90 ISSN 1120-1770 online, DOI 10.15586/ijfs.v35i3.2354 P U B L I C A T I O N S CODON Italian Journal of Food Science, 2023; 35 (3): 90–98 Ganoderma lucidum extract reverses hepatocellular carcinoma multidrug resistance via inhibiting the function of P-glycoprotein in vitro and in vivo Jing Li1, Lin Cao1, Cheng Yuan1, Zhaojian Jiang1, Hongfei Cai1, Wendong Xu1, Yaming Han1, Liang Chen1, Qin Zhang1, Renwang Jiang2*, Juyan Liu1,3* 1Guangzhou HanFang Pharmaceutical Company Limited, National Engineering Research Center of Pharmaceutical Processing Technology of Traditional Chinese Medicine and Drug Innovation, Guangdong Provincial Key Laboratory of Medicinal Lipid, Guangzhou, China; 2Jinan University, Guangzhou, China; 3Guangzhou Pharmaceutical Holdings Limited, Guangzhou, China *Corresponding Authors: Renwang Jiang, Jinan University, Guangzhou, China. Email: rwjiang2008@126.com; Juyan Liu, Guangzhou Pharmaceutical Holdings Limited, Guangzhou, China Email: LJYan65@163.com Received: 10 April 2023; Accepted: 12 July 2023; Published: 12 August 2023 © 2023 Codon Publications OPEN ACCESS PAPER Abstract Cancer is a leading cause of death globally. Chemotherapy still plays an indispensable role in the clinical treatment of cancer. However, the emergence of multidrug resistance (MDR) has greatly obstructed the further application of chemotherapy agents. Ganoderma lucidum (G. lucidum) is a traditional Chinese medicine as well as an edi- ble mushroom. In this study, we first explored the effect of six extract samples derived from G. lucidum on the cell viability of adriamycin-resistant human hepatocellular carcinoma multidrug-resistant cell subline (HepG-2/ ADM). All these samples showed no obvious toxicity to cells; however, only G. lucidum ethanol extract could reverse resistance to doxorubicin and paclitaxel. Then the P-glycoprotein (P-gp) function in hepatocellular car- cinoma G2 (HepG-2) and HepG-2/ADM cells was determined after incubated with these samples, and we found that G. lucidum ethanol extract could inhibit P-gp function in vitro. Furthermore, G. lucidum ethanol extract could reverse resistance to paclitaxel in HepG-2/ADM tumor-bearing mice in vivo, while the protein expression level of P-gp was unchanged. Taken together, these results indicated the potential role of G. lucidum ethanol extract in reversing MDR in the clinical treatment of hepatocellular carcinoma. Keywords: Ganoderma lucidum, hepatocellular carcinoma, multidrug resistance, paclitaxel, P-glycoprotein Introduction Cancer has become a leading cause of death globally, and has severely threatened the life expectancy of humans (Bray et  al., 2021). It is estimated that about 19.3 mil- lion new cases and 10 million cancer deaths occurred worldwide in 2020 (Sung et  al., 2021). Although differ- ent approach to treating cancer have developed suc- cessfully, the incidence and mortality are still increasing rapidly. The main cause of failure of anti-cancer therapy is the emergence of acquired resistance, among which multidrug resistance (MDR) has played a major role (Gottesman et al., 2002). MDR is a phenomenon in which cancer cells develop resistance to multiple chemotherapy agents with distinct structures or mechanisms (Kumar and Jaitak, 2019). P-glycoprotein (P-gp), encoded by ade- nosine triphosphate (ATP)-binding cassette (ABC) sub- family B member 1 (ABCB1) is the extensively studied factor in the development of MDR. ABCB1 could utilize the energy of ATP to actively transport drugs out of cyto- plasm, resulting in the survival of cancer cells (Kumar and Jaitak, 2019). P-gp has been confirmed to be involved in the resistance to several anti-cancer agents, such as pacl- itaxel, doxorubicin, cisplatin, etoposide, etc. (Gottesman et  al., 2002; Szakacs et  al., 2006; Zeino et  al., 2015). In spite of great efforts in exploring P-gp inhibitors, most of Italian Journal of Food Science, 2023; 35 (3) 91 Ganoderma lucidum extract reverses multidrug resistant HCC Cell lines and culture Human hepatocellular carcinoma (HCC) cell line HepG-2 and the multidrug-resistant P-gp overexpressing HCC cell line HepG-2/ADM were provided by Cancer Institute & Hospital, Chinese Academy of Medical Sciences (Beijing, China). All cell lines were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Corning, New York, US) containing 10% fetal bovine serum (FBS; Life Technologies, New York, US) and 1% penicillin– streptomycin solution (Life Technologies). Cell lines were maintained at 37°C in a humidified atmosphere with 95% air and 5% CO2. Cell viability assay Cells were seeded in 96-well plates at a density of 4×103 cells per well and cultured overnight. The cells were treated with various concentrations of samples 1–6 or compounds in the presence or absence of verapamil for 48 h. MTT, 20 μL, was added into each well and incu- bated for an additional 4 h. Finally, purple formazan crys- tals were dissolved in 150-μL dimethyl sulfoxide (DMSO) and the absorbance was observed at 490 nm by multi- mode microplate reader (Bio-Rad Laboratories, CA, US). Rhodamine-123 intracellular accumulation assay Cells were seeded in six-well plates at a density of 1×105 cells per well and cultured overnight. The cells were treated with 500 μg/mL of samples 1–6 for 48 h and incu- bated with 10-μM Rh-123 for an additional 4 h in dark at 37°C. Then the cells were collected, washed with phos- phate-buffered saline (PBS) thrice, and analyzed by flow cytometer (BD Bioscience, San Jose, CA, US). The data were analyzed using the FlowJo 7.6.1 software. Xenograft mouse model experiments BALB/c nude mice were purchased from Guangdong Province Medical Animal Center and monitored under specific pathogen-free conditions. All animal experi- ments complied with the National Institute of Health Guide for the Care and Use of Laboratory Animals, 8th edition (National Institutes of Health, 2011). HepG-2/ ADM cells (1×107 in 200 μL) were collected and injected subcutaneously into the right flank of mice. Treatments were initiated when tumors reached a mean volume of 100 mm3. Mice were randomly divided into the following four groups (n = 5): (1) control group (mice were given normal saline [NS]); (2) paclitaxel group (mice were given 18-mg/kg paclitaxel); (3) sample 6 group (mice were given 400-mg/kg sample 6); and (4) sample 6 + paclitaxel them failed to show ideal efficacy while possessing major toxicities (Chen et al., 2016). Therefore, discovering novel P-gp inhibitors with potent efficacy and little toxicity is required urgently. Ganoderma lucidum (G. lucidum) is a traditional Chinese medicine widely used in China (named Ling Zhi) for hundreds of years (Ahmad, 2018). As a medicinal and edible mushroom, G. lucidum has proved to possess diverse pharmacological activities, including antioxidant (Zhang et  al., 2021), antidiabetic (Ma et  al., 2015), anti- microbial (Cor et al., 2018), anti-inflammatory (Cai et al., 2016), immunomodulatory (Li et al., 2020), and antican- cer effects (Jin et al., 2016). Researchers have found that G. lucidum could exert its anticancer effects by directly killing cancer cells, inhibiting angiogenesis, inducing cell differentiation, and activating immune response of the host (Ahmad, 2018). However, whether constituents of G. lucidum could reverse P-gp-related MDR in vivo has not been studied amply. In this study, we explored the effect of six extract sam- ples derived from G. lucidum on the cell viability and MDR of adriamycin-resistant human hepatocellular carcinoma multidrug-resistant cell subline (HepG-2/ ADM). Then the difference in P-gp function between hepatocellular carcinoma G2 (HepG-2) and HepG-2/ ADM cells was compared, and we further investigated the effect of extract samples on P-gp function. Finally, the effect of G. lucidum ethanol extract alone and in combination with paclitaxel on tumor growth and expression level of P-gp was measured in HepG-2/ADM tumor-bearing mice in vivo. Materials and Methods Materials and chemicals Samples 1–6 were obtained from Guangzhou Hanfang Pharmaceutical Co. Ltd. (Guangzhou, China). Sample 1 was the major product of G. lucidum spore oil using supercritical fluid CO2. Sample 2 was the product of G. lucidum spore oil using supercritical fluid CO2 with modifier ethanol. Sample 3 was ethanol extract of the remains after using supercritical fluid CO2. Sample 4 was water extract of the remains after using supercritical fluid CO2. Sample 5 was the water extract of G. lucidum. Sample 6 was the ethanol extract of G.  lucidum. Verapamil, paclitaxel, 3-(4,5-Dimethylthiazol-2-yl)- 2,5-diphenyl-2H-tetrazolium bromide (MTT), and rhodamine-123 (Rh-123) were purchased from Sigma- Aldrich (Deisenhofer, Germany). Doxorubicin (Dox) was obtained from Zhejiang HISUN Pharmaceuticals Co. Ltd. (Zhejiang, China). 92 Italian Journal of Food Science, 2023; 35 (3) Li J et al. one-way ANOVA. Two-tailed Student's t-test was used to compare difference between two groups. Statistical analysis was performed using the SPSS 22.0 software; p < 0.05 were considered statistically significant. Results G. lucidum samples showed no obvious toxicity to HepG-2/ADM cells We measured the viability of HepG-2/ADM cells after incubation with various concentrations of sam- ples 1–6 using MTT assay to evaluate whether the six samples derived from G. lucidum possessed toxicity to multidrug- resistant HCC cells. As shown in Figure 1, all samples showed little toxicity for HepG-2/ADM cells. Over 90% cells were alive even when the concentration reached 1,000 μg/mL. The results indicated that these samples derived from G. lucidum had no obvious tox- icity to multidrug-resistant HCC cells, which could be candidates for investigating the effect of G. lucidum extract on MDR. G. lucidum ethanol extract reversed multidrug resistance in HepG-2/ADM cells In order to investigate the effect of G. lucidum extract on the MDR of HepG-2/ADM cells, we measured cell via- bility after incubation with doxorubicin or paclitaxel in combination with samples 1–6 or a classic inhibitor of P-gp (verapamil). As shown in Table 1, verapamil could potently suppress the function of P-gp and diminish cell viability at the 50% inhibitory concentration (IC50) of doxorubicin and paclitaxel, which proved the MDR characteristic of HepG-2/ADM cells. To our surprise, we also observed that only sample 6 could effectively reverse resistance to doxorubicin and paclitaxel at IC50 and 20% inhibitory concentration (IC20) and further reduced the viability of HepG-2/ADM cells. The fold reversal of resis- tance to doxorubicin and paclitaxel was almost three times at IC50, while that was nearly five to seven times at IC20. These data indicated that G. lucidum ethanol extract could reverse MDR in HCC cells. G. lucidum ethanol extract inhibited the function of P-gp in HepG-2/ADM cells Rh-123 is a specific fluorescent substrate of P-gp used to monitor the efflux function of P-gp. Therefore, we first treated HepG-2 cells and HepG-2/ADM cells with Rh-123 to validate their difference in P-gp function. As shown in Figure 2A, the fluorescent intensity of Rh-123 was significantly higher in HepG-2 cells than that in group (mice were given 400-mg/kg sample 6 + 18-mg/kg paclitaxel). Mice were daily administrated sample 6 orally. Mice were injected with paclitaxel intraperitone- ally every 2 days. Body weight and tumor volume were determined every 2 days, and tumor volume was calcu- lated using the following formula: V = π (length × width2)/6. Animals were euthanized with CO2 when the average tumor volume in the control group reached 1,300 mm3, and tumors were collected and weighed for the following experiments. Western blot analysis Tumor tissues were homogenized and total protein was quantified by Bradford assay. Protein extract was sepa- rated in 4–12% sodium dodecyl sulfate– polyacrylamide gel electrophoresis (SDS-PAGE) and transferred on polyvinylidene fluoride (PVDF) membranes. After blocking with 5% skimmed milk for 2 h, the membranes were incubated overnight at 4°C with primary antibodies against P-gp/ABCB1 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Cell Signaling Technology, Beverly, MA, US). Next, the membranes were incubated with a rabbit secondary horseradish peroxidase conju- gated antibody (CST) for 1 h at 37°C. Protein–antibody complexes were discovered using electrochemilumines- cence (ECL) kit (Thermo Fisher, IL, US), and the inten- sity of protein bands was quantitated by the ImageJ software. Hematoxylin and eosin (H&E) staining Tumor tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned with a thickness of 5 μm. The sections were stained with H&E according to a standard protocol, and observed using Leica DM750 microscope (Leica, Heidelberg, Germany). Assay for Alanine Aminotransferase (ALT) and Aspartate Aminotransferase (AST) The blood samples were collected and centrifuged at 3,500 rpm for 5 min at 4°C. The supernatant was col- lected and the levels of serum ALT and AST were analyzed using Drew Trilogy Analyzer (Diamond Diagnostics, Holliston, MA, US). Statistical analysis Data were expressed as mean ± SD. Comparison of dif- ferences between multiple groups was performed using Italian Journal of Food Science, 2023; 35 (3) 93 Ganoderma lucidum extract reverses multidrug resistant HCC G. lucidum ethanol extract reversed resistance to paclitaxel in HepG-2/ADM tumor-bearing mice without additional toxicity We established HepG-2/ADM tumor-bearing mice models and administrated with paclitaxel and sample 6. As shown in Figures 3A–3C, paclitaxel treatment alone failed to inhibit the growth of tumor, indicating that resis- tance to paclitaxel was maintained in the xenografted tumor model. Furthermore, although treatment with sample 6 alone did not inhibit the growth of HepG-2/ ADM tumors in vivo, it was apparent that sample 6 could HepG-2/ADM cells, indicating that there was less Rh-123 accumulation in multidrug-resistant HCC cells. We further treated HepG-2/ADM cells with 500 μg/mL of samples 1–6 to investigate their effect on P-gp func- tion. As shown in Figure 2B, samples 1–5 failed to change the accumulation of Rh-123 in HepG-2/ADM cells, while only sample 6 demonstrated a significant inhibitory effect on P-gp function and elevated fluorescent intensity, which was in consistent with the reversal effect assay. The results indicated that G. lucidum ethanol extract could inhibit the function of P-gp in multidrug-resistant HCC cells. Figure 1. Samples derived from G. lucidum showed no obvious toxicity to HepG-2/ADM cells. HepG-2/ADM cells were treated with samples 1–6, and the cell viability was measured by MTT assay (n = 5). Data are presented as mean ± SD. 150 100 50 0 0 31 .2 5 62 .5 12 5 25 0 50 0 10 00 Concentration (µg/mL) Sample 1 C e ll vi a b ili ty ( % c o n tr o l) 150 100 50 0 0 31 .2 5 62 .5 12 5 25 0 50 0 10 00 Concentration (µg/mL) Sample 2 C e ll vi a b ili ty ( % c o n tr o l) 150 100 50 0 0 31 .2 5 62 .5 12 5 25 0 50 0 10 00 Concentration (µg/mL) Sample 3 C e ll vi a b ili ty ( % c o n tr o l) 150 100 50 0 0 31 .2 5 62 .5 12 5 25 0 50 0 10 00 Concentration (µg/mL) Sample 4 C e ll vi a b ili ty ( % c o n tr o l) 150 100 50 0 0 31 .2 5 62 .5 12 5 25 0 50 0 10 00 Concentration (µg/mL) Sample 5 C e ll vi a b ili ty ( % c o n tr o l) 150 100 50 0 0 31 .2 5 62 .5 12 5 25 0 50 0 10 00 Concentration (µg/mL) Sample 6 C e ll vi a b ili ty ( % c o n tr o l) 94 Italian Journal of Food Science, 2023; 35 (3) Li J et al. Figure 2. G. lucidum ethanol extract inhibited the function of P-gp in HepG-2/ADM cells. (A) HepG-2 and HepG-2/ADM cells were incubated with Rh-123, and the fluorescent intensity was measured by flow cytometry. (B) HepG-2/ADM cells were treated with samples 1–6 and incubated with Rh-123, and the fluorescent intensity was measured by flow cytometry. Table 1. The reversal effect of G. lucidum ethanol extract on the MDR of HepG-2/ADM cells (n = 5). Cell viability ± SD (%) (fold-reversal) Doxorubicin (IC 50 ) 57.393 ± 4.889 +Sample 6 (500 μg/mL) 16.118 ± 4.093*** (3.561) +Sample 6 (250 μg/mL) 21.346 ± 8.690*** (2.689) +Verapamil (5 μmol) 4.646 ± 0.049*** (12.353) Doxorubicin (IC 20 ) 84.807 ± 3.378 +Sample 6 (500 μg/mL) 11.054 ± 3.366*** (7.672) +Sample 6 (250 μg/mL) 13.922 ± 11.810*** (6.092) Paclitaxel (IC 50 ) 47.507 ± 10.849 +Sample 6 (500 μg/mL) 15.402 ± 3.538### (3.085) +Sample 6 (250 μg/mL) 16.279 ± 4.660### (2.918) +Verapamil (5 μmol) 0.942 ± 0.121### (50.43) Paclitaxel (IC 20 ) 83.588 ± 11.879 +Sample 6 (500 μg/mL) 14.311 ± 2.058### (5.841) +Sample 6 (250 μg/mL) 16.989 ± 4.604### (4.920) Data are presented as mean ± SD, and significant differences are indicated as ***p < 0.001 vs the doxorubicin group, ###p < 0.001 vs the paclitaxel group. reverse resistance to paclitaxel and decrease tumor vol- ume and weight combined with paclitaxel. H&E staining of tumor sections showed that after treatment with pacl- itaxel + sample 6, tumor tissues demonstrated large areas of nuclear consolidation, cell vacuolization, and necro- sis, with lymphocyte-infiltrated hyperemia in Figure 3E. We also measured the body weight and serum levels of ALT and AST in mice, and the results showed that nei- ther sample 6 nor paclitaxel was toxic to mice as shown in Figures 3D and 3F. The results showed that G. lucidum ethanol extract could reverse resistance to paclitaxel in HepG-2/ADM tumor-bearing mice without additional toxicity. G. lucidum ethanol extract showed no effect on the protein expression level of P-gp In order to investigate the underlying mechanism of the reversal effect of sample 6 on MDR of HCC tumor, we extracted the total protein of tumor tissues and carried out Western blot assay. As shown in Figure 4, adminis- tration of paclitaxel could slightly increase the expression level of P-gp; however, sample 6 showed no additional effect on P-gp expression level. The results indicated that the reversal effect of G. lucidum ethanol extract on MDR (A) (B) HepG-2 blank control Rh-123 accumulation in HepG-2/ADM Rh-123 accumulation in HepG-2 C o u n t C o u n t 0 0 100 101 102 103 104 100 101 102 103 104 Sample 6 500 �g/mL Sample 5 500 �g/mL Sample 4 500 �g/mL Sample 3 500 �g/mL Sample 2 500 �g/mL Sample 1 500 �g/mL HepG-2/ADM control HepG-2/ADM blank control Italian Journal of Food Science, 2023; 35 (3) 95 Ganoderma lucidum extract reverses multidrug resistant HCC Control Paclitaxel Sample 6 Before After Sample 6 + Paclitaxel Control Paclitaxel Sample 6 Sample 6 + Paclitaxel HepG-2/ADM xenograft Co nt ro l Pa cl ita xe l Sa m pl e 6 Sa m pl e 6 + Pa cl ita xe l Co nt ro l Pa cl ita xe l Sa m pl e 6 Sa m pl e 6 + Pa cl ita xe l ALT AST U /L Co nt ro l Pa cl ita xe l Sa m pl e 6 Sa m pl e 6 + Pa cl ita xe l B o d y W e ig h t (g ) Tu m o r W e ig h t (g ) 0 2 4 6 8 10 Treatment period (Days) Tu m o r V o lu m e (m m 3 ) 12 14 16 Control saline 18 mg/kg 400 mg/kg Paclitaxel Sample 6 Sample 6 + Paclitaxel 1500 1200 900 600 300 0 30 25 20 15 10 5 0 1.0 0.8 0.6 0.4 0.2 0.0 60 45 30 15 0 Figure 3. G. lucidum ethanol extract reversed resistance to paclitaxel in HepG-2/ADM tumor-bearing mice without additional toxicity. (A) Tumors from nude mice in each treatment group. (B) Tumor volume and (C) tumor weight in each treatment group (n = 5). (D) Body weight of mice in each treatment group (n = 5). (E) H&E staining of paraffin-embedded tumor tissue sections in each treatment group (n = 5). (F) Serum levels of ALT and AST in each treatment group (n = 5). Data are presented as mean ± SD., and significant differences are indicated as **p < 0.01 vs the control group, ##p < 0.01 vs the sample 6 + paclitaxel group. could be related to the direct inhibition of P-gp function, rather than down-regulation of protein expression level. Discussion In the current study, the effect of six extract samples derived from G. lucidum on the viability of HepG-2/ADM human hepatocellular carcinoma multidrug-resistant cells was determined, and they exhibited no obvious toxicity. However, G. lucidum ethanol extract could reverse resistance to doxorubicin and paclitaxel. Then the P-gp function in HepG-2 and HepG-2/ADM cells was determined after incubated with these samples, and it was found that G. lucidum ethanol extract could inhibit P-gp function in vitro. Furthermore, G. lucidum ethanol extract could reverse resistance to paclitaxel in HepG-2/ADM tumor-bearing mice in vivo, without (A) (C) (E) (B) (D) (F) 96 Italian Journal of Food Science, 2023; 35 (3) Li J et al. P-gp, a member of the ABC transporter family, has been identified as a major factor that leads to MDR via utiliz- ing the energy of two ATP molecules. P-gp could fulfill one catalytic cycle and the following conformational change, and pump the substrate out of the cytoplasm, resulting in resistance to several cytotoxic chemotherapy agents (Zeino et  al., 2015). The overexpression of P-gp has been confirmed in different malignancies, such as hepatocellular carcinoma (Komori et  al., 2014), breast carcinoma (Ding et  al., 2021), colorectal carcinoma (Hu et  al., 2014), and chronic myeloid leukemia (Ammar et  al., 2020). Although great efforts have been invested in the research of P-gp inhibitors, most candidates failed because of inevitable toxicity and frustrating efficacy. Nowadays, natural products have demonstrated promis- ing capabilities in regulating the function of P-gp while providing safer outcomes (Kumar and Jaitak, 2019). In this study, we first applied a classic P-gp inhibitor, ver- apamil, and found that it could dramatically reverse the resistance of HepG-2/ADM cells to doxorubicin or pacl- itaxel, implying the role of P-gp in the MDR of HepG-2/ ADM cells. Interestingly, we further measured the rever- sal effect of G. lucidum extract samples and discovered that only the ethanol extract sample 6 could signifi- cantly reverse resistance to doxorubicin and paclitaxel. Therefore, in order to investigate the effect of G. lucidum components on P-gp function, we applied Rh-123, a spe- cific fluorescent substrate of P-gp, and performed flow cytometry experiments. Our results showed that, com- pared to HepG-2 cells, the multidrug-resistant HepG-2/ affecting the protein expression level of P-gp, indicating that G. lucidum ethanol extract might directly inhibit the function of P-gp. In the past decades, cancer has become one of the major threats to human health worldwide, with increased inci- dence and mortality (Sung et al., 2021). Currently, in the clinical treatment of cancer, chemotherapy still plays an indispensable role because of good response and wide utility. Unfortunately, the development of MDR has severely limited the use of chemotherapeutic drugs, such as paclitaxel, doxorubicin, etoposide, and cisplatin (Robey et al., 2018). G. lucidum, a basidiomycete, has been recognized as a traditional Chinese medicine with a history of hundreds of years in ancient China. It was believed that G. lucidum could promote immunity and extend life expectancy (Chan et al., 2021). Recently, researchers have discovered that G. lucidum exhibits multiple biological activities, including antioxidant, anticancer, anti-inflammatory, and immunomodulatory effects (Cor et al., 2018). In this study, to further investigate the anticancer char- acteristics of G. lucidum, we prepared six samples with different isolation methods, and measured their effect on the viability of HepG-2/ADM cancer cells. The results revealed that, within a range of experimental dose, none of the six samples showed any inhibitory effects on cell growth, indicating that they posed no direct toxicity to HepG-2/ADM cancer cells. Figure 4. G. lucidum ethanol extract showed no effect on the protein expression level of P-gp in vivo. The protein expression level of P-gp was assessed in HepG-2/ADM xenografts (n = 3). Data are presented as mean ± SD. Co nt ro l P-gp GAPDH Co nt ro l Pa cl ita xe l Sa m pl e 6 Sa m pl e 6 + Pa cl ita xe l Sa m pl e 6 + Pa cl ita xe l Co nt ro l Sa m pl e 6 Sa m pl e 6 + Pa cl ita xe l Co nt ro l R e la ti ve P -g p e xp re ss io n le ve ls R e la ti ve P -g p e xp re ss io n le ve ls Pa cl ita xe l Sa m pl e 6 + Pa cl ita xe l 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 Italian Journal of Food Science, 2023; 35 (3) 97 Ganoderma lucidum extract reverses multidrug resistant HCC in Guangdong Province (No. 2020B1212070024), the Guangdong Province Key Areas R&D Program Project (No. 2020B1111120002), and the National Key R&D Program of China (No. 2022YFC3500302). Authors of this research are in deep gratitude toward Professor Renwang Jiang of Jinan University for his guid- ance and support to this work. Conflict of interest The authors stated that they had no conflict of interest to declare. Author Contributions Renwang Jiang, Juyan Liu, and Jing Li designed the study. Lin Cao wrote the manuscript. Jing Li, Cheng Yuan, Wendong Xu, Yaming Han, and Juyan Liu revised the manuscript. Jing Li, Hongfei Cai, Liang Chen, and Qin Zhang carried out the experiments. Lin Cao, Zhaojian Jiang, and Hongfei Cai analyzed the data. All authors approved the final version of the paper. References Ahmad, M.F., 2018. Ganoderma lucidum: persuasive biologically active constituents and their health endorsement. Biomed Pharmacother. 107: 507–519. https://doi.org/10.1016/j. biopha.2018.08.036 Ammar, M., Louati, N., Frikha, I., Medhaffar, M., Ghozzi, H., Elloumi, M., Menif, H., Zeghal, K. and Ben Mahmoud, L., 2020. Overexpression of P-glycoprotein and resistance to Imatinib in chronic myeloid leukemia patients. J Clin Lab Anal. 34: e23374. https://doi.org/10.1002/jcla.23374 Bray, F., Laversanne, M., Weiderpass, E. and Soerjomataram, I., 2021. The ever-increasing importance of cancer as a leading cause of premature death worldwide. Cancer. 127: 3029–3030. https://doi.org/10.1002/cncr.33587 Cai, Z., Wong, C.K., Dong, J., Jiao, D., Chu, M., Leung, P.C., Lau,  C.B.S., Lau, C.P., Tam, L.S. and Lam, C.W.K., 2016. Anti- inflammatory activities of Ganoderma lucidum (Lingzhi) and San-Miao-San supplements in MRL/lpr mice for the treat- ment of systemic lupus erythematosus. Chin Med. 11: 23. https://doi.org/10.1186/s13020-016-0093-x Chan, S.W., Tomlinson, B., Chan, P. and Lam, C.W.K., 2021. The beneficial effects of Ganoderma lucidum on cardiovascu- lar and metabolic disease risk. Pharm Biol. 59: 1161–1171. https://doi.org/10.1080/13880209.2021.1969413 Chen, Z., Shi, T., Zhang, L., Zhu, P., Deng, M., Huang, C., Hu, T., Jiang, L. and Li, J., 2016. Mammalian drug efflux transport- ers of the ATP binding cassette (ABC) family in multidrug ADM cells accumulated less Rh-123 in cytoplasm, indi- cating that the elevated function of P-gp contributed to MDR in HepG-2/ADM cells. Moreover, G. lucidum eth- anol extract sample 6 could inhibit P-gp and elevate the level of Rh-123 in HepG-2/ADM cells, which further val- idated our previous results in the assay of reversal effect. Based on the above results, xenograft HepG-2/ADM tumor-bearing mouse models were created and admin- istered with paclitaxel and sample 6 to investigate whether G. lucidum ethanol extract could reverse MDR in HepG-2/ADM cells in vivo. The results demonstrated that paclitaxel had little effect on the growth of HepG-2/ ADM tumors, confirming the MDR characteristic of xenografted tumor model. Further, G. lucidum ethanol extract sample 6 could reverse the MDR of xenografted tumors in combination with paclitaxel, resulting in the suppression of tumor volume and weight, accompanied with deteriorated tumor pathologic conditions. The results indicated the reversal effect of G. lucidum eth- anol extract on MDR in vivo. Meanwhile, we also mea- sured the body weight and serum levels of ALT and AST, and the data showed that the administration of sample 6 did not alter body weight and liver functions of mice, implying that G. lucidum ethanol extract had no obvi- ous toxicity in vivo. Further, we extracted the total pro- tein of tumor tissues and determined the expression level of P-gp in each treatment group. However, the results showed that sample 6 did not change the protein expres- sion level of P-gp, while paclitaxel could slightly increase the expression of P-gp in vivo. Based on these results, we deduced that sample 6 could exert the reversal effect of MDR by directly inhibiting the function of P-gp, rather than affecting protein expression level. Therefore, our results indicated that G. lucidum ethanol extract could exert the reversal effect on MDR in HCC cells by directly inhibiting the function of P-gp both in vitro and in vivo. Conclusion Our study explored the effects of G. lucidum extract on multidrug-resistant HCC cells, and established that G.  lucidum ethanol extract could reverse MDR in HCC cells by inhibiting the function of P-gp both in vitro and in vivo without severe adverse reactions. These findings implied the promising role of G. lucidum ethanol extract in reversing MDR because of chemotherapy agents in the clinical treatment of HCC. Acknowledgments This work was supported by the Ministry of Science and Technology of China (No. 2017YFC1703104), the Key Laboratory Project of Pharmaceutical Lipids 98 Italian Journal of Food Science, 2023; 35 (3) Li J et al. Kumar, A. and Jaitak, V., 2019. Natural products as multidrug resis- tance modulators in cancer. Eur J Med Chem. 176: 268–291. https://doi.org/10.1016/j.ejmech.2019.05.027 Li, J., Gu, F., Cai, C., Hu, M., Fan, L., Hao, J. and Yu, G., 2020. Purification, structural characterization, and immunomodula- tory activity of the polysaccharides from Ganoderma lucidum. Int J Biol Macromol. 143: 806–813. https://doi.org/10.1016/j. ijbiomac.2019.09.141 Ma, H.T., Hsieh, J.F. and Chen, S.T., 2015. Anti-diabetic effects of Ganoderma lucidum. Phytochemistry. 114: 109–113. Robey, R.W., Pluchino, K.M., Hall, M.D., Fojo, A.T., Bates, S.E. and Gottesman, M.M., 2018. Revisiting the role of ABC transporters in multidrug-resistant cancer. Nature Rev Cancer. 18: 452–464. https://doi.org/10.1038/s41568-018-0005-8 Sung, H., Ferlay, J., Siegel, R.L., Laversanne, M., Soerjomataram,  I., Jemal, A. and Bray, F., 2021. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. Cancer J Clin. 71: 209–249. https://doi.org/10.3322/caac.21660 Szakacs, G., Paterson, J.K., Ludwig, J.A., Booth-Genthe, C. and Gottesman, M.M., 2006. Targeting multidrug resis- tance in cancer. Nature Rev. Drug Discov. 5: 219–234. https://doi.org/10.1038/nrd1984 Zeino, M., Paulsen, M.S., Zehl, M., Urban, E., Kopp, B. and Efferth,  T., 2015. Identification of new P-glycoprotein inhibi- tors derived from cardiotonic steroids. Biochem Pharmacol. 93: 11–24. https://doi.org/10.1016/j.bcp.2014.10.009 Zhang, Y., Cai, H., Tao, Z., Yuan, C., Jiang, Z., Liu, J., Kurihara,  H. and Xu, W., 2021. Ganoderma lucidum spore oil (GLSO), a novel antioxidant, extends the average life span in Drosophila melanogaster. Food Sci Hum Wellness. 10: 38–44. https://doi.org/10.1016/j.fshw.2020.05.011 resistance: a review of the past decade. Cancer Lett. 370: 153–164. https://doi.org/10.1016/j.canlet.2015.10.010 Cor, D., Knez, Z. and Knez Hrncic, M., 2018. Antitumour, antimicrobial, antioxidant and antiacetylcholinesterase effect of Ganoderma lucidum terpenoids and polysaccha- rides: a review. Molecules. 23: 649. https://doi.org/10.3390/ molecules23030649 Ding, Y., Fan, J., Fan, Z. and Zhang, K., 2021. Gamma- tocotrienol reverses multidrug resistance of breast cancer cells through the regulation of the gamma-tocotrienol- NF- kappaB-P-gp axis. J Steroid Biochem Mol Biol. 209: 105835. https://doi.org/10.1016/j.jsbmb.2021.105835 Gottesman, M.M., Fojo, T. and Bates, S.E., 2002. Multidrug resis- tance in cancer: role of ATP-dependent transporters. Nature Rev Cancer. 2: 48–58. https://doi.org/10.1038/nrc706 Hu, T., To, K.K., Wang, L., Zhang, L., Lu, L., Shen, J., Chan, R.L., Li, M., Yeung, J.H. and Cho, C.H., 2014. Reversal of P-glycoprotein (P-gp)-mediated multidrug resistance in colon cancer cells by cryptotanshinone and dihydrotanshinone of Salvia mil- tiorrhiza. Phytomedicine. 21: 1264–1272. https://doi.org/ 10.1016/j.phymed.2014.06.013 Jin, X., Ruiz Beguerie, J., Sze, D.M. and Chan, G.C., 2016. Ganoderma lucidum (Reishi mushroom) for cancer treatment. Cochrane Database Syst Rev. 4: CD007731. https://doi.org/10.1002/14651858.CD007731.pub3 Komori, Y., Arisawa, S., Takai, M., Yokoyama, K., Honda, M., Hayashi, K., Ishigami, M., Katano, Y., Goto, H., Ueyama, J., Ishikawa, T. and Wakusawa, S., 2014. Ursodeoxycholic acid inhibits overexpression of P-glycoprotein induced by doxo- rubicin in HepG2 cells. Eur J Pharmacol. 724: 161–167. https://doi.org/10.1016/j.ejphar.2013.12.023