Primary liver cancer (PLC) ranks sixth in cancer incidence and third in cancer-related mortality worldwide (Mak and Kramvis, 2021; Sung et al., 2021). Hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA) are the most common types of PLC (Mejia and Pasko, 2020; McGlynn et al., 2021). Liver stem cells and mature hepatocytes genetically predisposed or affected by cirrhosis resulting from viral hepatitis infections, increased alcohol consumption, aflatoxin or other unfavourable factors, give rise to HCC (Llovet et al., 2021). CCA arises at bile or hepatic ducts or their junctions, and such types of malignancies often emerge in the non-cirrhotic liver (Banales et al., 2020) (Lee and Lee, 2017).

Treatment options for advanced HCC and CCA remain extremely limited. Moreover, anticancer therapy is often exhaustive for the patients due to a high toxicity of the standard chemotherapeutics, e.g., sorafenib for HCC (Raoul et al., 2019) and gemcitabine for CCA (Hryciuk et al., 2018). Combination therapy, which would allow a decrease in the doses of chemotherapeutics, would be of advantage in treatment of liver cancer (Walcher et al., 2020).

Traditional Chinese Medicine (TCM) and Traditional Tibetan Medicine (TTM) has a long and successful history for treating cancer in China (Luo et al., 2015; Li et al., 2018), and to date, the Guidelines of Diagnosis and Therapy in Oncology using TCM and TTM was generated with international standards consistent with modern clinical practice (Li et al., 2018; Tang et al., 2020). In this study, we concentrated on a TCM representative, a sandy beige mushroom Trametes robiniophila Murr, known in Chinese as “槐耳” or “Huaier”. Huaier grows on Sophora japonica L. tree trucks and belongs to Hymenomycetes, Basidiomycotina (Anithworth et al., 1973; Rogerson, 1974; Li et al., 2007; Sun et al., 2013). Huaier is commonly used in China for cancer complementary therapy including HCC treatment (Shan et al., 2017; Zhao et al., 2017; Pan et al., 2019; Li et al., 2020a; Tang et al., 2020). For TTM, we focused on: 1) a 25 component representative known in Chinese as “二十五味松石丸” or “Ershiwuwei Songshi Wan”; and 2) a 7 component representative known in Chinese as “七味红花殊胜丸” or “Qiwei Honghua Shusheng Wan”. Both TTM representatives include a rich variety of plants and minerals, among them are Aristolochia debilis Siebold & Zucc., Meconopsis cabrica Vig, Terminalia chebula Retz., Bambusa testilis McClure, and others, which all are depicted in Supplementary Figures S1, S2 and liquid chromatography mass spectrometry (LC-MS/MS) profiles for the compounds are provided in Supplementary Figure S3.

Importantly, Ershiwuwei Songshi Wan and Qiwei Honghua Shusheng Wan have been in use for the treatment of liver injuries such as hepatitis, cholecystitis, cirrhosis and other diseases (Qi et al., 2000; Yang, 2000; Ci et al., 2018; Ma et al., 2020; Yan Xi et al., 2020). The long history of these compounds in TCM and TTM for the treatment of liver diseases encouraged us to evaluate the potential of Huaier [designated later on in this study as compound 1 (C1)], Ershiwuwei Songshi Wan (designated as C2) and Qiwei Honghua Shusheng Wan (designated as C3) in two types of PLC, HCC and CCA, in an in vitro setting.

Importantly, along with monotherapeutic regimes using C1, C2 and C3 alone, we also tested their combinations with standard chemotherapeutics, sorafenib for HCC and gemcitabine for CCA. Standard chemotherapeutics applied at their plasma concentrations (defined for human) as well as carriers served as controls. We searched for synergistic effects and inhibition mechanisms while investigating the efficacy of TCM and TTM in combination with standard chemotherapeutics. We also explored the opportunity to reduce the dose of chemotherapeutics and evaluated the synergistic effects with TCM and TTM in HCC and CCA.

We first established HCC and CCA cells lines from primary murine HCC and CCA, both with known genotype (see an experimental setup in Figure 1A). The obtained HCC and CCA cells were seeded 16 h prior to the addition of compounds (Figure 1A). Thereafter, the freshly prepared DMEM extracts from all three compounds (C1, C2, C3) were added in triplicates at different concentrations as 1) monotherapy, or as 2) combination with standard chemotherapeutics (sorafenib for HCC and gemcitabine for CCA) (Figure 1B; Supplementary Table S2). After 24 and 48 h of incubation, the diverse readouts such as: microscopy, CVSA, CCK-8 and SA-β-Gal assays, as well as FACS were performed to assess the inhibitory property of therapeutic regimes and to define a main mechanism beyond the inhibition (Figure 1C).

In addition, we performed a detailed characterization of C1, C2 (Supplementary Figure S1) and C3 (Supplementary Figure S2) using LC-MS/MS as depicted in the Supplementary Figure S3. In order to characterize the constituents of C1, C2 and C3 water extracts of the samples were analyzed by reversed phase ultrahigh-performance liquid chromatography coupled to trapped ion mobility quadrupole time-of-flight mass spectrometry. All extracts were featured by highly complex compositions (Supplementary Figure S3). They mainly contained hydrophilic to mid-polar compounds, with C2 containing most of the mid-polar compounds. In order to get a first overview on individual metabolites, we performed a feature extraction to generate peak tables for each sample. After blank subtraction, we were able to detect 2330 features in C1, 3678 in C2 and 3377 features in C3. Using lockmass-calibrated high resolution masses and the smartFormula algorithm of Metaboscape 2022, we determined the most probable sum formulas where possible (Supplementary Table S3).

In this study, we investigated the inhibitory properties of TCM (C1) and TTM (C2 and C3) in two types of PLC, HCC and CCA, in vitro. Standard chemotherapeutics which are currently used in the clinic (sorafenib for HCC and gemcitabine for CCA) were used as positive controls. A detailed LC-MS/MS analysis of C1–C3 provided an overview of the mainly hydrophilic to mid-polar character of all compounds with C2 having the most mid-polar complexity. The chromatographic trace of C1 was similar compared to a previous study (Hu et al., 2016). Importantly, to our knowledge, LC-MS/MS characterizations of extracts from C2 and C3 have not been described in literature so far, and are presented for the first time in this study.

In our research, we investigated the compounds applied as monotherapies or in combination with reduced doses of chemotherapeutics. Importantly, both chemotherapeutics induce strong side effects in patients. Sorafenib, a standard medicine for HCC treatment since 2007, is accompanied by adverse side effects including fatigue, diarrhea, and skin dermatologic toxicity. Combination therapy aimed to lower side effects seems to be an approach with high perspectives (Hochnadel et al., 2017; Raoul et al., 2019; Walcher et al., 2020). Moreover, combination therapies using sorafenib and TCM were often hypothesized as promising (Cao et al., 2020; Zhou et al., 2020).

A study comparing the safety and efficacy of Huaier and sorafenib monotherapies following HCC resection demonstrated no significant differences between both tested drugs. However, the study showed fewer side effects in patients treated with Huaier (Jianyong et al., 2014). Importantly, in the sorafenib-treated group even a discontinuation of sorafenib was required due to severe adverse reactions in patients in comparison to Huaier-treated patients (Jianyong et al., 2014). Further, a multicenter randomized clinical trial has proven the effectiveness of Huaier granules as an adjuvant therapy after curative liver resection and resulted in significant prolongation of recurrence-free survival and reduced extrahepatic recurrence rates. The average concentration of sorafenib in plasma after standard administration reaches 13.8 μM (Fucile et al., 2015). The results of our investigation have shown that in combination C1 or C3 with even a half dose of sorafenib (6.9 μM) was sufficient to inhibit the growth of HCC cells. Such combination approaches can be especially important for patients who do not tolerate the full dose of sorafenib and take only a half dose due to strong side effects of the drug (Dufour et al., 2010). C3 applied at 1 mg/ml also showed efficacy alone without sorafenib. C2 did not inhibit the HCC cell line alone but in combination with sorafenib (3.45 and 6.9 μM). This TTM formulation increased the therapeutic effect of standard anti-cancer medicine.

Previous studies reported, that traditional Chinese herbal medicine on example of a multi-component Long-Dan-Xie-Gan-Tang formulation does not affect sorafenib metabolism in animal models (Ting et al., 2017). This allows for the hypothesis that combination therapy will benefit both from the curative effect of sorafenib and traditional preparations. Thus, in clinical practice, transarterial chemoembolization (TACE) combined with C1 treatment has already been used in HCC therapy (Zhao et al., 2017). Huaier extract has been reported to improve the health status of HCC patients after resection or liver transplantation (Lei et al., 2015; Chen et al., 2018). Our research hypothesis is further supported by other studies, showing promising results of combinations of Huaier in HCC and other cancers. Such, combination treatment of oxaliplatin and Huaier had a significant synergistic anti-cancer effect and inhibited expression of Yes-associated protein (Tao et al., 2018). In another study, Huaier granules combined with Tegafur Gimeracil Oteracil Potassium could promote patient prognosis with a better disease-free and overall survival rate and induced apoptosis in gastric cancer (Qi et al., 2020a). Additionally, Huaier aqueous extract combined with routine chemotherapeutic drugs showed a synergistic effect on human acute lymphoblastic leukemia cells in vitro (Qu et al., 2020).

Huaier is known to decrease the proliferative and migratory potential of HCCs in murine models partially by down-regulation of Yes-associated protein 1 (Shan et al., 2017; Tao et al., 2018), to decrease the levels of phosphorylated AKT and mTOR (Bao et al., 2016), and to interfere in liver tumor angiogenesis (Li et al., 2015; Qi et al., 2020b). Still, the molecular mechanisms of HCC inhibition need further investigation.

In our study, we detected that two compounds C1 and C3 were the most efficacious in HCC, whereas in CCA C2 and C3 compounds demonstrated strong inhibitory properties and C1 unexpectedly proved to be inefficient. In contrast to our observation on C1 in CCA, a case study report of a CCA patient receiving hepatectomy and subsequent treatment with C1 showed an improvement in recurrence-free survival, indicating that C1 has the potential to be used for the treatment of CCA (Feng et al., 2022). Although C2 showed no effect on HCC in our study, Ngamkitidechakul et al. (2010), Achari et al. (2011), and Zhang et al. (2021) reported the effect of the single components of C2, showing its potency to inhibit HCC. Interestingly, C2 and C3 share four common ingredients (T. chebula Retz., A. debilis Siebold & Zuccarini, Meconopsis cambrica Vig., B. testilis McClure). We assume that these four components were responsible for the efficacy of C2 and C3 in CCA, where both compounds showed strong efficacy. In line with our research, several studies investigated these four components as a single agent in different types of cancer and confirmed their efficacy in the inhibition of cancer cell growth (Saleem et al., 2002; Achari et al., 2011; Fan et al., 2015a; Fan et al., 2015b; Ravi Shankara et al., 2016). C3 showed to be effective in both types of cancer in our study. It´s efficacy in HCC is probably mediated by the combination of three ingredients that are exclusive for C3 (are not present in C2 which was inefficient in HCC) and comprise: Carthamus tinctorius L., Swertia bimaculata, and Ephedra sinica Stapf. Notably, the efficacy of those three single components of C3 was reported in other types of cancer (Park et al., 2016; Sharula and Wu, 2017; Hyuga et al., 2020) and is further supported by our study.

Importantly, combinations of the successful compounds with standard chemotherapeutics demonstrated the most pronounced efficacy in comparison to human plasma concentrations of standard chemotherapeutics. We observed that the results of CVSA and CCK-8 assays detecting the inhibition of cell growth strongly correlated. We could show that late apoptosis was the main mechanism beyond the inhibition of C1 and C3 in HCC and early and late apoptosis represented the main protective mechanism of C2 and C3 in CCA. It is worth to mention, that our data on the mechanism of sorafenib standard chemotherapy in HCC and gemcitabine standard chemotherapy in CCA is fully in line with previous reports from Lin and colleagues (Lin et al., 2017) and Pauwels et al. (2009) and Li et al. (2020b), respectively, as well as the recent report by Pylypchuk et al. (2022).

In line with our studies, treatment of human HCC cell lines HepG2 and Huh7 with Huaier extracts resulted in apoptosis development (Bao et al., 2016; Xu et al., 2020).

We further demonstrated that cellular senescence and necroptosis were not associated with the inhibition of HCC and CCA cells in our study. Cycle arrest of the cells and subsequently entering the cellular senescence program is beneficial for limiting liver cancer development, as we have previously shown (Kang et al., 2011; Yevsa et al., 2012; Eggert et al., 2016; Hochnadel et al., 2017; Walcher et al., 2020). Senescent cells are characterized by a special senescence-associated secretory phenotype (so called “SASP”) including production and secretion of pro-inflammatory cytokines and chemokines influencing cell-cell interactions and tissue homeostasis (Lee and Schmitt, 2019) and attracting immune cells towards senescence sites (Kang et al., 2011; Eggert et al., 2016). Therapy-induced senescence plays an important role in suppression of cancer development (Yevsa et al., 2012; Hochnadel et al., 2017; Walcher et al., 2020; Wyld et al., 2020). There are several reports demonstrating that Huaier is able to induce senescence in cancerous cells. Huaier extract increased amount of cells in S and G2/M phases in gastric cancer cell lines (Wang et al., 2019), induced G0/G1 arrest in endocrine resistant breast cancer cell lines (Gao et al., 2017) and HCC cell lines (Bao et al., 2016), and S phase arrest in hepatoblastoma cells (Xu et al., 2020). Our study is in line with previous reports, as we observed, that C1 as a monotherapy and a combination therapy with sorafenib induced cellular senescence in HCC and CCA cell lines. However, we did not observe, that senescence correlated with protective capacity and inhibition of HCC and CCA in vitro. We do not exclude that induction of senescence in vivo might result in a very strong immune response towards senescent cancerous cells, which will further increase protection against liver cancer, as recently reviewed (Walcher et al., 2020).

It is important to mention that molecular mechanisms of multicomponent C2 and C3 in HCC and CCA are highly unanswered and there is no information in scientific literature about the application of C2 and C3 for CCA treatment. A few studies showing their effects on cholestatic hepatitis (Yang, 2000; Ci et al., 2018), alcoholic liver injury and chronic liver injury induced by CCl₄ (Ma et al., 2020) have been published. Although several single constituents of C2 and C3 have been reported to inhibit the growth of cancerous cells in different types of cancer (as reviewed by us in this study in Supplementary Tables S5–S7), the reports for multicomponent C2 and C3 are still very scarce. Our research has deepened the knowledge on C2 and C3. We defined the mechanism and found out that C3 as a monotherapy or in combination with 3.45 or 6.9 μM of sorafenib decreased the HCC cell line growth, thereby inducing cellular senescence and late apoptosis. However, C2 was efficient only in combination with sorafenib and induced late apoptosis and necroptosis in HCC.

Our study provides further knowledge on mechanisms beyond the efficacy of C1 in CCA. It was reported that a combination of Huaier with 5-fluorouracil (5-FU) in vitro on the CCA cell line Huh28 resulted in growth inhibition, apoptosis and arrest of cells in the S phase (Fu et al., 2019). Inhibition of proliferation, metastasis and invasion of CCA cells by activating apoptosis and downregulating TP73-AS1 in CCA after the treatment with Huaier have been described (Ji et al., 2020). In contrast to other reports, C1 extract in our experiments did not reduce the amount of CCA cells. The low efficacy of C1 in CCA is probably associated with the induction of cellular senescence as well as necroptosis phases, whereas protective early and late apoptosis were less efficient than in the gemcitabine group.

Side effects of gemcitabine include fatigue, nausea and vomiting, diarrhea, leuco- and thrombopenia and allergic reactions. Severe cases including cardio-pulmonary insufficiency with fatal consequences have been also described (Tonato et al., 1995; Shan et al., 2017). Taking into account the poor prognosis for CCA patients (Banales et al., 2020), various possibilities of combination therapy have to be urgently investigated. Successful combination therapy with Huaier and gemcitabine was described for pancreatic cancer but not for CCA (Chen et al., 2021). In contrast, we found that C1 was not efficient in CCA inhibition. However, we observed a strong inhibitory effect of C2 and C3 on CCA cell line growth and the efficacy was dependent on the induction of early and late apoptosis.

Summarizing everything mentioned above, we could state for the first time that the mechanism of inhibitory effect of C2 and C3 on CCA is explained by triggering early and late apoptosis, whereas cellular senescence and necroptosis did not correlate with protection.

Combination therapy of C2 or C3 with gemcitabine on CCA allowed decreasing the effective concentration of this standard medicine from 50 μM (human plasma concentration) to 0.1, 0.05 or even 0.025 μM.

One of the most important outcomes from combination therapy is a possibility to reduce the therapeutic doses of toxic anti-cancer preparations. Addition of TCM and TTM to schemes of the standard therapy could allow decreasing the consumption of anticancer drugs with the same efficacy of treatment. To confirm this suggestion, further experiments in vivo using C1, C2 and C3 in autochthonous HCC and CCA murine models need to be performed.

In this research, we studied the inhibitory properties of TCM (C1) and TTM (C2 and C3) in two types of PLC, HCC and CCA, in vitro. We detected, that two compounds C1 and C3 were the most efficacious in HCC, whereas in CCA C2 and C3 compounds demonstrated strong inhibitory properties and C1 was unexpectedly shown as inefficient. Importantly, combinations of the successful compounds with standard chemotherapeutics (sorafenib for HCC and gemcitabine for CCA) demonstrated the most pronounced efficacy in comparison to human plasma concentrations of standard chemotherapeutics. Moreover, application of combination therapy allowed for a strong reduction of standard chemotherapeutics: a half dose of sorafenib or even a dose 500 fold lower of gemcitabine applied were more efficacious in combination with compounds than human plasma concentrations of standard chemotherapeutics. The mechanism behind the successful inhibition was associated with the induction of late apoptosis in HCC and early/late apoptosis in CCA (see graphical abstract). Cellular senescence and necroptosis do not seem to be involved in the inhibition of HCC and CCA cell lines in vitro. Further in vivo studies using autochthonous HCC and CCA models are required to evaluate the efficacy of TCM/TTM and mechanism behind it. Special importance has to be given to combination therapies which show synergistic effects, allowing for a reduction of toxic chemotherapeutics and have therefore a strong perspective for patients with HCC and CCA.