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Red ginseng has been used in traditional Asian medicine for hundreds of years. In this study, we evaluated the ability of four types of red ginseng (Chinese red ginseng, Korean red ginseng A, Korean red ginseng B, and Korean red ginseng C) grown in different regions to inhibit the formation and growth of carcinogen-induced lung tumors. A benzo(a)pyrene (B(a)P) test was conducted on A/J mice, and Korean red ginseng B was found to be the most effective in reducing tumor burden among the four red ginseng varieties. In addition, we analyzed the contents of various ginsenosides (Rg1, Re, Rc, Rb2, Rb3, Rb1, Rh1, Rd, Rg3, Rh2, F1, Rk1 and Rg5) in four red ginseng extracts and found that Korean red ginseng B had the most high levels of ginsenoside Rg3 (G-Rg3), suggesting that G-Rg3 may play an important role in its therapeutic efficacy. This work shows that G-Rg3 has relatively low bioavailability. However, when G-Rg3 was coadministered with the P-gp inhibitor verapamil, the efflux of G-Rg3 into Caco-2 cells was decreased, the rate of intestinal absorption of G-Rg3 was increased in a rat model, and G-Rg3 was increased. In Caco-2 cells, the outflow of Rg3 decreases, and the level of Rg3 concentration decreases. G-Rg3 is increased in the intestine and plasma, and its ability to prevent tumors is also enhanced in a rat model of B(a)P-induced tumorigenesis. We also found that G-Rg3 reduced B(a)P-induced cytotoxicity and DNA adduct formation in human lung cells, and restored the expression and activity of phase II enzymes through the Nrf2 pathway, which may be related to the potential mechanism of action of G inhibition -Rg3. . About the occurrence of lung tumors. Our study demonstrates a potentially important role for G-Rg3 in targeting lung tumors in mouse models. The oral bioavailability of this ginsenoside is enhanced by targeting P-glycoprotein, allowing the molecule to exert anticancer effects.
The most common type of lung cancer is non-small cell lung cancer (NSCLC), which is one of the leading causes of cancer deaths in China and North America1,2. The main factor that increases the risk of developing non-small cell lung cancer is smoking. Cigarette smoke contains more than 60 carcinogens, including benzo(a)pyrene (B(a)P), nitrosamines, and radioactive isotopes from the decay of radon.3 Polycyclic aromatic hydrocarbons B(a)P are the main cause of toxicity in cigarette smoke. Upon exposure to B(a)P, cytochrome P450 converts it to B(a)P-7,8-dihydrodiol-9,10-epoxide (BPDE), which reacts with DNA to form BPDE-DNA adduct 4. Additionally, these adducts induce lung tumorigenesis in mice with tumor stage and histopathology similar to human lung tumors5. This feature makes the B(a)P-induced lung cancer model a suitable system for evaluating compounds with possible anticancer properties.
One possible strategy to prevent the development of lung cancer in high-risk groups, especially smokers, is the use of chemopreventive agents to suppress the development of intraepithelial neoplastic lesions and thereby prevent their subsequent progression to malignancy. Animal studies show that various chemopreventive agents are effective6. Our previous report7 highlighted the good preventive effects of red ginseng on lung cancer. This herb has been used for centuries in traditional Asian medicine to prolong life and health, and has been documented to have antitumor effects8.
The active factor of ginseng is ginsenoside, which is used as a composite marker to evaluate the quality of ginseng extracts. Quantitative analysis of crude ginseng extracts typically involves the use of several ginsenosides, including RK1, Rg1, F1, Re, Rb1, Rb2, Rb3, Rd, Rh1, Rh2, Rg3, Rg5, and Rc9,10. Ginsenosides have little clinical use due to their very poor oral bioavailability11. Although the mechanism for this poor bioavailability is not clear, the efflux of ginsenosides caused by P-glycoprotein (P-gp)12 may be the cause. P-gp is one of the most important efflux transporters in the ATP-binding cassette transporter superfamily, which uses the energy of ATP hydrolysis to release intracellular substances into the external environment. P-gp transporters are typically widely distributed in the intestine, kidney, liver and blood-brain barrier13. P-gp plays a critical role in intestinal absorption, and inhibition of P-gp increases the oral absorption and availability of some anticancer drugs12,14. Examples of inhibitors previously used in the literature are verapamil and cyclosporine A15. This work involves establishing a mouse system for studying B(a)P-induced lung cancer to evaluate the ability of different red ginseng extracts from China and Korea to affect malignancies. The extracts were individually analyzed to identify specific ginsenosides that may affect carcinogenesis. Verapamil was then used to target P-gp and improve the oral bioavailability and therapeutic efficacy of cancer-targeting ginsenosides.
The mechanism by which ginseng saponins exert therapeutic effects on carcinogenesis remains unclear. Research has shown that various ginsenosides can reduce DNA damage caused by carcinogens by reducing oxidative stress and activating phase II detoxification enzymes, thereby preventing cell damage. Glutathione S-transferase (GST) is a typical phase II enzyme that is required to reduce DNA damage caused by carcinogens17. Nuclear erythroid 2-related factor 2 (Nrf2) is an important transcription factor that regulates redox homeostasis and activates the expression of phase II enzymes and cytoprotective antioxidant responses18. Our study also examined the effects of identified ginsenosides on reducing B(a)P-induced cytotoxicity and BPDE-DNA adduct formation, as well as inducing phase II enzymes by modulating the Nrf2 pathway in normal lung cells.
The establishment of a mouse model of B(a)P-induced cancer is consistent with previous work5. Figure 1A shows the experimental design of a 20-week treatment of a mouse cancer model induced by B(a)P, water (control), Chinese red ginseng extract (CRG), Korean red ginseng extract A (KRGA), and Korean red ginseng. Extract B (KRGB) and Korean Red Ginseng Extract C (KRGC). After 20 weeks of red ginseng treatment, mice were sacrificed by CO2 asphyxiation. Figure 1B shows macroscopic lung tumors in animals treated with different types of red ginseng, and Figure 1C shows a representative light micrograph of a tumor sample. The tumor burden of KRGB-treated animals (1.5 ± 0.35) was lower than that of control animals (0.82 ± 0.2, P < 0.05), as shown in Figure 1D. The average degree of tumor load inhibition was 45%. Other red ginseng extracts tested did not show such significant changes in tumor burden (P > 0.05). No obvious side effects were observed in the mouse model during 20 weeks of red ginseng treatment, including no change in body weight (data not shown) and no liver or kidney toxicity (Figure 1E,F).
Red ginseng extract treats lung tumor development in A/J mice. (A) Experimental design. (B) Large lung tumors in a mouse model. Tumors are indicated by arrows. a: Chinese red ginseng group. b: group A of Korean red ginseng. c: Korean red ginseng group B. d: Korean red ginseng group C. d: Control group. (C) Light micrograph showing a lung tumor. Magnification: 100. b: 400. (D) Tumor load in the red ginseng extract group. (E) Plasma levels of the liver enzyme ALT. (F) Plasma levels of the renal enzyme Cr. Data are expressed as mean ± standard deviation. *P <0.05.
The red ginseng extracts identified in this study were analyzed by ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) to quantify the following ginsenosides: Rg1, Re, Rc, Rb2, Rb3, Rb1, Rh1, Rd, Rg3 , Rh2, F1, Rk1 and Rg5. The UPLC and MS conditions used to measure the analytes were described in a previous report19. UPLC-MS/MS chromatograms of four red ginseng extracts are shown in Figure 2A. There were significant differences in total ginsenoside content, with the highest total ginsenoside content in CRG (590.27 ± 41.28 μmol/L) (Figure 2B). When evaluating individual ginsenosides (Figure 2C), KRGB showed the highest level of G-Rg3 compared to other ginsenosides (58.33 ± 3.81 μmol/L for G-Rg3s and 41.56 ± 2.88 μmol/L for G -Rg3r).L). red ginseng type (P < 0.001). G-Rg3 occurs as a pair of stereoisomers G-Rg3r and G-Rg3s, which differ in the position of the hydroxyl group at carbon 20 (Fig. 2D). The results indicate that G-Rg3r or G-Rg3 may have important anticancer potential in a B(a)P-induced cancer mouse model.
Content of ginsenosides in various red ginseng extracts. (A) UPLC-MS/MS chromatograms of four red ginseng extracts. (B) Estimation of total ginsenoside content in the indicated extracts. (C) Detection of individual ginsenosides in labeled extracts. (D) Structures of ginsenoside stereoisomers G-Rg3r and G-Rg3s. Data are expressed as the mean ± standard deviation of triplicate determinations. ***P <0.001.
The UPLC-MS/MS study required the quantification of ginsenosides in intestinal and blood samples after 20 weeks of treatment. Treatment with KRGB showed the presence of only 0.0063 ± 0.0005 μg/ml Rg5 in the blood. No remaining ginsenosides were detected, indicating poor oral bioavailability and therefore reduced exposure to these ginsenosides.
The colon adenocarcinoma cell line Caco-2 is morphologically and biochemically similar to human intestinal epithelial cells, demonstrating its utility in assessing enterocyte transport across the intestinal epithelial barrier. This analysis was based on an earlier study 20 . Figures 3A,B,C,D,E,F show representative images of transcellular transport of G-Rg3r and G-Rg3 using a Caco-2 monolayer model. Transcellular transport of G-Rg3r or G-Rg3 across Caco-2 monolayers from the basolateral to apical side (Pb-a) was significantly higher than from the apical to basolateral side (Pa-b). For G-Rg3r, the mean Pa-b value was 0.38 ± 0.06, which increased to 0.73 ± 0.06 after treatment with 50 μmol/L verapamil and to 1.14 ± 0.09 after treatment with 100 μmol/L verapamil (p < 0.01 and 0.001, respectively; Figure 2). 3A). Observations for G-Rg3 followed a similar pattern (Fig. 3B), and the results showed that verapamil treatment enhanced the transport of G-Rg3r and G-Rg3. Verapamil treatment also resulted in a significant decrease in mean Pb-a and G-Rg3r and G-Rg3s efflux ratios (Figure 3C,D,E,F), indicating that verapamil treatment reduces ginsenoside content in Caco-2 efflux cells. .
Transcellular transport of G-Rg3 in Caco-2 monolayers and intestinal absorption in a rat perfusion assay. (A) Pa-b value of G-Rg3r group in Caco-2 monolayer. (B) Pa-b value of G-Rg3s groups in Caco-2 monolayer. (C) Pb value of G-Rg3r group in Caco-2 monolayer. (D) Pb value of G-Rg3s groups in Caco-2 monolayer. (E) Yield ratio of G-Rg3r groups in a Caco-2 monolayer. (F) Yield ratio of G-Rg3 groups in a Caco-2 monolayer. (G) Percentage of intestinal absorption of G-Rg3r in a perfusion assay in rats. (H) Percentage of intestinal absorption of G-Rg3 in a perfusion assay in rats. Permeability and absorption were compared without the addition of verapamil. Data are expressed as the mean ± standard deviation of five independent experiments. *P <0.05, **P <0.01, ***P <0.001.
Consistent with earlier work20, orthotopic intestinal perfusion of rats was performed to determine whether G-Rg3 absorption in the intestine increases after verapamil treatment. Figures 3G,H show representative perfusion assays to evaluate the percentage of intestinal absorption of G-Rg3r and G-Rg3 in cancer model rats during the above time periods. The initial percentage of weak G-Rg3r uptake of approximately 10% increased to more than 20% after treatment with 50 μM verapamil and to more than 25% after treatment with 100 μM verapamil. Likewise, G-Rg3, which had an initial uptake of 10%, also showed a peak of over 20% after treatment with 50 μM verapamil and nearly 30% after treatment with 100 μM verapamil, suggesting that inhibition of P-gp by verapamil enhances intestinal G-absorption Rg3 in a mouse model of lung cancer.
According to the above method, B(a)P-induced cancer model mice were randomly divided into six groups, as shown in Figure 4A. No significant weight loss or clinical signs of toxicity were observed in the G-Rg3 treatment group compared to the control group (data not shown). After 20 weeks of treatment, the lungs of each mouse were collected. Figure 4B shows macroscopic lung tumors in mice in the above treatment groups, and Figure 4C shows a representative light micrograph of a representative tumor. Regarding the tumor burden in each group (Fig. 4D), the values for mice treated with G-Rg3r and G-Rg3s were 0.75 ± 0.29 mm3 and 0.81 ± 0.30 mm3, respectively, while the values for G Mice treated with -Rg3s were 1.63 respectively ±0.40 mm3. control mice (p < 0.001), indicating that G-Rg3 treatment reduced tumor burden in mice. Administration of verapamil further enhanced this reduction: values in verapamil+ G-Rg3r mice decreased from 0.75 ± 0.29 mm3 to 0.33 ± 0.25 mm3 (p < 0.01), and values for verapamil+ from 0.81 ± 0.30 mm3 decreased to 0.29 ± 0.21 mm3 in G. -Rg3s-treated mice (p < 0.05), indicating that verapamil may enhance the inhibitory effect of G-Rg3 on tumorigenesis. Tumor burden showed no significant differences between the control group and the verapamil group, the G-Rg3r group and the G-Rg3s group, and the verapamil+G-Rg3r group and the verapamil+G-Rg3s group. Moreover, there were no significant liver or kidney toxicities associated with the evaluated treatments (Figure 4E,F).
Tumor burden after G-Rg3 treatment and plasma or intestinal G-Rg3r and G-Rg3 levels in the indicated groups. (A) Experimental design. (B) Macroscopic tumors in a mouse model. Tumors are indicated by arrows. a: G-Rg3r. b: G-Rg3s. c: G-Rg3r in combination with verapamil. d: G-Rg3 in combination with verapamil. d: Verapamil. e: control. (C) Optical micrograph of the tumor at magnification. Answer: 100x. b: 400X. (D) Effect of G-Rg3 + verapamil treatment on tumor burden in A/J mice. (E) Plasma levels of the liver enzyme ALT. (F) Plasma levels of the renal enzyme Cr. (G) Plasma levels of G-Rg3r or G-Rg3 of the indicated groups. (H) Levels of G-Rg3r or G-Rg3s in the intestines of the indicated groups. Data are expressed as the mean ± standard deviation of triplicate determinations. *P <0.05, **P <0.01, ***P <0.001.
G-Rg3 levels in the B(a)P-induced cancer model mice were assessed by UPLC-MS/MS after a 20-week treatment period according to the method described in the Methods section. Figures 4G and H show plasma and intestinal G-Rg3 levels, respectively. Plasma G-Rg3r levels were 0.44 ± 0.32 μmol/L and increased to 1.17 ± 0.47 μmol/L with concomitant administration of verapamil (p < 0.001), while intestinal G-Rg3r levels were 0.53 ± 0.08 µg/l. When combined with verapamil, g increased to 1.35 ± 0.13 μg/g (p < 0.001). For G-Rg3, the results followed a similar pattern, indicating that verapamil treatment increased the oral bioavailability of G-Rg3 in A/J mice.
Cell viability assay was used to evaluate the cytotoxicity of B(a)P and G-Rg3 on hEL cells. The cytotoxicity induced by B(a)P in hEL cells is shown in Figure 5A, while the nontoxic properties of G-Rg3r and G-Rg3 are shown in Figures 5A and 5B. 5B, C. To evaluate the cytoprotective effect of G-Rg3, B(a)P was co-administered with various concentrations of G-Rg3r or G-Rg3 into hEL cells. As shown in Figure 5D, G-Rg3r at concentrations of 5 μM, 10 μM, and 20 μM restored cell viability to 58.3%, 79.3%, and 77.3%, respectively. Similar results can also be seen in the G-Rg3s group. When the concentrations of G-Rg3s were 5 µM, 10 µM and 20 µM, cell viability was restored to 58.3%, 72.7% and 76.7%, respectively (Figure 5E) . ). The presence of BPDE-DNA adducts was measured using an ELISA kit. Our results showed that BPDE-DNA adduct levels were increased in the B(a)P-treated group compared with the control group, but compared with G-Rg3 co-treatment, BPDE-DNA adduct levels in the B(a)P group B in the treated group, DNA adduct levels were significantly reduced. The results of treatment with B(a)P alone are shown in Figure 5F (1.87 ± 0.33 vs. 3.77 ± 0.42 for G-Rg3r, 1.93 ± 0.48 vs. 3.77 ± 0.42 for G -Rg3s, p < 0.001).
Cell viability and BPDE-DNA adduct formation in hEL cells treated with G-Rg3 and B(a)P. (A) Viability of hEL cells treated with B(a)P. (B) Viability of hEL cells treated with G-Rg3r. (C) Viability of hEL cells treated with G-Rg3. (D) Viability of hEL cells treated with B(a)P and G-Rg3r. (E) Viability of hEL cells treated with B(a)P and G-Rg3. (F) Levels of BPDE-DNA adduct in hEL cells treated with B(a)P and G-Rg3. Data are expressed as the mean ± standard deviation of triplicate determinations. *P <0.05, **P <0.01, ***P <0.001.
GST enzyme expression was detected after co-treatment with 10 μM B(a)P and 10 μM G-Rg3r or G-Rg3s. Our results showed that B(a)P suppressed GST expression (59.7 ± 8.2% in the G-Rg3r group and 39 ± 4.5% in the G-Rg3s group), and B(a)P was associated with either with G-Rg3r, or with G-Rg3r, or with G-Rg3r. Co-treatment with G-Rg3s restored GST expression. GST expression (103.7 ± 15.5% in the G-Rg3r group and 110 ± 11.1% in the G-Rg3s group, p < 0.05 and p < 0.001, respectively, Fig. 6A, B, and C). GST activity was assessed using an activity assay kit. Our results showed that the combination treatment group had higher GST activity compared to the B(a)P only group (96.3 ± 6.6% vs. 35.7 ± 7.8% in the G-Rg3r group vs. 92.3 ± 6.5 in the G-Rg3r group). % vs 35.7 ± 7.8% in the G-Rg3s group, p < 0.001, Figure 6D).
Expression of GST and Nrf2 in hEL cells treated with B(a)P and G-Rg3. (A) Detection of GST expression by Western blotting. (B) Quantitative expression of GST in hEL cells treated with B(a)P and G-Rg3r. (C) Quantitative expression of GST in hEL cells treated with B(a)P and G-Rg3s. (D) GST activity in hEL cells treated with B(a)P and G-Rg3. (E) Detection of Nrf2 expression by Western blotting. (F) Quantitative expression of Nrf2 in hEL cells treated with B(a)P and G-Rg3r. (G) Quantitative expression of Nrf2 in hEL cells treated with B(a)P and G-Rg3s. Data are expressed as the mean ± standard deviation of triplicate determinations. *P <0.05, **P <0.01, ***P <0.001.
To elucidate the pathways involved in G-Rg3-mediated suppression of B(a)P-induced tumorigenesis, Nrf2 expression was assessed by Western blotting. As shown in Figures 6E,F,G, compared with the control group, only the level of Nrf2 in the B(a)P treatment group was decreased; however, compared with the B(a)P treatment group, B(a) Nrf2 levels in the PG-Rg3 group were increased (106 ± 9.5% for G-Rg3r vs. 51.3 ± 6.8%, 117 ± 6. 2% for G-Rg3r vs. 41 ± 9.8% for G-Rg3s, p < 0.01).
We confirmed the preventive role of Nrf2 by suppressing Nrf2 expression using specific small interfering RNA (siRNA). Nrf2 knockdown was confirmed by Western blotting (Fig. 7A,B). As shown in Figures 7C,D, co-treatment of hEL cells with B(a)P and G-Rg3 resulted in a decrease in the number of BPDE-DNA adducts (1.47 ± 0.21) compared to treatment with B(a)P alone in the control siRNA group.) G-Rg3r was 4.13 ± 0.49, G-Rg3s was 1.8 ± 0.32 and 4.1 ± 0.57, p < 0.01). However, the inhibitory effect of G-Rg3 on BPDE-DNA formation was abolished by Nrf2 knockdown. In the siNrf2 group, there was no significant difference in BPDE-DNA adduct formation between B(a)P and G-Rg3 co-treatment and B(a)P treatment alone (3.0 ± 0.21 for G-Rg3r vs. 3.56 ± 0.32). for G-Rg3r versus 3.6 for G-Rg3s versus ±0.45 versus 4.0±0.37, p > 0.05).
Effect of Nrf2 knockdown on BPDE-DNA adduct formation in hEL cells. (A) Nrf2 knockdown was confirmed by Western blotting. (B) Quantification of Nrf2 band intensity. (C) Effect of Nrf2 knockdown on BPDE-DNA adduct levels in hEL cells treated with B(a)P and G-Rg3r. (D) Effect of Nrf2 knockdown on BPDE-DNA adduct levels in hEL cells treated with B(a)P and G-Rg3. Data are expressed as the mean ± standard deviation of triplicate determinations. *P <0.05, **P <0.01, ***P <0.001.
This study evaluated the preventive effects of various red ginseng extracts on a mouse model of B(a)P-induced lung cancer, and KRGB treatment significantly reduced tumor burden. Considering that G-Rg3 has the highest content in this ginseng extract, the important role of this ginsenoside in inhibiting tumorigenesis has been studied. Both G-Rg3r and G-Rg3 (two epimers of G-Rg3) significantly reduced tumor burden in a mouse model of B(a)P-induced cancer. G-Rg3r and G-Rg3 exert anticancer effects by inducing apoptosis of tumor cells21, inhibiting tumor growth22, arresting the cell cycle23 and affecting angiogenesis24. G-Rg3 has also been shown to inhibit cellular metastasis25, and the ability of G-Rg3 to enhance the effects of chemotherapy and radiotherapy has been documented26,27. Poon et al demonstrated that G-Rg3 treatment could reduce the genotoxic effects of B(a)P28. This study demonstrates the therapeutic potential of G-Rg3 in targeting environmental carcinogenic molecules and preventing cancer.
Despite their good prophylactic potential, the poor oral bioavailability of ginsenosides poses a challenge for the clinical use of these molecules. Pharmacokinetic analysis of oral administration of ginsenosides in rats showed that its bioavailability is still less than 5%29. These tests showed that after the 20-week treatment period, only blood levels of Rg5 decreased. Although the underlying mechanism of poor bioavailability remains to be elucidated, P-gp is thought to be involved in the efflux of ginsenosides. This work demonstrated for the first time that administration of verapamil, a P-gp blocker, increases the oral bioavailability of G-Rg3r and G-Rg3s. Thus, this finding suggests that G-Rg3r and G-Rg3s act as substrates of P-gp to regulate its efflux.
This work demonstrates that combination treatment with verapamil increases the oral bioavailability of G-Rg3 in a mouse model of lung cancer. This finding is supported by increased intestinal transcellular transport of G-Rg3 upon P-gp blockade, thereby increasing its absorption. Assays in Caco2 cells showed that verapamil treatment reduced the efflux of G-Rg3r and G-Rg3s while improving membrane permeability. A study by Yang et al. Studies have shown that treatment with cyclosporine A (another P-gp blocker) increases the bioavailability of ginsenoside Rh2 from a baseline value of 1%20 to more than 30%. Ginsenosides compounds K and Rg1 also showed similar results30,31. When verapamil and cyclosporin A were co-administered, the efflux of compound K in Caco-2 cells was significantly reduced from 26.6 to less than 3, while its intracellular levels increased 40-fold30. In the presence of verapamil, Rg1 levels increased in rat lung epithelial cells, suggesting a role for P-gp in ginsenoside efflux, as shown by Meng et al.31. However, verapamil did not have the same effect on the efflux of some ginsenosides (such as Rg1, F1, Rh1 and Re), indicating that they are not affected by P-gp substrates, as shown by Liang et al. 32 . This observation may be related to the involvement of other transporters and alternative ginsenoside structures.
The mechanism of the preventive effect of G-Rg3 on cancer is unclear. Previous studies have shown that G-Rg3 prevents DNA damage and apoptosis by reducing oxidative stress and inflammation16,33, which may be the underlying mechanism for preventing B(a)P-induced tumorigenesis. Some reports indicate that genotoxicity induced by B(a)P can be reduced by modulating phase II enzymes to form BPDE-DNA34. GST is a typical phase II enzyme that inhibits BPDE-DNA adduct formation by promoting the binding of GSH to BPDE, thereby reducing DNA damage induced by B(a)P35. Our results show that G-Rg3 treatment reduces B(a)P-induced cytotoxicity and BPDE-DNA adduct formation in hEL cells and restores GST expression and activity in vitro. However, these effects were absent in the absence of Nrf2, suggesting that G-Rg3 induces cytoprotective effects through the Nrf2 pathway. Nrf2 is a major transcription factor for phase II detoxification enzymes that promotes the clearance of xenobiotics36. Activation of the Nrf2 pathway induces cytoprotection and reduces tissue damage37. Moreover, several reports have supported the role of Nrf2 as a tumor suppressor in carcinogenesis38. Our study shows that induction of Nrf2 pathway by G-Rg3 plays an important regulatory role in B(a)P-induced genotoxicity by causing B(a)P detoxification by activating phase II enzymes, thereby inhibiting the tumorigenesis process.
Our work reveals the potential of red ginseng in preventing B(a)P-induced lung cancer in mice through the important involvement of ginsenoside G-Rg3. The poor oral bioavailability of this molecule hampers its clinical application. However, this study shows for the first time that G-Rg3 is a substrate of P-gp, and administration of a P-gp inhibitor increases the bioavailability of G-Rg3 in vitro and in vivo. G-Rg3 reduces B(a)P-induced cytotoxicity by regulating the Nrf2 pathway, which may be a potential mechanism for its preventive function. Our study confirms the potential of ginsenoside G-Rg3 for the prevention and treatment of lung cancer.
Six-week-old female A/J mice (20 ± 1 g) and 7-week-old male Wistar rats (250 ± 20 g) were obtained from The Jackson Laboratory (Bar Harbor, USA) and the Wuhan Institute of Zoology. University (Wuhan, China). The Chinese Type Culture Collection Center (Wuhan, China) provided us with Caco-2 and hEL cells. Sigma-Aldrich (St. Louis, USA) is a source of B(a)P and tricaprine. Purified ginsenosides G-Rg3r and G-Rg3s, dimethyl sulfoxide (DMSO), CellTiter-96 proliferation assay kit (MTS), verapamil, minimal essential medium (MEM), and fetal bovine serum (FBS) were purchased from Chengdu Must Bio-Technology. Co.,Ltd. (Chengdu, China). The QIAamp DNA mini kit and BPDE-DNA adduct ELISA kit were purchased from Qiagen (Stanford, CA, USA) and Cell Biolabs (San Diego, CA, USA). GST activity assay kit and total protein assay kit (standard BCA method) were purchased from Solarbio (Beijing, China). All red ginseng extracts are stored in Mingyu Laboratory 7. Hong Kong Baptist University (Hong Kong, China) and Korea Cancer Center (Seoul, Korea) are commercial sources of CRG extract and various red ginseng extracts of various Korean origins (including KRGA, KRGB and KRGC). Red ginseng is made from the roots of 6-year-old fresh ginseng. Red ginseng extract is obtained by washing ginseng with water three times, then concentrating the aqueous extract, and finally drying at low temperature to obtain ginseng extract powder. Antibodies (anti-Nrf2, anti-GST, and β-actin), horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (IgG), transfection reagent, control siRNA, and Nrf2 siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA ). ), USA).
Caco2 and hEL cells were cultured in 100 mm2 cell culture dishes with MEM containing 10% FBS at 37 °C in a humidified atmosphere of 5% CO2. To determine the effect of treatment conditions, hEL cells were incubated with different concentrations of B(a)P and G-Rg3 in MEM for 48 h. Cells can be further analyzed or collected to prepare cell-free extracts.
All experiments were approved by the Experimental Animal Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology (Approval No. 2019; Registration No. 4587TH). All experiments were performed in accordance with relevant guidelines and regulations, and the study was conducted in accordance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines. Eight-week-old A/J mice were first intraperitoneally injected with B(a)P in tricaprine solution (100 mg/kg, 0.2 ml). After a week, the mice were randomly divided into control groups and different treatment groups, 15 mice in each group, and gavaged once a day. After 20 weeks of treatment, animals were sacrificed by CO2 asphyxia. Lungs were collected and fixed for 24 hours. The number of superficial tumors and individual tumor sizes were quantified for each lung under a dissecting microscope. Tumor volume estimates (V) were calculated using the following expression: V (mm3) = 4/3πr3, where r is the tumor diameter. The net sum of all tumor volumes in the lungs of mice represented the total tumor volume, and the average total tumor volume in each group represented the tumor load. Whole blood and intestinal samples were collected and stored at −80°C for UPLC-MS/MS determination. Serum was collected and an automated chemistry analyzer was used to analyze alanine aminotransferase (ALT) and serum creatinine (Cr) levels to assess liver and kidney function.
Collected samples were removed from cold storage, thawed, weighed, and placed into tubes as described above. To this was added 0.5 μM phlorizin (internal standard) in 0.8 ml methanol solution. The tissue was then homogenized using Tissue-Tearor and the homogenate was subsequently transferred to a 1.5 ml microcentrifuge tube. The mixture was centrifuged at 15500 rpm for 15 minutes. After removing 1.0 ml of supernatant, dry with nitrogen. Two hundred microliters of methanol was used for recovery. The blood is collected and processed on one line and is used as a reference for all measurements.
24-well Transwell plates were seeded with 1.0 × 105 Caco-2 cells per well to evaluate the potential enhancement of G-Rg3 transport by the addition of verapamil. After 3 weeks of culture, cells were washed with HBSS and preincubated at 37°C. 400 μL of 10 μM G-Rg3 (G-Rg3r, G-Rg3s, or a mixture with 50 or 100 μM verapamil) was injected onto the basolateral or apical side of the monolayer, and 600 μL of HBSS solution was added to the other side. Collect 100 µl of culture medium at the designated times (0, 15, 30, 45, 60, 90 and 120 minutes) and add 100 µl of HBSS to make up this volume. Samples were stored at −4 °C until detection by UPLC-MS/MS. The expression Papp = dQ/(dT × A × C0) is used to quantify the apparent unidirectional apical and basolateral permeability and vice versa (Pa-b and Pb-a, respectively); dQ/dT is the change in concentration, A (0.6 cm2) is the surface area of the monolayer, and C0 is the initial donor concentration. The efflux ratio is calculated as Pb-a/Pa-b, which represents the efflux rate of the study drug.
Male Wistar rats were fasted for 24 hours, drank only water, and anesthetized with an intravenous injection of 3.5% pentobarbital solution. The intubated silicone tube has the end of the duodenum as the entrance and the end of the ileum as the exit. Use a peristaltic pump to pump the inlet with 10 µM G-Rg3r or G-Rg3s in isotonic HBSS at a flow rate of 0.1 ml/min. The effect of verapamil was assessed by adding 50 μM or 100 μM of the compound to 10 μM G-Rg3r or G-Rg3s. UPLC-MS/MS was performed on perfusion extracts collected at time points 60, 90, 120, and 150 minutes after the start of perfusion. The percentage of absorption is quantified by the formula % absorption = (1 – Cout/Cin) × 100%; the concentration of G-Rg3 at the outlet and inlet is expressed by Cout and Cin, respectively.
hEL cells were seeded in 96-well plates at a density of 1 × 104 cells per well and treated with B(a)P (0, 1, 5, 10, 20, 30, 40 μM) or G-Rg3 dissolved in DMSO. The drugs were then diluted with culture medium to various concentrations (0, 1, 2, 5, 10, 20 μM) over 48 hours. Using a commercially available MTS assay kit, cells were subjected to a standard protocol and then measured using a microplate reader at 490 nm. The cell viability level of the groups co-treated with B(a)P (10 μM) and G-Rg3 (0, 1, 5, 10, 20 μM) was assessed according to the above method and compared with the untreated group.
hEL cells were seeded in 6-well plates at a density of 1 × 105 cells/well and treated with 10 μMB(a)P in the presence or absence of 10 μM G-Rg3. After 48 hours of treatment, DNA was extracted from hEL cells using the QIAamp DNA Mini Kit according to the manufacturer’s protocol. The formation of BPDE-DNA adducts was detected using a BPDE-DNA adduct ELISA kit. Relative levels of BPDE-DNA adduct were measured using a microplate reader by measuring absorbance at 450 nm.
hEL cells were seeded in 96-well plates at a density of 1 × 104 cells per well and treated with 10 μMB(a)P in the absence or presence of 10 μM G-Rg3 for 48 h. GST activity was measured using a commercial GST activity assay kit according to the manufacturer’s protocol. Relative GST activation was measured by absorbance at 450 nm using a microplate reader.
hEL cells were washed with ice-cold PBS and then lysed using radioimmunoprecipitation assay buffer containing protease inhibitors and phosphatase inhibitors. After protein quantification using a total protein assay kit, 30 μg of protein in each sample was separated by 12% SDS-PAGE and transferred to a PVDF membrane by electrophoresis. Membranes were blocked with 5% skim milk and incubated with primary antibodies overnight at 4°C. After incubation with horseradish peroxidase-conjugated secondary antibodies, enhanced chemiluminescence reagents were added to visualize the binding signal. The intensity of each protein band was quantified using ImageJ software.
GraphPad Prism 7.0 software was used to analyze all data, expressed as mean ± standard deviation. Variation between treatment groups was assessed using Student’s t test or one-way analysis of variance, with a P value <0.05 indicating statistical significance.
All data obtained or analyzed during this study are included in this published article and supplementary information files.
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Post time: Sep-17-2023