Abstract

Objective To investigate the mechanism of Cistanche phenylethanol glycosides (CPhGs) on innate immune function, tumor growth, and tumor cell apoptosis in nude mice bearing drug-resistant osteosarcoma. Methods Human osteosarcoma MG-63 drug-resistant cell line was established by gradient concentration method, and a nude mouse model of drug-resistant osteosarcoma was constructed (36 mice in total, 6 per group). The model group was given normal saline by gavage; the methotrexate (MTX) group received intraperitoneal injection of MTX (20 mg/kg) every three days; the combination group received intraperitoneal injection of MTX every three days combined with daily gavage of medium-dose CPhGs (250 mg/kg); the high-, medium-, and low-dose CPhGs groups were given daily gavage of CPhGs at high (500 mg/kg), medium (250 mg/kg), and low (125 mg/kg) doses, respectively. Tumor size changes in nude mice were observed, curves of mean tumor volume over time were plotted, and tumor inhibition rate was calculated; serum levels of IL-2 and TNF-α in nude mice were detected by ELISA; tumor cell apoptosis was measured by flow cytometry; Western blot was used to detect the expression of apoptosis-related proteins Bcl-2, Bax and the expression of p-AKT, p-mTOR, p-PI3K in tumor tissues. Results Compared with the model group, tumor volume and weight in all treatment groups were significantly reduced (P < 0.001); compared with the model group and MTX group, serum levels of IL-2 and TNF-α in nude mice of all CPhGs groups and the combination group were significantly increased (P < 0.01); compared with the model group, Bax expression in all treatment groups was significantly up-regulated (P < 0.001), and Bcl-2 expression in high- and medium-dose CPhGs groups and the combination group was significantly down-regulated (P < 0.001); compared with the model group and MTX group, tumor apoptosis rate in nude mice of all CPhGs groups and the combination group was significantly increased, while the levels of p-AKT, p-mTOR, and p-PI3K were significantly decreased (P < 0.05). Conclusion CPhGs can significantly improve immune function in nude mice bearing drug-resistant osteosarcoma, and promote tumor cell apoptosis by regulating the PI3K/AKT/mTOR signaling pathway and the expression of Bax and Bcl-2 apoptotic proteins, thereby effectively inhibiting tumor growth.

Key words osteosarcoma; Cistanche phenylethanol glycosides; MG-63; tumor cells; apoptosis; mechanism of action; PI3K/AKT/mTOR signaling pathway

 

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Osteosarcoma (OS) is a highly malignant primary bone tumor, with the highest incidence among adolescent primary malignant bone tumors, accompanied by extremely high disability and mortality rates. At present, high-dose methotrexate (MTX) alone or combined with other chemotherapeutic agents after surgery can effectively improve the survival rate of OS patients [1]. However, some OS patients are insensitive to chemotherapy due to drug resistance, leading to tumor progression. In addition, T lymphocyte subsets in tumor patients are often in a state of immune imbalance, and chemotherapy may further aggravate immune disorders and immunosuppression in the short term [2]. Studies have shown that enhancing human immune responses may improve the prognosis of OS patients from the perspective of biological characteristics of OS [3]. Chemotherapy resistance is one of the most common and intractable problems leading to treatment failure in OS patients, and immunosuppression is also one of the key factors causing rapid tumor progression [4].

The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway is considered one of the most important oncogenic pathways in human cancers. This pathway is often overactivated in OS, which can drive OS cell proliferation, invasion, metastasis and angiogenesis, inhibit apoptosis, and further lead to OS resistance to chemotherapeutic drugs [5]. Studies have confirmed that Cistanche phenylethanol glycosides (CPhGs), the main component of Cistanche deserticola, can enhance immune function and inhibit tumor growth in hepatoma-bearing mice [6]. In vitro studies have shown that CPhGs can significantly inhibit the proliferation of HepG2 hepatoma cells and induce their apoptosis [7]. Inducing cell apoptosis is the main mechanism of current antitumor drugs and also a research hotspot in OS treatment in recent years. Nude mice share many similarities with humans in anatomy, physiology and biology, and have high acceptability for transplanted tumors, allowing xenotransplantation of certain heterologous tumor cells, thus being widely used in establishing tumor models [8-9]. This study intends to construct a nude mouse model of drug-resistant osteosarcoma, observe the effects of CPhGs on immune function and the PI3K/AKT/mTOR pathway in this model, as well as its inhibitory effect on tumor growth and pharmacological mechanism, so as to provide references for broadening clinical treatment ideas and basic research of OS.

 

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1 Materials and Methods

1.1 Experimental Animals

Thirty-six SPF-grade nude mice (6–8 weeks old, weighing 19–25 g) were purchased from Changzhou Cavens Laboratory Animal Co., Ltd., with animal certificate No.202367524. This experiment was approved by the Laboratory Animal Ethics Committee of Northwest Minzu University (Xbmu-3m-202306).

1.2 Cell Line

MG-63 cells were purchased from the Cell Bank of the Chinese Academy of Sciences in Shanghai.

1.3 Drugs and Reagents

Fetal bovine serum, high-glucose DMEM medium, 0.25% Trypsin-EDTA, MTX (Dalian Meilun Biotechnology Co., Ltd., batch numbers PWL001, MA0212, MA0233-1, MB1156-1, respectively); CPhGs (Shaanxi Senyuan Biotechnology Co., Ltd., batch number SY20231212605); anti-p-mTOR antibody, anti-p-PI3K antibody, anti-p-AKT antibody, anti-Bcl-2 antibody, anti-Bax antibody (Hangzhou Huaan Biotechnology Co., Ltd., batch numbers HA600094, HA721673, ET1607-73, ET1702-53, ET1603-34, respectively); ELISA kits for TNF-α and IL-2 (Hangzhou Lianke Biotechnology Co., Ltd., batch numbers EK282, EK202, respectively).

1.4 Instruments

CT14RD desktop high-speed refrigerated centrifuge (Shanghai Tianmei Biochemical Instrument Co., Ltd.); SW-CJ-1FD clean bench (Suzhou Purification Co., Ltd.); CO-5AC cell incubator (Sanyo, Japan); AE31 fluorescence inverted microscope (Xiamen Motic Industrial Group Co., Ltd.); RT-6100 microplate reader (Shenzhen Rayto Life Sciences Co., Ltd.); PowerPoc gel electrophoresis system, ChemiDoc™ gel imaging analyzer (Bio-Rad, USA).

 

1.5 Experimental Methods

1.5.1 Establishment of human osteosarcoma MG-63 drug-resistant cell line (MG-63/MTX)

MG-63 cells were cultured to logarithmic growth phase with 80% cell confluence. Pre-prepared cell culture medium containing 100 ng/mL MTX was added, and the medium was changed after 48 h. When cells reached 80% confluence again, the same concentration of MTX-containing medium was added. The above steps were repeated to enable MG-63 osteosarcoma cells to grow stably in medium containing 100 ng/mL MTX. Then the concentration of MTX in the medium was gradually increased by the same method, using 200 ng/mL, 500 ng/mL, 1000 ng/mL, 2000 ng/mL, 5000 ng/mL and 10000 ng/mL in sequence. After 5 months of culture, osteosarcoma drug-resistant cell line (MG-63/MTX) stably growing in 10000 ng/mL MTX was selected for subsequent experiments.

 

 

1.5.2 Establishment of nude mouse model bearing drug-resistant osteosarcoma

The osteosarcoma animal model was established referring to literature [10]. MG-63/MTX cells were prepared into a cell suspension of 4×10⁶/100 μL, and 100 μL was injected subcutaneously into the axilla of nude mice to observe tumor formation.

1.5.3 Animal grouping and intervention

Tumor-bearing nude mice were randomly divided into model group, MTX group, MTX combined with CPhGs (MTX-CPhGs) group, high-dose CPhGs (CPhGs-H) group, medium-dose CPhGs (CPhGs-M) group and low-dose CPhGs (CPhGs-L) group, with 6 mice in each group. Gavage was started on day 6 after modeling. Mice in the model group were given 10 μL/g normal saline daily by gavage; the MTX group received intraperitoneal injection of 20 mg/kg MTX every three days; the MTX-CPhGs group received daily gavage of 250 mg/kg CPhGs combined with intraperitoneal injection of 20 mg/kg MTX every three days; the CPhGs-H group received daily gavage of 500 mg/kg CPhGs; the CPhGs-M group received daily gavage of 250 mg/kg CPhGs; the CPhGs-L group received daily gavage of 125 mg/kg CPhGs. All nude mice were treated continuously for 15 days.

1.5.4 Measurement of tumor volume and weight in nude mice bearing drug-resistant osteosarcoma

Tumor volume of nude mice in each group was observed and recorded, and tumor growth curves were plotted. Twenty-four hours after the last administration, mice were sacrificed by cervical dislocation, tumors were dissected and weighed, and tumor inhibition rate was calculated. The formulas are as follows:Tumor volume = 1/2 (long tumor diameter × short tumor diameter²)Tumor inhibition rate = [(mean tumor weight of model group − mean tumor weight of treatment group)/mean tumor weight of model group] × 100%

1.5.5 Detection of serum IL-2 and TNF-α levels by ELISA

Serum was extracted from nude mice in each group, and serum levels of IL-2 and TNF-α were detected by ELISA according to the manufacturer's instructions.

1.5.6 Detection of tumor cell apoptosis by flow cytometry

Tumor tissues from each group were minced, ground, filtered, centrifuged, and red blood cells were lysed to prepare single-cell suspensions in centrifuge tubes. Cell concentration was adjusted to 1×10⁶/mL, cells were resuspended in 100 μL Binding Buffer, added with 5 μL AV-FITC and PI, incubated in the dark for 15 min, centrifuged, resuspended, and added with 400 μL Binding Buffer. After mixing, samples were detected by flow cytometry.

1.5.7 Detection of p-AKT, p-mTOR, p-PI3K and apoptotic proteins Bcl-2, Bax by Western blot

Tumor tissues from each group were minced and placed in a homogenizer, lysed with protein lysis buffer and homogenized. After lysis, supernatant was collected by centrifugation at 12000 r/min. Electrophoretic gel was prepared for protein separation, followed by membrane transfer and blocking. Membranes were incubated with primary antibodies at 4°C overnight, washed, and incubated with secondary antibodies at room temperature for 1 h. After washing with TBST buffer on a shaker, ECL luminescent solution was added for development. After fixation, gel image analysis was performed, and protein band gray values were analyzed using Image J software.

1.6 Statistical Analysis

Statistical analysis was performed using GraphPad Prism 7 and SPSS 25.0 software. Measurement data were expressed as mean ± standard deviation (x ± s). One-way ANOVA was used for comparison between groups with normal distribution and homogeneous variance, and nonparametric test was used for non-normal distribution data. Paired t-test was used to analyze tumor inhibition rate. P < 0.05 was considered statistically significant.

 

 

Figure 1. Morphological comparison of MG-63 and MG-63/MTX cells (×200)

2 Results

2.1 Osteosarcoma MG-63 drug-resistant cell line (MG-63/MTX)

Original MG-63 cells grew adherently with medium size, mostly spindle-shaped, occasionally irregular, with large nuclei and clear nucleoli (Figure 1-A). MG-63/MTX cells were elongated, spindle or irregular, with obvious pseudopodia (Figure 1-B).

2.2 Comparison of tumor growth in mice of each group

From day 6 of gavage, tumor volume in treatment groups was significantly smaller than that in the model group. On day 21 of gavage, tumors from 3 nude mice in each group were compared (Figure 2-A). Tumor volume in all treatment groups was smaller than that in the model group (P < 0.001). Tumor volume in CPhGs-H, CPhGs-M, CPhGs-L and MTX-CPhGs groups was smaller than that in the MTX group (P < 0.001). Among CPhGs groups, tumor volume gradually decreased with increasing drug concentration, and the difference from the model group became more significant (Figure 2-B). The results indicated that CPhGs could significantly inhibit tumor growth in drug-resistant tumor-bearing mice, and the inhibitory effect was positively correlated with CPhGs concentration.

Compared with the model group, the mean tumor weight in MTX, CPhGs-H, CPhGs-M, CPhGs-L and MTX-CPhGs groups was significantly decreased (P < 0.001). Compared with the MTX group, the tumor inhibition rate in CPhGs-H, CPhGs-M, CPhGs-L and MTX-CPhGs groups was significantly increased, and in CPhGs-H, CPhGs-M and CPhGs-L groups, the tumor inhibition rate increased in a dose-dependent manner with increasing drug concentration (Table 1).

2.3 Effects of CPhGs on serum IL-2 and TNF-α levels in drug-resistant tumor-bearing nude mice

Compared with the model group, serum TNF-α level in the MTX group was significantly increased (P < 0.05), and serum TNF-α and IL-2 levels in the MTX-CPhGs group were significantly increased (P < 0.01). In addition, serum TNF-α levels in CPhGs-H, CPhGs-M and CPhGs-L groups were significantly higher than those in the model group (P < 0.001), serum IL-2 level in the CPhGs-M group was increased (P < 0.01), and serum IL-2 levels in CPhGs-H and CPhGs-L groups were increased more significantly (P < 0.001). Compared with the MTX group, serum TNF-α and IL-2 levels in CPhGs-H, CPhGs-M, CPhGs-L and MTX-CPhGs groups were significantly increased (P < 0.001) (Table 2). This suggests that CPhGs may improve innate immune function in drug-resistant OS tumor-bearing mice by regulating the expression of TNF-α and IL-2.

 

Figure 2. Comparison of tumor volumes in nude mice with tumors in each group after intervention (x ± s, n=3, mm3)

Note: Model group; MTX group; CPhGs-L group; low-dose Cistanche deserticola phenylethanol total glycosides group; CPhGs-M group; medium-dose Cistanche deserticola phenylethanol total glycosides group; CPhGs-H group; high-dose Cistanche deserticola phenylethanol total glycosides group; MTX-CPhGs group; compared with the Model group, ***P<0.001; compared with the MTX group, ###P<0.001

Table 1. Effect of Cistanche phenylethanoid glycosides on tumor weight in tumor-bearing nude mice (n=3)

Group	Mean tumor weight (g)	Inhibition rate (%)
Model	0.79 ± 0.04	-
MTX	0.49 ± 0.03	35.73 ± 4.61
CPhGs–L	0.33 ± 0.01	58.55 ± 4.02
CPhGs–M	0.31 ± 0.02	60.50 ± 0.98
CPhGs–H	0.28 ± 0.01	64.58 ± 1.77
MTX–CPhGs	0.18 ± 0.01	75.77 ± 1.78

*Note: The "Model" group has no inhibition rate reported in the source table (shown as em dash). If you want, I can also add the significance markers (e.g., *, ##, etc.) as footnotes exactly as in the figure.

2.4 Effects of CPhGs on tumor cell apoptosis in drug-resistant tumor-bearing nude mice

Compared with the model group, tumor apoptosis rate in MTX and CPhGs-L groups was significantly increased (P < 0.05), and that in CPhGs-H, CPhGs-M and MTX-CPhGs groups was increased more significantly (P < 0.001). Compared with the MTX group, tumor apoptosis rate in CPhGs-H, CPhGs-M and MTX-CPhGs groups was increased (P < 0.001) (Figure 3, Table 3). The above results show that CPhGs can promote tumor cell apoptosis in drug-resistant tumor-bearing nude mice.

2.5 Effects of CPhGs on the expression of p-AKT, p-mTOR and p-PI3K in tumor tissues of drug-resistant tumor-bearing nude mice

Compared with the model group, p-AKT expression in tumor tissues of drug-resistant nude mice in the MTX group was significantly decreased (P < 0.001). The expression levels of p-AKT, p-mTOR and p-PI3K in tumor tissues of nude mice in MTX-CPhGs, CPhGs-H and CPhGs-M groups were significantly decreased (P < 0.001), p-AKT expression in the CPhGs-L group was decreased (P < 0.01), and p-mTOR and p-PI3K expression was decreased more significantly (P < 0.001). Compared with the MTX group, the expression levels of p-AKT, p-mTOR and p-PI3K in tumor tissues of nude mice in MTX-CPhGs and CPhGs-H groups were also significantly decreased (P < 0.001), p-AKT expression in the CPhGs-M group was slightly decreased (P < 0.05), p-mTOR and p-PI3K expression was decreased more significantly (P < 0.001), p-AKT expression in the CPhGs-L group was decreased (P < 0.01), and p-mTOR and p-PI3K expression was decreased more significantly (P < 0.001) (Figure 4-A to 4-D).

 

Table 2. Serum TNF-α and IL-2 expression levels in each group (n = 3)

Group	TNF-α (pg/mL)	IL-2 (pg/mL)
Model	79.90 ± 3.60	28.36 ± 2.35
MTX	93.71 ± 2.86*	28.87 ± 1.60
MTX–CPhGs	118.00 ± 3.78***###	40.92 ± 1.54***###
CPhGs–H	122.76 ± 2.97***###	45.03 ± 4.95***###
CPhGs–M	117.52 ± 3.60***###	41.44 ± 3.87***###
CPhGs–L	124.19 ± 8.37***###	44.77 ± 2.31***###

 

Notes (as shown in the figure): Model = model group; MTX = methotrexate group; CPhGs–L/M/H = low/medium/high-dose Cistanche phenylethanoid glycosides group; MTX–CPhGs = combination group. Significance vs. Model: * P<0.05P<0.05P<0.05, ** P<0.01P<0.01P<0.01, *** P<0.001P<0.001P<0.001. Significance vs. MTX: ### P<0.001P<0.001P<0.001.

2.6 Effects of CPhGs on the expression of Bcl-2 and Bax in tumor tissues of drug-resistant tumor-bearing nude mice

Compared with the model group, Bax expression in tumor tissues of drug-resistant nude mice in all treatment groups was significantly increased (P < 0.001), Bcl-2 expression in the MTX group was increased (P < 0.05), and Bcl-2 expression in MTX-CPhGs, CPhGs-H and CPhGs-M groups was significantly decreased (P < 0.001). Compared with the MTX group, Bcl-2 expression in tumor tissues of CPhGs-H, CPhGs-M, CPhGs-L and MTX-CPhGs groups was significantly decreased (P < 0.001), and Bax expression in CPhGs-H and CPhGs-M groups was significantly increased (P < 0.001) (Figure 4-A, 4-E, 4-F). It further indicates that CPhGs can promote tumor cell apoptosis in drug-resistant tumor-bearing nude mice by regulating the expression of Bcl-2 and Bax.

 

 

3 Discussion

Chemotherapy resistance and immunosuppression in OS patients are bottlenecks in clinical treatment of OS. At present, the main mechanism of antitumor drugs is to promote tumor cell apoptosis, which directly reflects the efficacy of chemotherapy; on the other hand, body immunity is a strong guarantee for patients to tolerate chemotherapeutic drugs. Therefore, it is crucial to study how to effectively promote apoptosis of drug-resistant OS cells and reduce chemotherapy-induced immune impairment.

Figure 3. The apoptosis rate of tumor tissue cells in each group of mice (n=3, %)

Note: A. Model group; B. Methotrexate (MTX) group; C. Methotrexate combined with Cistanche deserticola phenylethanol total glycosides (MTX-CPhGs) group; D. High-dose Cistanche deserticola phenylethanol total glycosides (CPhGs-H) group; E. Medium-dose Cistanche deserticola phenylethanol total glycosides (CPhGs-M) group; F. Low-dose Cistanche deserticola phenylethanol total glycosides (CPhGs-L) group.

Table 3. Apoptosis rate of tumor tissue cells in each group (n = 3)

Group	Apoptosis rate (%)
Model	14.47 ± 1.10
MTX	19.77 ± 1.32*
MTX–CPhGs	29.21 ± 0.77***###
CPhGs–H	31.66 ± 1.93***###
CPhGs–M	30.13 ± 2.16***###
CPhGs–L	22.49 ± 3.02**

Notes (as shown in the figure): Model = model group; MTX = methotrexate group; CPhGs–L/M/H = low/medium/high-dose Cistanche phenylethanoid glycosides group; MTX–CPhGs = combination group. Significance vs. Model: * P<0.05P<0.05P<0.05, ** P<0.01P<0.01P<0.01, *** P<0.001P<0.001P<0.001. Significance vs. MTX: ### P<0.001P<0.001P<0.001.

 

 

Cistanche deserticola has pharmacological effects of warming the kidney and replenishing essence, moistening the intestines, anti-oxidation and improving immunity. Zhang et al. [11] reported in 1988 that aqueous extract of Cistanche deserticola could improve immune function and stimulate the development of immune organs such as thymus in mice. Subsequent in vivo studies further found that Cistanche polysaccharides had anti-lung cancer and anti-sarcoma effects [12]. Studies have shown that CPhGs, the main component of Cistanche deserticola, has pharmacological effects such as anti-oxidation, anti-aging, neuroprotection, immune enhancement, liver protection and treatment of spermatogenic disorders [13], and also exerts antitumor and immune-enhancing effects on various malignant tumors including liver cancer, lung cancer, breast cancer and ovarian cancer [14]. Our previous study found that Cistanche deserticola could promote apoptosis of drug-resistant OS cells in vitro by reducing the expression of MRP1 and mutant P53 proteins [15]. This study found that tumor volume and weight in treatment groups were smaller than those in the model group, and tumor volume in different CPhGs concentration groups and MTX-CPhGs group was also smaller than that in the MTX group, with increased tumor inhibition rate. This indicates that CPhGs can significantly inhibit tumor growth in drug-resistant OS tumor-bearing mice, and the inhibitory effect is positively correlated with CPhGs concentration, which is basically consistent with the study by Hou et al. [16] on the inhibitory effect of CPhGs on subcutaneous transplanted tumors in H22 hepatoma-bearing mice.

Immune function is closely related to tumor occurrence and development. IL-2 is a type of cell growth factor in the immune system, which can improve body immune function by promoting T cell proliferation and differentiation, and produce immune cells with antitumor activity [17]. TNF-α exerts antitumor effects by binding to receptors to induce tumor cell apoptosis, enhance host immune function, act on tumor vascular endothelial cells, increase vascular permeability, induce programmed death, and subsequently cause tumor hemorrhage and necrosis [18]. Analysis of serum IL-2 and TNF-α expression in mice of each group showed that the expression levels of serum TNF-α and IL-2 in tumor-bearing model mice of different CPhGs concentration groups and MTX-CPhGs group were significantly increased. This suggests that CPhGs may improve immune function in drug-resistant OS tumor-bearing mice by regulating the expression of TNF-α and IL-2, inhibit the proliferation of drug-resistant osteosarcoma cells, and thus exert antitumor effects.

 

Figure 4. Electrophoresis result diagram of p-mTOR, p-PI3K, p-AKT, Bcl-2, and Bax proteins in tumor tissue of nude mice (x ± s, n=3, mm3)

Note: Model, model group; MTX, methotrexate group; MTX-CPhGs, methotrexate combined with Cistanche deserticola phenylethanol total glycosides group; CPhGs-L, low-dose Cistanche deserticola phenylethanol total glycosides group; CPhGs-M, medium-dose Cistanche deserticola phenylethanol total glycosides group; CPhGs-H, high-dose Cistanche deserticola phenylethanol total glycosides group; A. Western Blot results of p-mTOR, p-PI3K, p-AKT, Bcl-2, and Bax proteins; B. p-mTOR; C. p-PI3K; D. p-AKT; E. Bcl-2; F. Bax; Compared with the model group, * P < 0.05, ** P < 0.01, *** P < 0.001; Compared with the MTX group, # P < 0.05, ## P < 0.01, ### P < 0.001.

 

AKT phosphorylation in the PI3K/AKT/mTOR signaling pathway can directly or indirectly regulate a series of apoptosis-related factors such as Bcl-2 family, X-linked inhibitor of apoptosis protein (XIAP), mouse double minute 2 (MDM-2) and Bcl-2-associated X protein (Bax) [19]. Abnormally activated PI3K/AKT/mTOR signaling pathway can up-regulate the expression of membrane transporters ABCB1 and ABCC1, thereby reducing chemotherapy efficacy and mediating OS resistance to chemotherapeutic drugs [20]. A variety of traditional Chinese medicine monomers can promote OS cell apoptosis by inhibiting the PI3K/AKT/mTOR signaling pathway, such as procyanidin B2, quercetin, luteolin and Lactarius deliciosus polysaccharides [21]. This study found that under the single action of MTX, p-AKT protein expression was significantly decreased, while p-mTOR and p-PI3K protein expression showed no significant difference, indicating that MTX has little effect on the PI3K/AKT/mTOR signaling pathway in tumor tissues of drug-resistant tumor-bearing nude mice. However, the expression of p-AKT, p-mTOR and p-PI3K proteins in tumor tissues of drug-resistant OS tumor-bearing nude mice in different CPhGs concentration groups and MTX-CPhGs group was lower than that in the model group, and also significantly lower than that in the MTX group, revealing that CPhGs has a significant inhibitory effect on the PI3K/AKT/mTOR signaling pathway in drug-resistant OS cells. Flow cytometry detection of tumor cell apoptosis showed that tumor apoptosis rate in each group was significantly increased after drug intervention. Detection of Bax and Bcl-2 protein expression showed that Bax expression in tumor tissues of drug-resistant OS tumor-bearing nude mice in different CPhGs concentration groups and MTX-CPhGs group was significantly increased, while Bcl-2 expression in tumor tissues of model mice in MTX-CPhGs, CPhGs-H and CPhGs-M groups was significantly decreased. This suggests that a certain concentration of CPhGs may promote apoptosis of drug-resistant OS cells in vivo by inhibiting the PI3K/AKT/mTOR signaling pathway, down-regulating Bcl-2 expression and up-regulating Bax expression.

In conclusion, CPhGs can promote apoptosis of drug-resistant OS cells and inhibit tumor growth in vivo, and its mechanism may be achieved by inhibiting the PI3K/AKT/mTOR signaling pathway and regulating the expression of Bcl-2 and Bax. Meanwhile, CPhGs can improve immune function in drug-resistant OS tumor-bearing mice by up-regulating the expression levels of TNF-α and IL-2, thereby exerting synergistic antitumor effects. However, its clinical application potential still needs further exploration and research.

 

References

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