogenic), in vivo, shows that the LZ-8 protein has immunomodulatory activity. Not only that, when rLZ-8 stimulates human peripheral blood lymphocytes (PBL), it causes the production of cytokines. Studies have shown that LZ-8 causes IL-2 production in the signaling pathway triggered by protein hormones in human T cells. In addition, LZ-8 also enhances the activity and maturation of immune dendritic cells through the NF-NB and MAPK pathways. However, the role of LZ-8 as an anticancer factor and the message delivery pathways it participates in are still unknown.

In human cancer cells, the p53 tumor suppressor gene is the most frequently mutated gene. In cancer cells in which the p53 gene is not mutated, the p53 pathway is usually not activated. Many stress signals, such as genotoxic DNA damage, interfere with the MDM2-p53 feedback pathway, thus activating the p53-induced response.

Stressors, also known as stress molecules, are caused by the interference of ribosome formation or biogeneration. In the ribosome pressure reaction, some ribosomal proteins (RPs), such as L11, L23, L5, S7 and L26, are released from the nucleolus and are linked to MDM2 in the cytoplasm. These RPs-MDM2 interactions inhibit the p53 protein ubiquitination and p53 protein breakdown involved in MDM2. Thus, cell cycle arrest or apoptosis caused by p53 protein is activated.

The anti-cancer mechanism caused by LZ-8 is still unknown, so finding out this anti-cancer mechanism is a major problem to be solved in the medical field.

In view of the above, an object of the present invention is to provide a mechanism for inhibiting the growth of cancer cells, and thereby providing a pharmaceutical compound or method for regulating the mechanism to inhibit the growth of cancer cells.

The invention provides an application for inhibiting the growth of a cancer cell by the component of the extract of Ganoderma lucidum, wherein the Ganoderma lucidum extract component is LZ-8 protein.

Preferably, the aforementioned protein line comprises the protein of SEQ ID: NO. (The sequence is: S D TA L I F R L AW DV K K L S F DY T P N W G R G N P NN F I D T V T F P K V LT D K AY T Y R VAV S G R N L G VK P S YAV E S D G S Q K V N F L E Y N S G Y G I A D T N TI Q V F V V D P D T N N D F I I A Q W N).

Preferably, the aforementioned protein system is Ling Zhi-8 (LZ-8).

Preferably, the aforementioned Ganoderma lucidum is Ganoderma lucidum.

Preferably, the aforementioned Ganoderma lucidum is G. lucidum or G. sinense .

Preferably, the aforementioned Ganoderma lucidum is G. lucidum.

Preferably, the cancer cell line is a p53 unmutated cancer cell, and the p53 contained in the cancer cell is not mutated, but is in a state of being inhibited by murine double minute 2 (MDM2), so the cancer cell usually exists in the wild. With wild-type p53 and high-performance MDM2, when unmutated p53 binds to MDM2, p53 proceeds to ubiquitination and degradation. Preferably, the cancer cell line which is not mutated by p53 is a breast cancer cell, a cervical cancer cell, an ovarian cancer cell, a liver cancer cell, a lung cancer cell, a rectal cancer cell, an oral cancer cell, and a stomach. cancer cell.

Preferably, the aforementioned application prevents the murine double minute 2 (MDM2) protein in the cancer cells from binding to the p53 protein by causing ribosome pressure in the cancer cells to achieve the effect of activating the p53 protein in the cancer cells.

The present invention provides a mechanism for inhibiting the growth of cancer cells, and thereby providing a pharmaceutical compound or method for regulating this mechanism to inhibit the growth of cancer cells. The invention provides the application of the Ganoderma lucidum extract component for inhibiting the growth of cancer cells, wherein the Ganoderma lucidum extract component is LZ-8 protein. In a preferred embodiment, the protein is a variety of Ganoderma lucidum extract proteins, and in a preferred embodiment, is the protein of SEQ ID NO: 1. The aforementioned protein system is Ling Zhi-8 (LZ-8).

Ganoderma system of the present invention is the G.lucidum or G.sinense, in the preferred embodiment, is based Ganoderma G.lucidum.

The cancer cell line of the present invention is a variety of p53 unmutated cancer cells, including, but not limited to, breast cancer cells, cervical cancer cells, ovarian cancer cells, liver cancer cells, lung cancer cells, rectal cancer cells, oral cancer cells, gastric cancer cells. . In a preferred embodiment, it is a lung cancer cell, a cervical cancer cell, or a breast cancer cell.

The application provided by the present invention includes, but is not limited to, transmission to cause ribosome pressure in the aforementioned cancer cells. In a preferred embodiment, the aforementioned application is to activate the p53 protein in the aforementioned cancer cells by preventing the murine double minute 2 (MDM2) protein in the aforementioned cancer cells from binding to the aforementioned p53 protein.

The mechanism of inhibiting the growth of cancer cells caused by LZ-8 developed by the present invention is as follows: Ling-Zhi-8 (LZ-8) causes ribosome pressure in cancer cells, thereby preventing murine double minute 2 in the aforementioned cancer cells ( The MDM2) protein binds to the aforementioned p53 protein to activate the p53 protein in the aforementioned cancer cells, causing apoptosis of the cancer cells or arrest of the cell cycle G1.

The cell lines used in the following examples belong to the cancer cells of p53 non-mutation (wild-type p53):

Example 1. rLZ-8 inhibits proliferation of human lung cancer cell A549

Human lung cancer cell A549 was added to different concentrations (0, 5, 10, 15 μg/mL) of rLZ-8 to MTT The assay was observed for cell survival at 12, 24, and 72 hours, and the results are shown in the first panel. This result showed that cell growth was significantly inhibited by treatment with cells at a higher concentration (10, 15 μg/mL) of rLZ-8 for 48 and 72 hours. When the cells were treated with rLZ-8 at a lower concentration (5 μg/mL), the effect of inhibiting cell growth was not observed until 72 hours.

Example 2. rLZ-8 prolongs the G1 stagnation phase of human lung cancer cell A549 and enhances the transcriptional activity of p53 (transactivational activity)

A549 cells were treated with rLZ-8 for 24 hours, stained with propidium Iodide (PI) and analyzed by flow cytometry. The results are shown in Figure AA. When A549 cells were treated with 15 μg/mL of rLZ-8 for 24 hours, the G1 arrested cells increased from 64.9% in the control group to 80.4%. Under the same treatment, the sub G1 population increased only from 1.1% to 5.9%.

Protein expression was then analyzed by Western blotting and the results are shown in Figure B. The three proteins p53, p21/Cip1 and p27/Kip1 were all increased by rLZ-8 treatment. Both p21 and p27 proteins are inhibitors of cyclin-dependent kinase (CDK) and are linked to kinases. Not only that, but p21 is directly regulated by p53.

The cells were co-transformed with the p53 luciferase reporter and the Renilla luciferase vector for 24 hours, and then treated with rLZ-8 for 6 hours. The results are shown in Figure C, When UV is irradiated to cells, DNA damage is caused to activate p53, and cells treated with rLZ-8 also have the effect of activating p53.

Example 3. rLZ-8 prolongs the G1 stagnation phase of human lung cancer cell A549, but only if p53 is present (p53 dependent) and is not mutated

The cells are incubated into the sh-p53 gene (viral lentiviruses expression gene) or the sh-luc gene (control group) to knock down the p53 gene (Panel 3); or the p53 mutant gene of R248W or V143A is introduced. A549 cells were obtained to obtain isogenic A549 cells (Fig. 3B). After the above two treatments were treated with rLZ-8 for 2 hours, the protein content in the cells was observed by Western blotting, and the results are shown in Figures A and B. The results showed that the amount of p53 protein in A549 cells was significantly decreased after sh-p53 transfection, while the amount of p53 protein in R248W or V143A mutants was significantly increased, but the p53-regulated CDK inhibitor protein p21 was observed. The amount of expression was not increased or decreased compared with the control group. It can be seen that the increased p53 protein has been Mutated and not functional.

The cells treated with the above two treatments (p53 gene degradation or mutation) were treated with 10 μg/mL of rLZ-8 for 24 hours, and then the cell cycle distribution was observed by flow cytometry. The results are shown in the third C and D diagrams. From the control group (sh-luc and Vector), it was found that when the p53 gene was not degraded or mutated, the G1 stagnation period was significantly prolonged after the action of rLZ-8. Once the p53 gene is degraded (sh-p53) or mutated (R248W or V143A), the prolongation of the G1 stagnation period caused by rLZ-8 is no longer present, thus demonstrating the growth caused by rLZ-8 in A549 cells. Stalling must rely on the p53 gene (p53 dependent).

Example 4. rLZ-8 inhibits xenograft growth of Lewis rat lung cancer cell LLC1

LLC1 cells were injected subcutaneously into both sides of each C57BL/6 mouse, and on the fourth day after transplantation, each mouse was intraperitoneally injected with rLZ-8 every four days. Control group mice were injected with the same amount of PBS instead of rLZ-8. These mice were randomly divided into three groups.

The body weight distribution of the mice is as shown in Fig. 4A, and the mice injected with rLZ-8 and the mice injected with PBS have no significant difference in body weight.

In contrast, the size of the tumor produced was as shown in Figure 4B, and the tumor volume of mice injected with rLZ-8 was significantly smaller than that of mice injected with PBS.

When the tumor volume of the control mice reached 2000 cubic centimeters (about 20 days), the mice were sacrificed to obtain their tumor tissues, and the cell lysates of their tumor tissues were analyzed by Western blotting. The amount of p53, p21, p27 and MDM2 protein was as shown in the fourth C-graph. The results showed that the amount of p53, p21, p27 and MDM2 protein in the cell lysate of the tumor treated with rLZ-8 increased.

Example 5. Changes of p53 protein in cervical cancer cells HeLa and breast cancer cells MCF-7 after rLZ-8 treatment

Cervical cancer cells (HeLa) and breast cancer cells (MCF-7) were treated with 10 μg/mL of rLZ-8 for 6 hours, and the amount of change in p53 protein was observed. The results are shown in Fig. 5 and Fig. 6, respectively. muscle The protein content was used as a control group. From the results, the expression of p53 protein was increased by 3-4 fold in HeLa cells after treatment with rLZ-8 (figure 5), while the expression of p53 protein was increased by 1.3-fold in MCF-7 cells (Fig. Figure 6). Since p53 protein is usually associated with cell growth and apoptosis, it is known that rLZ-8 also inhibits the growth of cervical cancer cells (HeLa) and breast cancer cells (MCF-7).

The first panel shows the survival rate of A549 cells after treatment with rLZ-8; the second panels A, B and C show that rLZ-8 increases G1 arrest of A549 cells and enhances the transcription factor activity of p53; B, C, D showed that rLZ-8 can prolong the G1 stagnation phase of human lung cancer cell A549, but only if p53 is present (p53 dependent) and is not mutated; the fourth panel A, B, C shows rLZ- 8 can inhibit the xenograft growth of Lewis rat lung cancer cell LLC1; the fifth panel shows the change of p53 protein in cervical cancer cell HeLa after rLZ-8 treatment; the sixth figure shows the breast cancer cell MCF-7 after rLZ-8 treatment The change in p53 protein.

SEQUENCE LISTING

<110> National Yangming University

<120> An application of Ganoderma lucidum extract to inhibit cancer cell growth

<130> 2011RDPA031

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 110

<212> PRT

<213> G.Iucidum

<400> 1

Claims (10)

An application of a Ganoderma lucidum extract to inhibit the growth of a cancer cell, the Ganoderma lucidum extract comprising a protein. The use of claim 1, wherein the protein comprises the protein of SEQ ID NO: 1. The application of claim 2, wherein the protein is Ling Zhi-8 (LZ-8). The application of claim 1, wherein the aforementioned Ganoderma lucidum is Ganoderma lucidum. The application of claim 1, wherein the aforementioned Ganoderma lucidum is G. lucidum or G. sinense . The application of claim 5, wherein the aforementioned Ganoderma lucidum is G. lucidum. The application of claim 1, wherein the cancer cell line is a p53 unmutated cancer cell. The application according to claim 7, wherein the p53 unmutated cancer cell line is a breast cancer cell, a cervical cancer cell, an ovarian cancer cell, a liver cancer cell, a lung cancer cell, a rectal cancer cell, an oral cancer cell, and a gastric cancer cell. . The application of claim 1 is directed to causing ribosome pressure in the cancer cells. The application of claim 1, wherein the p53 protein in the cancer cell is activated by preventing the murine double minute 2 (MDM2) protein from binding to the p53 protein in the cancer cell.