Use of ganoderma triterpene in preparation of medicine for treating cervical cancer

Abstract

The present application relates to the technical field of medicine, and particularly relates to application of ganoderma triterpene in preparation of medicine for treating cervical cancer. Ganoderma triterpene (Ganodecalone A, referred to as GDA) can inhibit migration, proliferation and clonogenic capacity of cervical cancer cells; GDA can arrest the cell cycle of cervical cancer cells in G0 / G1 phase, and with the increase of the concentration of GDA, the proportion of cells in G0 / G1 phase is significantly increased; GDA can promote apoptosis of cervical cancer cells, and with the increase of the concentration of GDA, the proportion of late apoptotic cells is significantly increased; GDA can promote apoptosis of cervical cancer cells by reducing the phosphorylation level of Akt in the cervical cancer cells. GDA and Akt phosphorylation agonist SC79 are mutually antagonistic, and when GDA is combined with Akt phosphorylation inhibitor MK-2206, the apoptosis of two strains of cervical cancer cells is promoted. GDA can induce apoptosis of cells by inhibiting phosphorylation of Akt, up-regulating pro-apoptotic protein Bad and down-regulating anti-apoptotic protein Bcl-2, and thus the anti-tumor effect is exerted.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to the application of a Ganoderma lucidum triterpenoid in the preparation of a drug for treating cervical cancer. Background Technology

[0002] Cervical cancer is one of the most common and deadliest cancers among women, ranking third in incidence and fourth in mortality among female malignant tumors. Human papillomavirus (HPV) infection is the main cause of cervical cancer. In recent years, with the widespread adoption of HPV screening and the maturation of vaccine technology, the mortality rate of cervical cancer has tended to stabilize. In the exploration of novel therapeutic drugs for cervical cancer, small molecule compounds from traditional Chinese medicine have shown broad prospects. These compounds are abundant in sources, have diverse targets, possess a basis for clinical application, and have relatively low toxicity and side effects, providing valuable resources for the development of low-toxicity and highly effective anti-cervical cancer drugs. Currently, traditional treatment for cervical cancer still mainly relies on platinum-based drugs combined with radiotherapy and chemotherapy. Some patients undergo surgical resection, but surgery is highly invasive (often requiring the removal of reproductive organs and part of the intestines and urethra), with a five-year survival rate of approximately 50%. Paclitaxel combined with chemotherapy has become a first-line treatment, but acquired resistance exists, reducing the five-year survival rate by 20%-30%. Newly introduced anti-angiogenic drugs (bevacizumab), PARP inhibitors, and immune checkpoint inhibitors, while causing less damage, have limited efficacy when used alone and often require combination therapy with cisplatin or paclitaxel. Furthermore, their high cost makes them inaccessible to women with low socioeconomic status. Therefore, treatment strategies for cervical cancer still require further development.

[0003] Natural products and their active ingredients play a crucial role in the discovery of cancer treatment drugs. Traditional Chinese medicine (TCM), with its thousands of years of development and clinical application, has accumulated rich experience and resources, becoming a vital resource for screening novel anticancer drugs. Its profound historical foundation and extensive practical application give TCM an irreplaceable position in anticancer drug research and development. With the advancement of science and technology, compound separation and extraction techniques are becoming increasingly mature. The exploration and analysis of TCM components not only promotes the rapid development of the TCM industry, but the discovery of active lead compounds in TCM also provides more possibilities for the treatment of human diseases.

[0004] Heat-loving Ganoderma ( Ganoderma calidophilum Ganoderma lucidum, a rare and precious medicinal resource unique to Hainan Province, China, is used by the Li ethnic minority in Hainan to make medicinal wine for cancer prevention and treatment, possessing extremely high medicinal value. Addressing the issues of significant side effects, high recurrence rate, and unclear mechanisms of action in existing anti-cervical cancer drugs, the inventors have discovered for the first time that Ganodecalone A, a triterpenoid compound from the heat-loving Ganoderma lucidum of the Hainan Li ethnic minority, exhibits significant anti-cervical cancer activity, providing a new candidate molecule for developing highly effective, low-toxicity, and clearly defined naturally derived anti-cervical cancer drugs. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides the application of Ganodecalone A (GDA) in the preparation of drugs for treating cervical cancer. The Ganodecalone A described in this invention is a Ganodecalone compound, one of the main active ingredients isolated from the ethanol extract of the fruiting body of heat-loving Ganoderma lucidum. This invention systematically investigated the effects of GDA on the phenotype and related gene and protein expression of human cervical cancer cell lines C33A and HeLa using techniques such as scratch healing assays, plate colony assays, AO / EB fluorescence staining, flow cytometry, real-time label-free cell analysis (RTCA), RT-qPCR, and Western blot. This provides a new source for the development of anti-cervical cancer drugs.

[0006] To achieve the above objectives, the present invention provides the following technical solution: This invention provides the application of Ganoderma lucidum triterpenes in the preparation of drugs for treating cervical cancer.

[0007] Preferably, the structural formula of the Ganoderma lucidum triterpenoid is as follows: .

[0008] Preferably, the aforementioned Ganoderma lucidum triterpenoid treats cervical cancer by inhibiting the migration, proliferation, and colony formation of cervical cancer cells.

[0009] Preferably, the aforementioned Ganoderma lucidum triterpenoid treats cervical cancer by arresting the cell cycle of cervical cancer cells in the G0 / G1 phase.

[0010] Preferably, the aforementioned Ganoderma lucidum triterpenoid treats cervical cancer by promoting apoptosis of cervical cancer cells.

[0011] Preferably, the aforementioned Ganoderma lucidum triterpenoid promotes apoptosis of cervical cancer cells by reducing Akt phosphonation levels in cervical cancer cells.

[0012] Preferably, the cervical cancer cells include cervical cancer cell lines C33A and HeLa.

[0013] This invention also provides the application of Ganoderma lucidum triterpenes combined with Akt phosphorylation inhibitors in the preparation of drugs for treating cervical cancer.

[0014] Preferably, the Akt phosphorylation inhibitor includes MK-2206 at an effective concentration of 5 μM.

[0015] The effective concentration of the aforementioned Ganoderma lucidum triterpenoid is 20 μM.

[0016] The present invention also provides a drug for treating cervical cancer, comprising a Ganoderma lucidum triterpenoid and an Akt phosphorylation inhibitor.

[0017] The beneficial effects of this invention are: This invention discloses for the first time a novel use of GDA, a compound derived from *Ganoderma lucidum*, in the preparation of anti-cervical cancer drugs, providing a novel natural candidate molecule for the treatment of cervical cancer. GDA exhibits significant anti-cervical cancer activity, inhibiting the proliferation, migration, and colony formation of cervical cancer cells, arresting the cell cycle, and effectively inducing apoptosis. Its mechanism of action is clearly defined: it regulates the expression of downstream apoptosis-related proteins by inhibiting Akt protein phosphorylation. GDA is derived from the traditional Li medicine *Ganoderma lucidum*, possessing the advantages of being natural, low in toxicity, and highly effective, and has the potential for synergistic effects with existing Akt inhibitors. This invention provides a new drug source and strategy for the treatment of cervical cancer. Furthermore, GDA can be isolated from the traditional Li medicine *Ganoderma lucidum* using conventional chromatographic methods. The raw material has a basis in ethnic medicine applications, and the preparation process is suitable for industrial production. It can be further prepared into various dosage forms or used in combination with other anti-tumor drugs, showing broad prospects for clinical development and application. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0019] Figure 1 To illustrate the effect of GDA on the migration ability of C33A cells, the results of scratch healing experiments were obtained after treating C33A cells with different concentrations of GDA for 24 h (left) and 48 h (right). P <0.05, P <0.01 and P <0.001); Figure 2 To illustrate the effect of GDA on the migration ability of HeLa cells, the results of scratch healing experiments were obtained after treating HeLa cells with different concentrations of GDA for 24 h (left) and 48 h (right). P <0.05, P <0.01 and P <0.001); Figure 3 To illustrate the effect of GDA on the colony-forming ability of cervical cancer cell lines, the changes in colony-forming ability of cervical cancer cell lines C33A (left) and HeLa (right) after stimulation with different concentrations of GDA are shown. P <0.05, P <0.01 and P <0.001); Figure 4 To investigate the effect of GDA on the cell cycle of cervical cancer cell lines C33A and HeLa, cell cycle assays were performed on C33A and HeLa cells treated with different concentrations of GDA for 48 hours. P <0.05, P <0.01 and P <0.001); Figure 5 To investigate the effect of GDA on apoptosis in cervical cancer cell line C33A, the results of apoptosis detection in C33A cells treated with different concentrations of GDA for 48 hours were obtained. P <0.05, P <0.01 and P <0.001); Figure 6 To investigate the effect of GDA on apoptosis in the cervical cancer cell line HeLa, the results of apoptosis detection were obtained after treating HeLa cells with different concentrations of GDA for 48 hours. P <0.05, P <0.01 and P <0.001); Figure 7 To investigate the effect of GDA on the transcriptional level of the PI3K / Akt signaling pathway in C33A and HeLa cells, the relative expression levels of PI3K / Akt signaling pathway mRNA in C33A and HeLa cells treated with different concentrations of GDA for 24 h and 48 h were measured. P <0.05, P <0.01 and P <0.001); Figure 8 To investigate the effect of GDA on the protein levels of the PI3K / Akt signaling pathway in cervical cancer cells, the expression levels of PI3K / Akt signaling pathway proteins in C33A and HeLa cells were measured after treatment with different concentrations of GDA for 24 h and 48 h. Figure 9 The results of GDA combined with SC 79 on apoptosis in C33A (left) and HeLa (right) cells. P <0.05, P <0.01 and P <0.001 indicates a difference compared to the control group; # P <0.05, ## P <0.01 and ### P <0.001 indicates a comparison with the GDA group); Figure 10 The effect of combined GDA and SC 79 on the PI3K / Akt signaling pathway in cervical cancer cells; Figure 11 The results of GDA combined with MK-2206 on apoptosis in C33A (left) and HeLa (right) cells. P <0.05, P <0.01 and P <0.001 indicates a difference compared to the control group; # P <0.05, ## P <0.01 and ### P <0.001 indicates a comparison with the GDA group); Figure 12 The effect of the combination of GDA and MK-2206 on the PI3K / Akt signaling pathway in cervical cancer cells; Figure 13 A diagram illustrating the mechanism by which GDA exerts its anti-cervical cancer effect through the PI3K / AKT signaling pathway. Detailed Implementation

[0020] This invention provides the application of Ganoderma lucidum triterpenes in the preparation of drugs for treating cervical cancer.

[0021] In this invention, the structural formula of the Ganoderma lucidum triterpenoid is as follows: .

[0022] In this invention, the Ganoderma triterpenoid (GDA) is prepared according to Chinese patent CN201811347417.X.

[0023] In this invention, the aforementioned Ganoderma lucidum triterpenoid preferably treats cervical cancer by inhibiting the migration, proliferation, and colony formation of cervical cancer cells. In this invention, the aforementioned Ganoderma lucidum triterpenoid preferably treats cervical cancer by arresting the cell cycle of cervical cancer cells in the G0 / G1 phase. In this invention, the aforementioned Ganoderma lucidum triterpenoid preferably treats cervical cancer by promoting apoptosis of cervical cancer cells. In this invention, the aforementioned Ganoderma lucidum triterpenoid preferably promotes apoptosis of cervical cancer cells by reducing the level of Akt phosphonation in cervical cancer cells. In this invention, the cervical cancer cells preferably include cervical cancer cell lines C33A and HeLa. This invention does not impose specific limitations on the dosage form and preparation method of the drug; any medically acceptable dosage form and preparation method of the Ganoderma lucidum triterpenoid is acceptable.

[0024] This invention also provides the application of Ganoderma lucidum triterpenes combined with an Akt phosphorylation inhibitor in the preparation of a drug for treating cervical cancer. In this invention, the Akt phosphorylation inhibitor comprises MK-2206 at a concentration of 5 μM, and the Ganoderma lucidum triterpenes are present at a concentration of 20 μM. Preferably, the Akt phosphorylation inhibitor comprises MK-2206. This invention does not impose any particular limitations on the dosage form or preparation method of the drug.

[0025] This invention also provides a drug for treating cervical cancer, comprising a Ganoderma lucidum triterpenoid and an Akt phosphorylation inhibitor. This invention does not impose any particular limitations on the dosage form or preparation method of the drug.

[0026] To further illustrate the present invention, the following detailed description is provided in conjunction with embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0027] Example 1 1. Materials and Methods 1.1 Cell Culture 1.1.1 Cell resuscitation Remove the cells from the liquid nitrogen container, seal them tightly with plastic wrap, and place them in a 37°C water bath. Gently agitate the cryovials during heating to accelerate thawing. Add an appropriate amount of DMEM complete culture medium to a 15 mL centrifuge tube. Once the liquid in the cryovial is completely thawed, transfer it to the 15 mL centrifuge tube. Centrifuge at 1000 rpm for 5 min at room temperature. Discard the supernatant, add an appropriate amount of DMEM complete culture medium, and slowly pipette to mix thoroughly. Centrifuge again at 1000 rpm for 5 min to remove any residual DMSO. Add 1 mL of complete culture medium, mix thoroughly, and transfer to a prepared T25 culture flask containing 4 mL of complete culture medium. Gently agitate to ensure the medium covers the bottom of the flask. Transfer the flask to a 5% CO2 incubator and incubate at 37°C. During cell culture, regularly observe the cell growth status and perform timely medium changes, passages, and other related procedures.

[0028] 1.1.2 Cell passage When the cell confluence is approximately 80%, passage the cells. Place the culture flasks to be passaged in a clean bench, discard the old culture medium, and gently wash the cells twice with PBS. Add 1 mL of 0.25% trypsin and gently agitate to cover the bottom of the flask. Incubate at 37°C for 1-2 minutes. Observe the cell state under an inverted microscope. If most cells shrink and round, and easily detach in a flowing sand-like manner when the side of the culture flask is gently tapped, digestion is complete. Add fresh complete culture medium and gently pipette repeatedly to completely detach the cells adhering to the bottom. Transfer the cell mixture to a 15 mL centrifuge tube and centrifuge at 1000 rpm for 5 minutes at room temperature. Discard the supernatant. Resuspend the cell pellet in 3-5 mL of complete culture medium, and then transfer it to a new T25 culture flask at a 1:3 ratio. Incubate at 37°C in a cell culture incubator containing 5% CO2. Observe the cell adhesion at irregular intervals, change the medium or continue passage, and carry out subsequent experiments after passage 3 times.

[0029] 1.1.3 Cell cryopreservation Discard the old culture medium from the culture flask, wash the cells twice with PBS buffer, then add 0.25% trypsin solution and incubate at 37°C for 1-2 minutes. Observe the cell morphology changes during digestion. When the cells become bright, round, add complete culture medium to stop digestion. Gently pipette the bottom of the culture flask until the cells are completely detached, forming a cell suspension. Transfer the suspension to a 15 mL centrifuge tube and centrifuge at 1000 rpm for 5 minutes at room temperature. Discard the supernatant. Resuspend the cells in an appropriate amount of cell cryopreservation medium and aliquot into cryovials, labeling the cell type, passage number, and cryopreservation date.

[0030] 1.2 MTT assay for antitumor activity C33A and HeLa cells in logarithmic growth phase were divided into groups of 8 × 10⁸ cells per well. 3 Cells were seeded at a density of 100 μL per well into 96-well plates, with 3-4 replicates per experimental group. After cell adhesion, the original medium was replaced with fresh medium containing 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, and 0 μM GDA, respectively. After culturing for 24 and 48 hours in the dark, 20 μL of 5 mg / mL MTT was added to each well, and the cells were incubated for another 4 hours. Finally, 100 μL of 10% SDS-HCl was added, and the cells were incubated for another 6 hours at 37°C with 5% CO2. The OD value was measured at 570 nm using a microplate reader.

[0031] 1.3 Scratch healing assay to detect the effect of GDA on cell migration Take a 6-well plate and draw 6 horizontal lines on the back of the plate as reference lines using a marker pen; after digesting and counting the cells in the culture flask, divide them into 6 × 10⁶ cells per well. 5 Cells were seeded into labeled 6-well plates, gently shaken to mix, and incubated in a CO2 incubator for 24 h to allow complete adhesion. The culture medium was discarded, and a white pipette tip was used to make a scratch mark perpendicular to the horizontal reference line in each well. The cell surface was washed twice with PBS to remove detached cells. Different concentrations of GDA-containing media were prepared using fresh FBS-free medium to replace the original medium. Cell migration was observed by photographing at 0 h, 24 h, and 48 h after drug stimulation. Finally, ImageJ software was used to statistically analyze the scratch area, calculate the migration ratio of tumor cells at different time points, and perform data analysis.

[0032] 1.4 Determination of the role of GDA in cell plate colony assay Cells were cultured in 6cm culture dishes (1500 cells / dish). After stable cell adhesion, cells were treated with different concentrations of GDA. Cultured in a CO2 incubator for 2-3 weeks until clearly visible cell colonies were observed. The cells were then washed three times with PBS, fixed with paraformaldehyde for 20 min, and stained with crystal violet solution in the dark for 20 min. The bottom of the culture dish was slowly rinsed with ddH2O until the clones were clearly visible. Images were taken, cells were counted, and analyzed using ImageJ.

[0033] 1.5 Effects of GDA on the cell cycle of cervical cancer cells Cell cycle determination was performed using a DNA content assay kit. C33A and HeLa cells were cultured at 3 × 10⁻⁶ cells / year. 5Cells were cultured in six-well plates at a density of 1 / ml for 24 h, and then fresh complete culture medium containing 0 μM, 10 μM, 20 μM, and 40 μM MDA were added, respectively. After another 24 hours of culture, cells were collected, fixed overnight at 4°C with pre-chilled 70% ethanol solution, centrifuged, and the supernatant was discarded. The precipitate was washed once with PBS, and then 100 μL of RNase A was added. The mixture was heated in a water bath at 37°C for 30 min. 400 μL of LPI staining solution was added under light and incubated at 4°C for 30 min. After staining, the cell clusters were quickly filtered through a 200-mesh sieve. The cells were then analyzed by flow cytometry, and the red fluorescence at an excitation wavelength of 488 nm was recorded.

[0034] 1.6 Effect of GDA on apoptosis of cervical cancer cells The apoptosis rate of cervical cancer cells after GDA treatment was detected using the annexin V-FITC / PI staining kit. C33A and HeLa cells were cultured at 3 × 10⁻⁶ cells / year. 5 Cells were cultured at a density of 1 / ml in six-well plates for 24 h, then incubated for another 24 h with fresh complete culture medium containing 0 μM, 10 μM, 20 μM, and 40 μM MDA, respectively. The supernatant was discarded, and the cell pellet was collected. Cells were washed three times with pre-chilled PBS, and the cell pellet was collected again. 1× Binding buffer was added to the pellet to prepare a cell suspension. 5 μL of Annexin V-FITC and 5 μL of PI were added to the cell suspension, and the mixture was incubated at room temperature in the dark for 10 min, then gently mixed. The stained samples were analyzed by flow cytometry within 1 h.

[0035] 1.7 Effects of GDA on genes related to the PI3K / Akt signaling pathway 1.7.1 Effects of GDA on the transcriptional levels of genes related to the PI3K / Akt signaling pathway Total RNA was extracted using the TransZol Up Plus RNA Kit from TransGen Biotech, and then the mRNA was reverse transcribed into cDNA according to the HiScript II Q RT SuperMix for qPCR (+gDNA wiper) kit instructions. Using GAPDH mRNA as an internal control, real-time quantitative PCR amplification of the total cDNA was performed using SYBR Green PCR Super Mix (TransGen Biotech). The reaction conditions were: 95 ℃ for 30 s, 60 ℃ for 30 s, and 72 ℃ for 30 s, for a total of 40 cycles. After PCR, the cycle threshold (Ct) value was obtained and analyzed using 2^... -ΔΔCt The transcriptional level of the target gene relative to GAPDH was calculated.

[0036] 1.7.2 Effects of GDA on the translation levels of genes related to the PI3K / Akt signaling pathway Cells were treated with RIPA lysis buffer containing protease inhibitors, homogenized, and centrifuged at 12,000×g at 4°C for 15 minutes. The supernatant was collected. Protein concentration was detected using a BCA protein assay kit. An equal volume of 1× loading buffer was added, and the cells were briefly centrifuged and incubated at 100°C for 10 minutes. Proteins were separated using 8%–12% SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked with 5% skim milk at room temperature for 1 hour, and then incubated overnight at 4°C with a primary antibody specific to the target protein. After incubation with horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature for 2 hours, the PVDF membrane was immersed in ECL luminescent solution and incubated in the dark for 3 minutes. The membrane was developed using ImageLab software, and the grayscale values ​​were analyzed using ImageJ software.

[0037] 1.8 Effects of combined use of GDA and Akt protein phosphorylation agonist SC 79 on cervical cancer cells 1.8.1 SC 79 can reverse GDA-induced apoptosis The effect of combined use of GDA (20µM) and SC79 (20µM) on apoptosis of cervical cancer cells was detected by flow cytometry, using the same experimental method as in 1.6.

[0038] 1.8.2 Changes in Akt and related proteins Western blotting was used to detect changes in the expression levels of C33A and HeLa PI3K / Akt signaling pathway-related proteins in cervical cancer cells after the addition of SC 79. The experimental method was the same as in 1.7.2.

[0039] 1.9 Effects of combined use of GDA (20µM) and Akt protein phosphorylation inhibitor MK-2206 (5µM) on cervical cancer cells 1.9.1 Combined action of MK-2206 and GDA induces apoptosis in cervical cancer cells Flow cytometry was used to detect changes in apoptosis in two cervical cancer cell lines after combined use of GDA and MK-2206. The experimental method was the same as in 1.6.

[0040] 1.9.2 Changes in Akt and related proteins Western blotting was used to detect changes in the expression levels of PI3K / Akt signaling pathway-related proteins in cervical cancer cells C33A and HeLa after the addition of MK-2206. The experimental method was the same as in 1.7.2.

[0041] 2. Experimental Results 2.1 Effects of GDA on the proliferation of cervical cancer cells C33A and HeLa The results are shown in Table 1. In C33A cells, the IC50 values ​​after 24 h and 48 h of treatment with the positive control drug cisplatin were... 50 The IC50 values ​​for cisplatin in HeLa cells were 11.40 μM and 5.65 μM, respectively, after 24 h and 48 h of treatment. 50 The concentrations were 25.97 μM and 13.93 μM, respectively. The IC50 values ​​of GDA after 24 h and 48 h of treatment with C33A cells were [not specified]. 50 The concentrations were 23.00 μM and 14.35 μM, respectively. The IC50 values ​​of GDA in HeLa cells after 24 h and 48 h of treatment were... 50 The concentrations were 43.55 μM and 25.14 μM, respectively. Overall, GDA showed good inhibitory effects on the proliferation of cervical cancer cells C33A and HeLa.

[0042] Table 1. IC50 of GDA and cisplatin at 24h and 48h for cervical cancer cell lines. 50 value

[0043] 2.2 GDA's ability to inhibit the migration of cervical cancer cells The effect of GDA on the migration ability of two cervical cancer cell lines, C33A and HeLa, was detected using a scratch healing assay. After treatment with GDA, images were taken at the same scratch sites at 0h, 24h, and 48h. The scratch area at the same location at different time points was statistically analyzed using ImageJ software to calculate the migration rate. The scratch healing results for C33A cells showed (…). Figure 1 Among the various concentration groups treated with GDA for 24 hours, only the high-concentration group showed a significant difference from the control group. After 48 hours of GDA treatment, cell migration ability was significantly reduced in all treatment groups compared to the control group, and this reduction was concentration-dependent. HeLa cell scratch assay results showed... Figure 2 In the samples taken at 24 and 48 hours, no significant differences were found between the low-concentration group and the control group, while other concentration groups showed significant differences from the control group. Cell migration ability decreased with increasing concentration.

[0044] The above results indicate that GDA can significantly inhibit the migration ability of the two cervical cancer cell lines in a concentration- and time-dependent manner.

[0045] 2.3 GDA's ability to inhibit the cloning of cervical cancer cells The effect of GDA on the clonal potential of cervical cancer cell lines C33A and HeLa was examined using a plate colony assay. Results are as follows: Figure 3As shown, cell clone counts were performed on three petri dishes treated with the same concentration. The cell clone counts for C33A cells with increasing GDA concentrations were 858, 587, 432, and 92, respectively; for HeLa cells, the cell clone counts were 851, 550, 350, and 211, respectively. Compared with the control group, the clone counts of both cervical cancer cell lines showed a significant decrease in colony-forming potential across different GDA concentration treatment groups, indicating that GDA can reduce the clonal potential of cervical cancer cell lines C33A and HeLa in a concentration-dependent manner.

[0046] 2.4 GDA affects the cell cycle of cervical cancer cells Flow cytometry was used to detect the cell cycle arrest effect of GDA on two cervical cancer cell lines, C33A and HeLa. Cells stimulated by GDA were fixed overnight, stained with PI under light-protected conditions, and then analyzed by flow cytometry.

[0047] Cell cycle analysis of C33A cells after 48 hours of GDA stimulation showed that, compared with the control group, the number of G0 / G1 phase cells significantly increased with increasing drug concentration. The number of S phase cells decreased significantly in all groups except the low-concentration drug group, which showed no significant change. Flow cytometry analysis of HeLa cells after 48 hours of drug stimulation showed that, compared with the control group, the number of G0 / G1 phase cells significantly increased and the number of S phase cells significantly decreased with increasing drug concentration. The trends in HeLa cell analysis were consistent with those of C33A cell analysis. Figure 4 This indicates that GDA has a certain cell cycle arrest effect on cervical cancer cells C33A and HeLa. Compared with the control group, cell cycle arrest occurred at the G0 / G1 phase in a concentration-dependent manner.

[0048] 2.5 GDA promotes apoptosis in cervical cancer cells Cervical cancer cells were treated with different concentrations of GDA. After 48 hours, the collected cells were analyzed by Annexin V-FITC / PI double staining to detect the proportion of apoptotic cells. The results of flow cytometry are as follows: Figure 5 As shown, in C33A cells, the apoptotic cell ratios in each group with increasing GDA concentration were 24.35%, 48.67%, 54.13%, and 62.43%, respectively. Compared with the control group, the proportion of total apoptotic cells increased with increasing drug concentration, and the proportion of late-apoptotic cells also increased significantly in each concentration group. After treating HeLa cells with the same drug concentration gradient for 48 hours (… Figure 6The total apoptotic cell ratios were 10.03%, 20.57%, 30.06%, and 52.57%, respectively. Compared with the control group, the total apoptotic cell ratio and the proportion of late-apoptotic cells increased significantly, and the trend was consistent with the trend of apoptosis in C33A cells. The results indicate that GDA can promote apoptosis in cervical cancer cells C33A and HeLa in a concentration-dependent manner, with a more significant effect on late-apoptosis in cervical cancer cells.

[0049] 2.6 Effects of GDA on the gene levels of proteins related to the PI3K / Akt signaling pathway To investigate the molecular mechanism of GDA-induced apoptosis in cervical cancer cells, the changes in the mRNA levels of key proteins in the PI3K / Akt signaling pathway were explored. RT-PCR results in C33A cells showed that the expression levels of PI3K and Akt proteins at 24h and 48h were not significantly different from the control group in each treatment group. The downstream protein Bad showed significantly increased expression levels in the medium and high concentration groups, but no significant difference in the low concentration group compared to the control group. The expression level of Bcl-2 protein at the mRNA level was significantly decreased compared to the control group. RT-PCR results in HeLa cells showed that the expression levels of PI3K and Akt proteins at different treatment concentrations and time points were not significantly different among the treatment groups. The expression level of the downstream protein Bad at the mRNA level was significantly increased in all groups except the low concentration group compared to the control group. The expression level of Bcl-2 protein at the mRNA level was significantly decreased in all treatment groups compared to the control group. Figure 7 ).

[0050] 2.7 Effects of GDA on the levels of proteins related to the PI3K / Akt signaling pathway Western blot results showed that after 24 hours of treatment with different GDA concentrations, compared with the control group, the expression levels of PI3K and Akt proteins in each concentration group did not change significantly; p-Akt protein expression was significantly decreased; BDA protein expression increased significantly with increasing concentration; and Bcl-2 protein expression decreased. After 48 hours of stimulation with different concentrations, the expression levels of PI3K and Akt proteins in two cervical cancer cell lines also did not change significantly compared with the control group; p-Akt and Bcl-2 protein expression was significantly decreased; and BDA protein expression was significantly increased, consistent with the results after 24 hours of treatment. Figure 8 In summary, GDA can regulate the downstream proteins Bad and Bcl-2 of the PI3K / Akt signaling pathway at the gene level without affecting the expression levels of PI3K and Akt proteins. The results of RT-PCR and Western Blot are consistent. However, the expression level of p-Akt protein decreases significantly with increasing drug concentration, indicating that GDA can reduce the phosphorylation of Akt protein.

[0051] 2.8 Akt protein phosphorylation agonist SC 79 can reverse GDA-induced apoptosis. Flow cytometry was used to investigate changes in apoptosis in two cervical cancer cell lines after the introduction of SC 79. Flow cytometry apoptosis detection results for C33A cells showed that, 48 h after drug administration, the total apoptosis rates in the control group, SC 79 sample group, GDA group, and combined drug administration group were 8.27%, 7.49%, 32.83%, and 17.81%, respectively. Compared with the control group, the total apoptotic cell rate significantly increased in the other two groups, except for the SC 79 treatment group, which showed no significant difference. Compared with the GDA-only group, the total apoptotic cell rate significantly decreased in the combined drug administration group. Similar to the total apoptotic cell rate, the late apoptosis cell rate in the combined drug administration group was significantly lower than that in the GDA-only group. 48 h after HeLa cell administration, the total apoptosis rates in the control group, SC 79 sample group, GDA group, and combined drug administration group were 7.52%, 7.27%, 27.60%, and 13.30%, respectively. The results for the total apoptotic cell rate and the late apoptosis cell rate were consistent with those of C33A cells. Figure 9 ).

[0052] 2.9 Changes in p-Akt protein expression levels after combination of SC79 and GDA Western blot analysis was used to detect changes in the expression levels of C33A and HeLa PI3K / Akt signaling pathway-related proteins in cervical cancer cells after the introduction of SC 79. Figure 10 The results showed that, compared with the control group, there were no significant changes in Akt protein expression in any of the C33A cell treatment groups at 24h and 48h. In the SC 79 monotherapy group, p-Akt expression was increased, while in the other two groups, p-Akt expression was significantly decreased. In the SC 79 monotherapy group, Bad protein expression showed no significant difference, while in the other two groups, it was significantly increased. In the SC 79 monotherapy group, Bcl-2 protein expression showed no significant change. In the GDA monotherapy group and the combined treatment group, Bcl-2 protein expression was significantly decreased compared with the control group. Furthermore, compared with the GDA monotherapy group, there was no significant difference in Akt protein expression in the combined treatment group; p-Akt protein expression was increased; Bad protein expression was downregulated; and Bcl-2 protein expression was upregulated. The HeLa cell Western Blot results were consistent with the C33A cell Western Blot results, with no significant changes in Akt protein expression in all samples compared with the control group. Compared with the GDA treatment group, the 24h and 48h combined drug administration groups showed increased p-Akt protein expression, decreased Bad protein expression, and increased Bcl-2 protein expression.

[0053] Results from two cervical cancer cell lines showed that SC 79 promoted phosphorylation of Akt protein in cervical cancer cells C33A and HeLa, thereby affecting downstream proteins and blocking apoptosis. GDA significantly downregulated p-Akt in both cervical cancer cell lines, thereby regulating downstream proteins Bad and Bcl-2 and inducing apoptosis. SC 79 reversed the GDA-induced downregulation of p-Akt protein.

[0054] In summary, SC 79, as an agonist of Akt protein phosphorylation, can reverse the downregulation of p-Akt caused by GDA, thereby reversing the GDA-induced decrease in cell growth status and GDA-induced apoptosis in cervical cancer cells C33A and HeLa. The effects of SC 79 and GDA in the two cervical cancer cell lines are antagonistic.

[0055] 2.10 The combined action of Akt protein phosphorylation inhibitor MK-2206 and GDA induces apoptosis in cervical cancer cells. Flow cytometry was used to detect changes in apoptosis in two cervical cancer cell lines after the introduction of MK-2206. The results of C33A cell flow cytometry apoptosis detection showed that the total apoptosis rates in the control group, MK-2206 sample group, GDA group, and combined treatment group were 9.27%, 13.13%, 25.23%, and 32.83%, respectively. Compared with the control group, the total apoptosis rate in all sample groups was significantly increased. The combined GDA and MK-2206 treatment group showed a significantly higher total apoptosis rate compared to the GDA-only group, with a particularly significant increase in late-apoptotic cells; compared to the MK-2206-only group, the total apoptosis rate was significantly higher, and the number of late-apoptotic cells was significantly increased. Flow cytometry analysis of HeLa cells showed that the total apoptosis rates in the control group, MK-2206 sample group, GDA group, and combined treatment group were 7.52%, 19.20%, 27.60%, and 44.14%, respectively. The HeLa cell apoptosis results were consistent with those of C33A cells, and the total apoptosis rate in each group was significantly higher than that in the control group. The combined GDA and MK-2206 treatment group had the highest total apoptosis rate, significantly higher than both the GDA and MK-2206 groups, with a significantly increased proportion of late-apoptotic cells. In conclusion, the combined use of MK-2206 and GDA is more effective in promoting apoptosis in cervical cancer cells than either drug alone, and the combined administration significantly increased the proportion of late-apoptotic cells in both cervical cancer cell lines. Figure 11 ).

[0056] 2.11 Changes in p-Akt protein expression levels after combination therapy with MK-2206 and GDA Experimental results ( Figure 12The results showed that the expression of Akt protein in C33A cells was not significantly different from the control group after the combination of Akt protein phosphorylation inhibitor MK-2206 and GDA. Compared with the control group, the expression level of p-Akt was significantly reduced in the GDA-only group, while no p-Akt protein was expressed in the MK-2206-only group and the combination group. Compared with the control group, the expression level of Bad protein was increased and the expression level of Bcl-2 protein was decreased in all groups. Compared with the GDA-only group and the MK-2206-only group, the expression level of Bad protein was significantly upregulated and the expression level of Bcl-2 protein was significantly downregulated in the combination group. The Western blot results of HeLa cells were consistent with the Western blot results of C33A cells, and the expression level of Akt protein in each treatment group was not significantly different from the control group. No p-Akt protein was expressed in the combination group and the MK-2206-only group. Compared with the control group, the expression level of p-Akt was significantly reduced in the GDA-only stimulation group. The expression levels of Bad protein in all groups were increased compared to the control group, and the expression levels in the combined drug treatment group were upregulated compared to the GDA-only group and the MK-2206-only group. The expression levels of Bcl-2 protein in the combined drug treatment group were downregulated compared to the GDA-only group and the MK-2206-only group.

[0057] Results from two cervical cancer cell lines showed that MK-2206 inhibited the phosphorylation of Akt protein in cervical cancer cells C33A and HeLa, thereby affecting downstream proteins and promoting apoptosis. GDA and MK-2206 synergistically inhibited p-Akt expression, thereby regulating downstream proteins and inducing apoptosis. The combined use of MK-2206 and GDA resulted in a more significant apoptosis-inducing effect.

[0058] 3. Conclusion GDA can promote apoptosis in cervical cancer cell lines C33A and HeLa, and arrest the cell cycle at the G0 / G1 phase. It can also inhibit the migration, proliferation, and colony formation of cervical cancer cells. Furthermore, this study verified that GDA does indeed induce apoptosis in cervical cancer cells by reducing p-Akt expression using the Akt phosphorylation agonist SC79 and an Akt phosphorylation inhibitor. Although the upstream targets of GDA inducing apoptosis in cervical cancer cells still need further investigation (Figure 13), the combined use of GDA and an Akt phosphorylation inhibitor showed better efficacy and has the potential to become a new treatment for cervical cancer.

[0059] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of a Ganoderma lucidum triterpenoid in the preparation of a drug for treating cervical cancer.

2. The application according to claim 1, characterized in that, The structural formula of the Ganoderma triterpenoid is as follows: 。 3. The application according to claim 1, characterized in that, The aforementioned Ganoderma lucidum triterpenoid treats cervical cancer by inhibiting the migration, proliferation, and colony formation of cervical cancer cells.

4. The application according to claim 1, characterized in that, The aforementioned Ganoderma lucidum triterpenoid treats cervical cancer by arresting the cell cycle of cervical cancer cells in the G0 / G1 phase.

5. The application according to claim 1, characterized in that, The aforementioned Ganoderma lucidum triterpenoid treats cervical cancer by promoting apoptosis of cervical cancer cells.

6. The application according to claim 5, characterized in that, The aforementioned Ganoderma lucidum triterpenoid promotes apoptosis in cervical cancer cells by reducing Akt phosphonation levels.

7. The application according to any one of claims 4 to 6, characterized in that, The cervical cancer cells include cervical cancer cell lines C33A and HeLa.

8. Application of a Ganoderma lucidum triterpenoid combined with an Akt phosphorylation inhibitor in the preparation of a drug for treating cervical cancer.

9. The application according to claim 8, characterized in that, The Akt phosphorylation inhibitor includes MK-2206 at an effective concentration of 5 μM. The effective concentration of the aforementioned Ganoderma lucidum triterpenoid is 20 μM.

10. A drug for treating cervical cancer, characterized in that, It includes a Ganoderma triterpenoid and an Akt phosphorylation inhibitor.

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