Modulation of gut microbiota and its metabolite Equol by Huaier granule suppresses hepatocellular carcinoma via the gut-liver axis.

Huaier extract has been demonstrated to exhibit potent anti-tumor effects in various types of cancer cells. However, the clinical benefit of Huaier granule in breast cancer has not been reported. In this study, we aimed to evaluate the efficacy of Huaier granule in breast cancer patients. Our study included 284 breast cancer patients treated with or without Huaier granule between January 2005 and October 2016 at Qilu Hospital, Shandong University, Jinan, China. Retrospective data obtained included demographics, clinicopathological characteristics, disease-free survival (DFS), serum concentrations of tumor markers, the Karnofsky performance scale (KPS), and incidences of emotional symptoms. DFS was the main outcome measure. Of the patients included, 144 were classified into the control group and 140 into the Huaier group. Baseline characteristics were well balanced between the study arms. Median DFS was 91.43months for control group and 112.61months for Huaier group (hazard ratio (HR) = 2.97, 95% confidence interval (CI) = 1.57-5.61, p < 0.01). After Huaier granule treatment, the serum levels of tumor markers could be reduced to the normal range. In addition, breast cancer patients with Huaier granule treatment had higher KPS scores and less emotional symptoms. Our data demonstrated that patients orally administrated Huaier granule got longer DFS. Furthermore, Huaier granule could reduce serum tumor markers, improve the functional status, and decrease the incidences of emotional symptoms in breast cancer patients. Therefore, Huaier granule was an effective therapy for women with breast cancer.

There is basic research suggesting that Huaier granule can inhibit liver cirrhosis and hepatocellular carcinoma (HCC), but this conclusion has not been clinically verified. We analyzed the distant cancer tissue of two groups of hepatitis B virus (HBV) related HCC with/without Huaier granule, to clarify the effect of Huaier granule on liver inflammation, liver fibrosis, and postoperative recurrence. We collected clinicopathological data of HCC patients who received two surgery procedures at Mengchao Hepatobiliary Hospital of Fujian Medical University in China from January 2014 to December 2020. Patients according to taking/not taking Huaier granule after the first hepatectomy were divided into two groups, 51 patients with Huaier granule for more than 6 months after operation (Group A); 56 patients without Huaier granule (Group B). The effects on liver inflammation, fibrosis grade, and postoperative recurrence were compared between two groups. The results showed that liver inflammation improved significantly in the Group A [19 (37.3%) cases improved, 31 (60.8%) cases remained unchanged, and 1 (2.0%) case deteriorated] was significantly more than that in the Group B [7 (12.5%) cases improved, 32 (57.1%) cases remained unchanged, and 17 (30.4%) cases deteriorated] (P<0.001). The liver fibrosis in the Group A [17 (33.3%) cases improved, 32 (62.7%) cases remained unchanged, and 2 (3.9%) cases deteriorated] was significantly improved in the Group B [5 (8.9%) cases improved, 45 (80.4%) cases remained unchanged, and 6 (10.7%) cases deteriorated] (P=0.005). The recurrence interval (27.0±21.2 months) in the Group A was significantly longer than that in the Group B (19.0±14.2 months) (P=0.026). Huaier granule can improve liver inflammation, fibrosis, and liver function and prolong the time to recurrence in HBV-related HCC. Given the high rate of postoperative recurrence and poor prognosis of HBV-related HCC, our findings may have useful clinical significance in the prevention of tumor recurrence in these patients.

Hepatocellular carcinoma, a common malignancy of the digestive system, typically progresses through a sequence of hepatitis, liver fibrosis, cirrhosis and ultimately, tumor. The interaction between gut microbiota, the portal venous system and the biliary tract, referred to as the gut-liver axis, is crucial in understanding the mechanisms that contribute to the progression of hepatocellular carcinoma. Mechanisms implicated include gut dysbiosis, alterations in microbial metabolites and increased intestinal barrier permeability. Imbalances in gut microbiota, or dysbiosis, contributes to hepatocellular carcinoma by producing carcinogenic substances, disrupting the balance of the immune system, altering metabolic processes, and increasing intestinal barrier permeability. Concurrently, accumulating evidence suggests that gut microbiota has the ability to modulate antitumor immune responses and affect the efficacy of cancer immunotherapies. As a new and effective strategy, immunotherapy offers significant potential for managing advanced stages of hepatocellular carcinoma, with immune checkpoint inhibitors achieving significant advancements in improving patients' survival. Probiotics play a vital role in promoting health and preventing diseases by modulating metabolic processes, inflammation and immune responses. Research indicates that they are instrumental in boosting antitumor immune responses through the modulation of gut microbiota. This review is to explore the relationship between gut microbiota and the emergence of hepatocellular carcinoma, assess the contributions of probiotics to immunotherapy and outline the latest research findings, providing a safer and more cost-effective potential strategy for the prevention and management of hepatocellular carcinoma.

e16216 Background: Clinical trials had confirmed the efficacy and safety of Huaier granules in the treatment of hepatocellular carcinoma (HCC), but real-world evidence is still lacking. This prospective study aimed to evaluate the real-world effectiveness and safety of Huaier granules for HCC recurrence after ablation. Methods: Post-ablation patients with no residual lesions confirmed by imaging were enrolled in this study and divided into the Huaier granule group and the control group based on receiving Huaier granules or not. Propensity-score matching was performed according to age, gender, TACE within 3 months before ablation, alpha-fetoprotein level, time interval between ablation and enrollment, and Child-Pugh Grade in 1-(up)-to-2 ratio. The primary outcome was progression-free survival (PFS), and the secondary outcomes were local recurrence-free survival (LRFS) and adverse events (AEs). Results: There were 340 patients in the Huaier granule group and 141 patients in the control group. After propensity score matching, 189 patients and 111 patients were selected for the Huaier granule group and the control group, respectively. There was no significant difference in baseline characteristics between two groups after matching. The median follow-up time of the Huaier granule group and the control group were 49.14 weeks and 73.29 weeks, respectively. The number of PFS events were 29 (15.34%) and 27 (24.32%) in the Huaier granule group and the control group, respectively. The 48, 72, and 96-week PFS rates were 87.4%, 79.4% and 75.2% in the Huaier granule group, and 79.9%, 70.1% and 66.5% in the control group, respectively ( P=0.1649). The number of LRFS events were 27 and 25 in the Huaier granule group and the control group, respectively. In the pre-matching Huaier granule cohort, the incidence of AEs, serious AEs, and grade ≥ 3 AEs were 54 (16.56%), 30 (9.20%) and 14 (4.30%), respectively. Conclusions: This study suggested potential benefit in PFS and RFS and favorable safety profile of Huaier granules in patients with HCC after ablation. Longer-term follow-up is needed to confirm this finding.[Table: see text]

Simple SummaryHepatocellular carcinoma (HCC), the most common form of liver cancer, is the third leading cause of cancer-related mortality worldwide. Recent research indicates that altering the gut environment affects liver immune response and cancer progression, through what is known as the ‘gut–liver axis’. HCC patients have dampened anticancer responses, but through modulating the gut environment, there is potential to reinvigorate these ‘exhausted’ immune cells to target tumors. In promising research on melanoma, transplanting stool from healthy donors through faecal microbiota transplantation (FMT) resulted in increased response to immunotherapy treatment in patients who were previously nonresponding. However, manipulating the gut environment as a therapeutic option in HCC remains to be explored. In this review, we explore the mechanisms through which this occurs, including how the gut environment affects gut barrier function, bacterial sensing, and liver immune responses, and how FMT may be a potential therapy for HCC patients nonresponsive to immunotherapy.Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related mortality worldwide. Recently, the gut microbiota has been shown to be closely linked to modulation of the immune and inflammatory responses, hence its potential as a therapeutic target. Although still under intense investigation, there exists a ‘gut–liver axis’ that links changes in the gut to the liver. In this regard, composition of gut microbiota and related metabolites, such as bile acids and short-chain fatty acids, have been shown to orchestrate key immune–metabolic events in liver disease and liver cancer. As hepatic immune cells are important determinants of antitumor responses, it is now increasingly recognized that the gut–liver axis plays a key role in influencing the intrahepatic immune response in HCC to favor a pro- or antitumor immune milieu. Hence, modulation of gut microbiota is potentially an attractive option to reinvigorate the antitumor responses. In this regard, promising evidence from melanoma preclinical and clinical studies has demonstrated the efficacy of gut-based intervention in reinvigorating the antitumor responses and improving responses to immunotherapy. However, the role of gut-based interventions as a therapeutic option in HCC remains to be elucidated. This review details how the gut microbiota and bacterial metabolites affect gut barrier function and ultimately immune response in HCC and raises the question of the potential of gut-based interventions as an adjunct therapy for patients with HCC receiving immunotherapy.

Objective To investigate the auxiliary effects of huaier granule on hepatoeellular carcinoma (HCC)patients after liver transplantation.Methods Sixty HCC patients who had undergone liver transplantation from Julv 2004 to June 2006 and met the standard of UCSF were involved in this study.All patients were divided into huaier granule group(n=20),chemotherapy group(n=15),huaier granule+chemotherapy group (n=15)and control group(n=10).The white blood cell count,liver function,cell immunity and immunologieal reiection were detected.The 1-year tumor recurrence rate wag calculated.Results The white blood cell counts in chemotherapy group 1,3,and 6 months after treatment were significantly lower than that before treatment (F=62.053,58.472,49.807,P＜0.05).The changes of white blood cell counts of the other 3 groups before and aftertreatment were small.The difference on the white blood cell counts of the 4 groups had no statistical 8ignincanee(F=102.361,113.412,87.572,P＜0.05).The NK activity,CD4+/CD8+ ratio,IL-2 level in huaier granule group and huaier granule+chemotherapy group 1,3,6 months after treatment were significantly higher than those before treatment,and were significantly higher than those in chemotherapy group and control group(P＜0.05).No immunological rejection occurred in all the groups.Two patients in each group had recurrence and metastasis of HCC within 1 year after the treatment.and the incidence in control group was higher than the other 3 groups(P＜0.05). Conclusions Humer granule can increase the white blood cell count which is decreased after chemotherapy,impmve cellular immunity,and effectively suppress the recurrence and metastasis of HCC at the first year after operation. Key words: Liver neoplams; Liver transplantation; Huaier granule; Chemotherapy

[This corrects the article DOI: 10.1371/journal.pone.0148263.].

BackgroundHepatocellular carcinoma (HCC) associated with macroscopic vascular invasion and distant metastasis is an advanced-stage disease with an extremely poor prognosis and low survival rate. Therefore, there is an urgent need to develop novel therapeutic strategies to extend the lives of patients with advanced HCC.Case PresentationWe represent a case of HCC with macroscopic vascular invasion and pulmonary metastasis responding dramatically to the combination treatment with drug-eluting beads transarterial chemoembolization (DEB-TACE) and Huaier granule. A 64-year-old man with hepatitis B virus (HBV)-induced liver cirrhosis was diagnosed with advanced HCC involved renal vein and inferior vena cava accompanied by pulmonary metastasis. The patient received three cycles of on-demand DEB-TACE from 9th September 2016 to 22nd August 2017 and combined with Huaier granule 20 g three times a day orally. Eight months following the treatment, complete response occurred with regression of HCC and vascular thrombus and disappearance of pulmonary metastasis. The levels of AFP had decreased from 8165.8ng/mL to within the normal range (1.7 ng/mL). This is the first case report of complete response of HCC to the combination treatment with DEB-TACE and Huaier granule. At the most recent follow-up, he remained in remission 36 months after cessation of treatment without clinical or imaging evidence of disease recurrence. The current overall survival is 54 months since the initial treatment.ConclusionData from this clinical case report suggest that the combination treatment with DEB-TACE and Huaier granule is a promising therapeutic option for advanced HCC with macroscopic vascular invasion and distant metastasis.

The mortality of hepatocellular carcinoma (HCC) is high. Plant-derived bioactive compounds have emerged as potential therapies for HCC. Procyanidin (PAC) has been shown to possess immune-modulating and anti-tumor properties. However, the role and mechanism of total PAC in treating HCC remain unclear. We established subcutaneous and orthotopic HCC mouse models to assess the effect of PAC on tumor growth. Multi-omics analyses and in vitro experiments were conducted to investigate the changes in the gut microbiota, related-metabolites, and the tumor microenvironment (TME). 16S rDNA sequencing revealed that PAC could reshape the gut microbiota, notably increasing Lactobacillus murinus abundance. Furthermore, transplantation of Lactobacillus murinus reduced tumor volumes in mice. Single-cell RNA sequencing showed upregulation of the MAPK pathway in B cells within the TME. Metabolomic analysis suggests that 5-Hydroxytryptophan (5-HTP) derived from Lactobacillus murinus was significantly increased in B cells from mesenteric lymph nodes (MLNs) in the PAC-treated group. In vitro experiments revealed that 5-HTP could significantly upregulate the MAPK pathway in B cells. Additionally, 5-HTP-educated B cells could activate IFN-γ+CD8+T cells through B cell-T cell interactions, indicating that 5-HTP is a key metabolite in the therapeutic effect of PAC. Finally, feeding 5-HTP to HCC mice reduced tumor volume, upregulated the MAPK pathway in B cells from MLNs, and activated IFN-γ+CD8+T cells in the TME. PAC reshapes the gut microbiota and metabolites, upregulates the MAPK pathway in B cells from MLNs, and activates CD8+T cells in the TME through the gut-liver axis, thereby inhibiting HCC progression.

The Central Qi theory in traditional Chinese medicine: Gut microbiota modulation as a strategic target for hepatocellular carcinoma therapy.

Hepatocellular carcinoma (HCC) is the second most lethal malignancy globally and is increasing in incidence in the United States. Unfortunately, there are few effective systemic treatment options, particularly for disseminated disease. Glypican-3 (GPC3) is a proteoglycan cell surface receptor overexpressed in most HCCs and provides a unique target for molecular therapies. We have previously demonstrated that PET imaging using a 89Zr-conjugated monoclonal anti-GPC3 antibody (αGPC3) can bind to minute tumors and allow imaging with high sensitivity and specificity in an orthotopic xenograft mouse model of HCC and that serum alpha-fetoprotein (AFP) levels are highly correlated with tumor size in this model. In the present study, we conjugated 90Y, a high-energy beta-particle-emitting radionuclide, to our αGPC3 antibody to develop a novel antibody-directed radiotherapeutic approach for HCC. Luciferase-expressing HepG2 human hepatoblastoma cells were orthotopically implanted in the livers of athymic nude mice, and tumor establishment was verified at 6 weeks after implantation by bioluminescent imaging and serum AFP concentration. Tumor burden by bioluminescence and serum AFP concentration was highly correlated in our model. Yttrium-90 was conjugated to αGPC3 using the chelating agent 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and injected via the tail vein into the experimental mice at a dose of 200 μCi/mouse or 300 μCi/mouse. Control mice received DOTA-αGPC3 without radionuclide. At 30 days after a single dose of the radioimmunotherapy agent, mean serum AFP levels in control animals increased dramatically, while animals treated with 200 μCi only experienced a minor increase, indicating cessation of tumor growth, and animals treated with 300 μCi experienced a reduction in serum AFP concentration, indicating tumor shrinkage. Mean tumor-bearing liver weight in control animals was also significantly greater than that in animals that received either dose of 90Y-αGPC3. These results were achieved without significant toxicity as measured by body condition scoring and body weight. The results of this preclinical pilot demonstrate that GPC3 can be used as a target for radioimmunotherapy in an orthotopic mouse model of HCC and may be a target of clinical significance, particularly for disseminated HCC.

Mouse models are commonly used for studying hepatocellular carcinoma (HCC) biology and exploring new therapeutic interventions. Currently three main modalities of HCC mouse models have been extensively employed in pre-clinical studies including chemically induced, transgenic and transplantation models. Among them, transplantation models are preferred for evaluating in vivo drug efficacy in pre-clinical settings given the short latency, uniformity in size and close resemblance to tumors in patients. However methods used for establishing orthotopic HCC transplantation mouse models are diverse and fragmentized without a comprehensive comparison. Here, we systemically evaluate four different approaches commonly used to establish HCC mice in preclinical studies, including intravenous, intrasplenic, intrahepatic inoculation of tumor cells and intrahepatic tissue implantation. Four parameters—the latency period, take rates, pathological features and metastatic rates—were evaluated side-by-side. 100% take rates were achieved in liver with intrahepatic, intrasplenic inoculation of tumor cells and intrahepatic tissue implantation. In contrast, no tumor in liver was observed with intravenous injection of tumor cells. Intrahepatic tissue implantation resulted in the shortest latency with 0.5cm (longitudinal diameter) tumors found in liver two weeks after implantation, compared to 0.1cm for intrahepatic inoculation of tumor cells. Approximately 0.1cm tumors were only visible at 4 weeks after intrasplenic inoculation. Uniform, focal and solitary tumors were formed with intrahepatic tissue implantation whereas multinodular, dispersed and non-uniform tumors produced with intrahepatic and intrasplenic inoculation of tumor cells. Notably, metastasis became visible in liver, peritoneum and mesenterium at 3 weeks post-implantation, and lung metastasis was visible after 7 weeks. T cell infiltration was evident in tumors, resembling the situation in HCC patients. Our study demonstrated that orthotopic HCC mouse models established via intrahepatic tissue implantation authentically reflect clinical manifestations in HCC patients pathologically and immunologically, suggesting intrahepatic tissue implantation is a preferable approach for establishing orthotopic HCC mouse models.

This study evaluated the clinical efficacy of Huaier granules combined with targeted therapy plus immunotherapy in patients with unresectable hepatocellular carcinoma (HCC) who had not undergone systemic treatment. Patients with unresectable HCC and no prior treatments were recruited from the Hospital of Traditional Chinese Medicine of Xinjiang and the First Affiliated Hospital of Xinjiang Medical University between March 2022 and July 2023. Patients received targeted therapy and immunotherapy with (exposure group) or without Huaier Granules (non-exposure group). The primary endpoint was progression-free survival (PFS), with secondary endpoints including 6-month PFS rate, HCC Symptom Severity Quantitative Response, EORTC QLQ-HCC18 Score, and safety. The mPFS in the exposure group was 8.9 months compared to 5 months in the non-exposure group (P = 0.001; HR = 0.50). The 6-month PFS rates were 66.7% and 34.1% for the exposure and non-exposure groups, respectively (P = 0.001). The clinical efficacy rate of TCM symptom classification in HCC was higher in the exposure group (87.50% vs 59.09%; P = 0.001). The exposure group also showed improvement in fatigue (P = 0.023). Extrahepatic metastasis was an independent prognostic factor (HR = 1.77; P = 0.016), while Huaier granules reduced the risk of disease progression by 47% (HR = 0.53; P = 0.006). No significant differences were observed for adverse events. The most common adverse events were hypertension, proteinuria, abnormal liver function, and diarrhea. Huaier granules significantly prolong PFS and improve the 6-month PFS rate, reducing disease progression risk in HCC patients. Subgroup analysis showed more pronounced benefits in patients with vascular invasion and alcohol consumption, with mPFS extending beyond 1year.

Objective To evaluate the safety and efficacy of combined transcatheter intraarterial chemoembolization with 350-560 μm gelatin sponge particles and lobaplatin (GSPs-TACE) and oral Chinese herb medication Huaier granules in patients with hepatocellular carcinoma (HCC).Methods This is a case-control,single-institution clinical trial.31 HCC patients receiving GSPs-TACE (group A) were matched with 31 patients receiving GSPs-TACE and Huaier granules (group B).Tumor response was evaluated by CECT using response evaluation criteria in solid tumors at 1 month after initial GSPs-TACE.The overall survival was calculated from the data of initial GSPs-TACE using the Kaplan-Meier method.Results The overall mean follow-up period was 28.7 (range 12-42) months.The overall 6-and 12-month survival rates in group A and group B were 90.3％ and 80.6％ vs 100％ and 93.5％,respectively.There was statistically significant difference in 12-month survival rates (x2 =5.213,P ＜0.05).Median survival time respectively was 17.1 and 20.6 months(x2 =0.745,P ＞0.05).The average TACE times were (4.1 ±7.3) and (2.9 ±8.7) respectively in group A and group B (P =0.01).Tumor objective response rate was statistically different in 6-and 12-month in two arms(x2 =5.945,6.384,P ＜0.05).There was no statistically significant difference in complications (respectively x2 =0.435,0.485,2.037,0.137,0.325,all P ＞ 0.05).Conclusions Huaier granules combined with transcatheter intraarterial chemoembolization with 350-560 μm gelatin sponge particles and carboplatin for HCC is safe and effectively prolongs patients' survival. Key words: Carcinoma, hepatocellular; Drug therapy, combination; Gelatin sponge,absorbable; Huaier granules; Transcatheter intraarterial chemoembolization

<p indent="0mm">Gut microbiota is closely related to host health. The interactions between gut microbiota and hosts are complex, including the relationships between microbiota and the immune system, gut-brain axis, gut-lung axis etc. Gut microbiota disorders are related to the occurrence and development of some diseases, and some microbial strains are identified to be the cause of some diseases. Moreover, gut microbiota has effects on drug metabolism, and the individual differences of gut microbiota might lead to the different individual effects of the same drug. Therefore, recovering individual gut microbiota is essential for the implementation of individual precision medical treatment. Gut microbiota is alterable, and gut microbiota can be modulated at healthy state by dietary regulation, probiotics/prebiotics/synbiotics supplement, and fecal microbiota transplantation. Besides, microbiota editing techniques and synthetic microbiota have been applied in the modulation of gut microbiota. Currently, modulation of gut microbiota has become one of the effective strategies to improve or cure some diseases. This review summarized the interactions between gut microbiota and hosts, the correlations and causal relationships between gut microbiota and diseases, the ways to improve human health by modulating gut microbiota, and gave insights into the application of microbiome and synthetic biology on the modulation and synthesis of gut microbiota.