Hepatocellular carcinoma (HCC) is the fifth most common type of cancer and the fourth leading cause of cancer-related deaths in the world. HCC has a reported recurrence rate of 70%-80% after 5 years of follow-up. Controlling tumor recurrence is the most critical factor associated with HCC mortality. Conventional salvage therapies for recurrent HCC include re-hepatectomy or liver transplantation, transcatheter arterial chemoembolization, Y-90, target therapy, and immunotherapy; however, these conventional treatment modalities have yet to achieve consistently favorable outcomes. Meanwhile, previous studies have demonstrated that conventional therapies in combination with traditional Chinese medicine (TCM), acupuncture, moxibustion or dietary supplements could notably benefit patients with HCC recurrence by strengthening and augmenting the overall management strategy. However, systemic reviews related to the interactions between complementary therapies and conventional therapy in recurrent HCC are limited. In this review, we discuss the molecular mechanisms underlying the functions of complementary therapies for recurrent HCC, which include augmenting the local control to improve the congestion status of primary tumors and reducing multicentric tumor occurrence via inducing autophagy, apoptosis or cell cycle arrest. TCM and its derivatives may play important roles in helping to control HCC recurrence by inhibiting epithelial-mesenchymal transition, migration, invasion, and metastasis, inhibiting cancer stem cells, and ameliorating drug resistance.

Worldwide, hepatocellular carcinoma (HCC) is the fifth most common type of cancer and the fourth leading cause of cancer-related deaths. The highest prevalence rates of HCC are reported in East Asia, while the incidence rates are approximately 6.7/100000 among the age-adjusted population, 2.6% in nonalcoholic steatohepatitis cirrhosis patients, and 0.13% in nonalcoholic fatty liver disease patients[1,2]. The annual incidence rate increased by 2% to 3% between 2007 and 2016, while HCC notably has the second poorest 5-year survival rate of all cancer types (18%)[3]. Early-stage treatments for HCC include resection, liver transplantation, and radiofrequency ablation (RFA), while transcatheter arterial chemoembolization (TACE), chemotherapy, molecular target therapy, immunotherapy with immune checkpoint inhibitors may commonly be applied in later stages. Even in resected HCC, the recurrence rate remains over 10% after 1 year, and 70%-80% after 5 years[4]. Thus, controlling tumor recurrence is a primary concern to reduce HCC mortality rates.

Salvage therapies for recurrent HCC include re-hepatectomy or liver transplantation, TACE, Y-90, target therapy, and immunotherapy. Meanwhile, studies have reported that complementary therapies such as traditional Chinese medicine (TCM), acupuncture, and dietary supplements have demonstrated notable anti-tumor effects[5,6]. These complementary therapies affect multiple biological mechanisms such as promoting tumor cell apoptosis, autophagy, cell cycle arrest, anti-metastasis, anti-angiogenesis, anti-proliferation, anti-epithelial-mesenchymal transition (EMT), and control of cancer stem cell (CSC) proliferation[5,6]. In addition, TCM has been noted to prevent drug resistance and act to facilitate conventional therapies in cases of recurrence.

However, systemic reviews related to complementary therapies in recurrent HCC are limited. The aim of this review is to introduce and discuss the molecular mechanisms underlying the effects of complementary therapies in recurrent HCC.

Patients graded as Child-Pugh class A or B, presenting with three or fewer tumors of < 3 cm are commonly recommended local control methods such as hepatectomy and RFA[7]. Although hepatic resection has a lower reported rate of recurrence as compared to RFA or TACE, the recurrence rate remains relatively high (70%-80%)[4]. This could be due to incomplete treatment of the primary tumor related to poor tumor location or surgical factors. Studies have indicated two critical mechanisms which come into play after local control.

Reduced multicentric tumor recurrence

The second mechanism involves multicentric tumor occurrence in the liver which could lead to recurrence. To this end, TCM offers multiple compounds presenting anti-tumor effects, such as flavonoids[28], phenylpropanoids, quinones, and alkaloids[5]. Alkaloids act to induce autophagy and apoptosis to inhibit HCC proliferation[5]. Piperidine alkaloids have been reported to induce mitochondrial fission and to regulate the mammalian sterile 20-like kinase 1-c-Jun N-terminal kinase (JNK) pathway, the extracellular signal-regulated kinase (ERK) signaling pathway and the phosphatase and tensin homolog deleted on chromosome 10 (PTEN)/ protein kinase B (AKT) pathway[5]. In addition, isoquinoline alkaloids have been shown to affect the AKT pathway, the AKT/Forkhead box O (FoxO) 3a/S-phase kinase-associated protein 2 (Skp2) axis, the phosphatidylinositol 3-kinase (PI3K)/AKT mammalian target of rapamycin (mTOR) pathway, and the Wnt/β-catenin-mediated pathway, thereby hindering HCC cell growth[5]. Further studies have indicated that terpenoid alkaloids, including perillyl alcohol, geraniol and paclitaxel are effective antitumor agents[29]. In a study involving Hep G2 and BEL-7402 cell lines, terpenoid alkaloids regulated AKT, p53, caspase-3, mitogen-activated protein kinase (MAPK) and Ras which induced apoptosis and cell cycle arrest, and effectively inhibited proliferation[5]. Meanwhile, indole alkaloids have been noted to influence the nucleotide-binding oligomerization domain 1 pathway, the AKT pathway, and the WW domain-containing oxidoreductase-dependent pathway to induce apoptosis and cell cycle arrest, and thereby inhibit HCC proliferation[5]. In terms of TCM, herbs containing steroidal alkaloids include Solanaceae, Apocynaceae, and Liliaceae[30]. Steroidal alkaloids may induce necroptosis, apoptosis, and cell cycle arrest to inhibit cell proliferation. Yin et al[31] reported that the compound solamargine induced autophagy and apoptosis by affecting the microRNA (miR)-192-5p/CYR61/Akt signaling pathways[31]. Additionally, in a study involving a HepG2 cell line, quinoline alkaloids were reported to induce necroptosis and apoptosis[5]. Of further note, flavonoids have been shown to offer notable anti-HCC effects. Wogonin, one type of flavonoid, acts to induce apoptosis and cell cycle arrest by activating the MOB1-LATS1 signal pathway and over-expressing phospho-glycogen synthase kinase (GSK) 3beta Tyr216[32,33]. Additionally, wogonin has been reported to inhibit HCC proliferation by affecting nuclear factor kappa B (NF-κB)/B-cell lymphoma 2 (Bcl-2), epidermal growth factor receptor (EGFR) and the EGFR downstream ERK/AKT signal pathway[5]. Furthermore, baicalein has been reported to inhibit cancer progression by inducing autophagy, apoptosis and cell cycle arrest in HCC cell lines[34]. Similarly, the long non-coding RNAs (lncRNAs)-hsa-miR-4443-AKT1 pathway responds positively to baicalein treatment[35]. Studies have further revealed that silibinin induces autophagy through the adenosine monophosphate (AMP)-activated protein kinase (AMPK) pathway and induces apoptosis by up-regulated p21/cyclin-dependent kinases (CDK) 4 and p27/CDK4 complexes, and down-regulated Rb-phosphorylation and E2F1/DP1 complex[36,37]. Lee and Kwon[38] demonstrated that luteolin causes ER stress in p53-wild type HepG2 cells and Hep3B cells[38]. It has further been shown that luteolin induces apoptosis and cell cycle arrest by transforming the growth factor (TGF)-β1, p53, and Fas/Fas-ligand signaling pathway and increasing the BCL2-associated X protein (Bax)/Bcl-XL ratio[39,40]. Similarly, studies have reported that kaempferol induces autophagy by activating the AMPK signaling pathway[41]. Moreover, the combination of luteolin and kaempferol has been shown to increase caspase-3 and reactive oxygen species (ROS) reactions and induce apoptosis in a rat model[42]. Additionally, quercetin has been noted to inhibit cell proliferation by decreasing ROS and downregulating the PI3K pathway and induce apoptosis and autophagy by modulation of the PI3K/Akt/mTOR, Wnt/-catenin and MAPK/ERK1/2 pathways[43,44]. Additionally, studies have reported that phenylpropanoid (chlorogenic acid and 16-O-caffeoyl-16-hydroxylhexadecanoic acid) and quinone (thymoquinone and juglanthraquinone C) induce apoptosis in HCC cell lines[45-48]. Separate studies have demonstrated that 4-acetylantrocamol LT3 induces autophagy by activation of the AMPK pathway[49], while aloin and andrographolide induce apoptosis[50,51], and plantamajoside and sanguisorba Officinalis L. decrease proliferation in HCC cell lines[52,53]. Curcumin also offers antioxidant, apoptotic, and anti-inflammatory effects, and is thus applied in the treatment of HCC[54]. Meanwhile, several herbs associated with TCM have been reported to inhibit HCC proliferation by targeting miRNAs, these include Coptidis rhizoma (miR21 and miR23a), berberine (miR-23a), ginsenoside (miR-491), camptothecin (miR-122), and matrine (miR-21)[55]. Yang et al[56] reported on a randomized control trial of 291 patients who received the Fuzheng Jiedu Xiaoji formula and consequently exhibited a reduced mortality rate by the effective inhibition of liver cancer cell proliferation and migration via modulated AKT/CyclinD1/p21/p27 pathways[56]. The compounds associated with reducing multicentric tumor occurrence are shown in Table 1.

Compound or Chinese herbal medicine	Cell line	Molecular mechanism	Effect	Ref.
Piperidine alkaloids	HepG2, Hep3B	Modulate Mst1-JNK pathway, ERK pathway, PINK1/Parkin axis and PTEN/AKT pathway	↑Autophagy, apoptosis, mitochondrial fission. ↓Proliferation	Liu et al[5]
Isoquinoline alkaloids	SMMC-7721, HCCLM9, Huh7, HepG2	Modulate AKT pathway, AKT/FoxO3a/Skp2 axis, PI3K/AKT-mTOR pathway, Wnt/β-catenin-mediated pathway and anthranilic acid metabolic pathway	↑Autophagy, apoptosis, cell cycle arrest. ↓Proliferation	Liu et al[5]
Indole alkaloids	HepG2, SMMC-7721, Hepa1-6, BEL-7404, Hep3B, Huh7	Modulate NOD1 pathway, AKT pathway and WWOX-dependent pathway	↑Apoptosis, cell cycle arrest. ↓Proliferation	Liu et al[5]
Terpenoids alkaloids	HLE, L-02, BEL-7402, HepG2	Modulate AKT, p53, caspase-3, MAPK, AFP, Ras	↑Apoptosis, cell cycle arrest. ↓Proliferation	Liu et al[5]
Steroidal alkaloids	HepG2, SMMC-7721, Hep3B	↑Gene expression of human TNFR I	↑Necroptosis, apoptosis, cell cycle arrest. ↓Proliferation	Liu et al[5]
Quinoline alkaloids	HepG2, L-02, QGY-7703	Modulate MMP-9, PCNE, ANT3 and VEGF	↑Necroptosis, apoptosis	Liu et al[5]
Solamargine	HepG2, Huh7	Modulate miR-192-5p/CYR61/Akt pathway	↑Autophagy, apoptosis. ↓Proliferation	Yin et al[31]
Wogonin	HepG2, BEL-7402	Modulate NF-κB/Bcl-2, EGFR and EGFR/ERK/AKT pathway	↓Proliferation	Liu et al[5]
	SMMC-7721, HCCLM3	↑MOB1-LATS1 pathway. ↓YAP, WW domain–containing transcription regulator 1, and expression of Claspin	↑Apoptosis, cell cycle arrest	Wu et al[32]
	MHCC97-L, HepG2	↑Phospho-GSK-3β Tyr216. ↓Cyclin D1	↑Cell cycle arrest. ↓Proliferation	Hong et al[33]
Baicalein	Human HCC tissues	Modulate lncRNAs-hsa-miR-4443-AKT1 pathway	↓Proliferation	Zhao et al[35]
Silibinin	HepG2, Hep3B	Modulate AMPK pathway	↑Autophagy. ↓Glycolysis	Yang et al[36]
	Huh7, HepG2, Hep3B, PLC/PRF/5 human hepatoma cells	↑p21/CDK4 and p27/CDK4 complexes. ↑Caspase-3 and -9. ↓Rb-phosphorylation and E2F1/DP1 complex	↑Apoptosis. ↓Proliferation	Lah et al[37]
luteolin	p53-wild type HepG2 cells, Hep3B	↑Endoplasmic reticulum stress	↑Autophagy, apoptosis	Lee and Kwon[38]
	HepG2	Modulate TGF-β1, p53, Fas/Fas ligand pathway	↑Apoptosis, cell cycle arrest	Yee et al[39]
	HepG2, SK-Hep-1, PLC/PRF/5, Hep3B, HA22T/VGH	↑Bax/Bcl-XL ratio. ↑Caspase-3	↑Cell cycle arrest	Chang et al[40]
Kaempferol	HepG2, Huh7, BEL-7402, SMMC	↑AMPK pathway. ↑Melanoma antigen 6, AMPK ubiquitin ligase, AMPKα1	↑Autophagy	Han et al[41]
Luteolin and Kaempferol	DEN and 2-AAF induced rat model	↑Caspase-3 and ROS reaction	↑Apoptosis	Seydi et al[42]
Quercetin	HepG2	↑p53, BAX. ↓ROS, PI3K, COX-2, PKC	↓Proliferation	Maurya and Vinayak[43]
Chlorogenic acid	Hep-G2, Huh7	↑BH3-only protein Bcl-2 binding component 3. ↓Noncanonical NF-κB pathway	↑Apoptosis	Jiang et al[45]
Thymoquinone	Thioacetamide (TAA)-induced HCC, Sprague Dawley rats	↓Oxidative stress. ↓TGF-β1	↑Apoptosis	Helmy et al[47]
Juglanthraquinone C	HepG2, BEL-7402	↑Akt/Fox O pathway. ↑Intracellular ROS level	↑Apoptosis	Hou et al[48]
4-acetylantrocamol LT3	HepG2	↑AMPK pathway	↑Autophagy	Chen et al[49]
Aloin	--	Modulate circ_0011385/miR-149-5p/WT1 axis	↑Apoptosis and autophagy	Fu et al[50]
Andrographolide	Hep G2	↓EphB4	↑Apoptosis	Duan et al[51]
Sanguisorba Officinalis L.	HepG2 cells	Modulate EGFR, PI3K/AKT, NF-κB and MAPK pathways	↓Proliferation	Jiang et al[52]
Plantamajoside	Huh7, PLC/PRF 5, THLE-2	↓NF-κB and Cox-2	↓Proliferation	Luo et al[53]

Akt: Protein kinase B; AMPK: AMP-activated protein kinase; Bax: BCL2-Associated X Protein; Bcl-2: B-cell lymphoma 2; CDK: Cyclin-dependent kinases; COX-2: Cyclooxygenase-2; EGFR: Epidermal growth factor receptor; EPHB4: EPH Receptor B4; ERK: Extracellular signal-regulated kinase; FoxO: Forkhead box O; GSK: Glycogen synthase kinase; JNK: c-Jun N-terminal kinase; MAPK: Mitogen-activated protein kinase; MMP: Matrix metalloproteinase; Mst1: Mammalian sterile 20-like kinase 1; mTOR: Mammalian target of rapamycin; NF-κB: Nuclear factor kappa B; NOD: Nucleotide-binding oligomerization domain; PI3K: Phosphatidylinositol 3-kinase; PINK1: PTEN-induced kinase 1; PTEN: Phosphatase and tensin homolog deleted on chromosome 10; ROS: Reactive oxygen species; Skp2: S-phase kinase-associated protein 2; TGF: Transforming growth factor; TNFR: Tumor necrosis factor receptor; VEGF: Vascular endothelial growth factor; WWOX: WW domain-containing oxidoreductase; YAP: Yes-associated protein; DEN: Diethylnitrosamine; 2-AAF: 2-acetylaminofluorene.

In summary, TCM and acupuncture treatments have been reported to augment the local control by improving the congestion status of the primary tumor. The molecular mechanisms are related to attenuating nitric oxide production and inhibiting fibrosis progression (via reducing procollagen III), thereby effectively preventing HCC recurrence. Meanwhile, various anti-tumor compounds have been reported to reduce multicentric tumor occurrence by inducing autophagy, apoptosis and cell cycle arrest to inhibit HCC proliferation. With regards to the pathways associated with autophagy, multiple herbs and their derivates have been shown to primarily affect the AMPK/mTOR pathway and the Akt/Fox O pathway, and to regulate the JNK level. Additionally, several herbs and their derivates have been reported to induce apoptosis mainly via regulating the PTEN/AKT pathway, the BCL-2/Bax ratio and the NF-κB level, and by increasing the caspase 3/9 level. As for cell cycle arrest, several herbs and their derivates affect the PI3K/AKT-mTOR pathway, the AMPK/mTOR pathway and the NF-κB level, and act to up-regulate levels of p53 and phospho-GSK-3β (Figure 1).

Figure 1 Summary of molecular pathways involved in complementary therapies in recurrent hepatocellular carcinoma. Akt: Protein kinase B; AMPK: AMP-activated protein kinase; Bax: BCL2-Associated X Protein; Bcl-2: B-cell lymphoma 2; CD: Cluster of differentiation; EpCAM: Epithelial cell adhesion molecule; ERK: Extracellular signal-regulated kinase; FoxO: Forkhead box O; GSK: Glycogen synthase kinase; HCC: Hepatocellular carcinoma; HOTTIP: HOXA distal transcript antisense RNA; JNK: c-Jun N-terminal kinase; MAPK: Mitogen-activated protein kinase; MCL-1: Myeloid cell leukemia 1; MMP: Matrix metalloproteinase; mTOR: Mammalian target of rapamycin; MUC1: Mucin 1; NF-κB: Nuclear factor kappa B; PI3K: Phosphatidylinositol 3-kinase; PTEN: Phosphatase and tensin homolog deleted on chromosome 10; PTTG1: Pituitary tumor transforming gene 1; RAF: Rapidly accelerated fibrosarcoma; STAT3: Signal transducer and activator of transcription 3; TGF: Transforming growth factor; TUG1: Taurine upregulated 1.

The liver has a sinus structure, abundant blood flow, an immunosuppressive microenvironment, and is involved in regulating blood circulation and the lymphatic system[57]. The migration, invasion, and metastasis associated with HCC recurrence significantly influence mortality rates. HCC is prone to metastasis to the lungs (47%), lymph nodes (45%), bones (37%), and adrenal glands (12%). The prognosis of HCC patients presenting with extrahepatic metastasis is poor[58], and likewise linked to a poor survival rate. Of note, anti-metastasis drugs, including sorafenib, lenvatinib, and a combination of protocols (e.g., sintilimab plus bevacizumab) have not demonstrated favorable outcomes in metastatic HCC patients[59,60].

Research indicates that the progression of cancer metastasis involves a series of steps[61]. First, EMT occurs in the early stages of tumor-cell metastasis, which allows epithelial phenotypic cells to convert into mesenchymal-like cells[62]. EMT studies have observed the involvement of epithelial proteins (E-cadherin, claudins, occludins, and α-catenin) as well as mesenchymal phenotypic proteins (N-cadherin, β-catenin, and vimentin). There are various pathways, including Wnt/β-catenin, mesenchymal-epithelial transition factor (c-Met)/hepatocyte growth factor (HGF)/Snail, neurogenic locus notch homolog protein 1/NF-κB, TGF-β/suppressor of mothers against decapentaplegic (SMAD), and basic fibroblast growth factor (FGF)-related signaling which play roles in EMT[62]. A coordinated sequence of invasion and metastasis subsequently occurs, which involves parenchymal, nonparenchymal and immune cells related to cytokines, histone methyltransferase/demethylase [e.g., enhancer of zeste homolog 2, SETDB1 (KMT1E) and euchromatic histone lysine methyltransferase 2 (G9a, EHMT2)], and non-coding RNAs[63,64].

Multiple TCM herbs and their derivatives have been shown to possess inhibitory effects against EMT as related to HCC. Scutellariae baicalensis, and its derivative baicalin, have recognized hepatoprotective effects, acting to modulate the TGF-β/SMAD, MAPK and NF-κB pathways, and inhibit matrix metalloproteinase (MMP)-1 to hinder EMT in HCC[65]. In a study involving Huh7 and MHCC97-H cell lines, astragaloside IV modulated the Akt/GSK-3β/β-catenin pathway and inhibited EMT[66]. Additionally, camptothecin, a topoisomerase inhibitor, has been shown to inhibit EMT by upregulating the expressions of zonula occludens protein-1, E-cadherin, and claudin-1[57]. Isoviolanthin has been shown to inhibit TGF-β1, associated with the downregulation of the TGF-β/SMAD and PI3K/Akt/mTOR signaling pathways, which resulted in the inhibition of EMT[67]. 18β-Glycyrrhetinic Acid, an ingredient of Glycyrrhiza glabra L. root (licorice), has been found to inhibit EMT and metastasis by suppressing the Src (Sarcoma) homology 2 domain phosphatase (SHP)1&SHP2/signal transducer and activator of transcription 3 (STAT3)/Snail pathway[68]. One study revealed that Echinacea purpurea regulated the PI3K/Akt signaling pathway to inhibit EMT[69]. Separately, tetrandrine impeded the Wnt/β-catenin signaling pathway and decreased metastatic tumor antigen 1 expression in Huh7 and Hep3B cell lines, leading to the inhibition of EMT, invasion, and migration[70]. Other studies have reported that scorpion and myricetin regulated the epithelial/mesenchymal proteins ratio and inhibited EMT[71,72]. More recently, miRNA and lncRNA have been associated with impacting the EMT process and drug resistance in HCC. Hydroxygenkwanin (upregulation in miR320a)[73], oleanolic acid (upregulation in miR-122)[74], aloin (regulation in circ_0011385/miR-149-5p/WT1 axis)[50], and puerarin (regulation in miR-21/PTEN/EMT axis)[75] inhibited EMT, invasion and migration in HCC cell lines. Both in vivo and in vitro studies by Chen et al[76] demonstrated that corylin, a flavonoid compound extracted from Psoralea corylifolia L., upregulated lncRNA growth arrest-specific transcript 5 to inhibit EMT and decrease tumor size[76].

Researchers have revealed that TCM offers multiple herbs and formulas found to inhibit migration and invasion and may therefore be applied to prevent HCC recurrence. Both kaempferol and dulcitol have been noted to decrease MMP to impede migration[77,78]. Sanguisorba officinalis has been shown to modulate the PI3K/AKT, NF-κB and MAPK signaling pathways to inhibit HepG2 cell migration and invasion[52]. Zanthoxylum avicennae augemented PP2Acα, GSK-3β, adenomatous polyposis coli protein and β-transducin repeat-containing protein levels, and diminished β-catenin, p-GSK-3β, T-Box Transcription Factor 3 and interleukin (IL)-8 proteins to prevent metastasis[79]. A study by Feng et al[80] reported that bufalin upregulated tank-binding kinase 1 and the interferon regulatory factor 3 and NF-κB pathways to hinder migration and invasion[80]. In terms of specific TCM formulas linked to EMT inhibition, QHF [consisting of HuaChanSu, 20(R) ginseng saponin Rg3, notoginseng total saponin, and lentinan] activated p38/JNK/MAPK pathway and inactivated ERK pathway to inhibit migration and invasion in a study using HepG2 cells[81]. The main ingredients in QHF, including cinobufotalin, ginsenoside Rg3, panax notoginsenosides, and lentinan, act to downregulate the HGF/c-Met signaling pathway to prevent metastasis and invasion[82]. In addition, the Biejiajian pill and Jiedu recipe have separately been found to prevent EMT by suppressing the Akt/GSK-3β/Snail signaling cascade and modulating E-cadherin/N-cadherin ratio, respectively[83,84]. In a study involving multiple HCC cell lines, the Xiaoai Jiedu recipe regulated the miRNA-29a/transcription 3 axis and decreased metastasis[85]. The TCM compounds and formulas associated with reducing HCC recurrence via inhibition of EMT, migration, invasion and metastasis are summarized in Table 2.

Collectively, multiple investigations have demonstrated that TCM and its derivative compounds act to prevent HCC recurrence by inhibiting EMT, migration, invasion and metastasis. The possible mechanisms involved in this prevention include suppressing TGF-β and MMP-2/9, E-cadherin/N-cadherin ratio reversion, and microRNA modulation (Figure 1).

CSCs may be characterized as possessing features of self-renewal, differentiation potential, and colony-forming. Research indicates that CSCs may be a major cause of tumorigenesis, metastasis and antitumor agent resistance, and are thus a primary culprit in tumor relapse after therapy. Dai et al[86] suggested that HCC CSCs create an immunosuppressive microenvironment through both intrinsic and extrinsic mechanisms to escape immune surveillance[86]. In HCC cell lines, CSCs are primarily identified as CD133[87]; while other surface markers include epithelial cell adhesion molecule (EpCAM), CD44, CD13, CD90, CD24, CD47, oval cell marker OV6, K19, c-kit, breast cancer resistant protein, and aldehyde dehydrogenases[88]. Meanwhile, signaling pathways including the Wnt/β-catenin, AKT/GSK-3β/β-catenin, ERK/Snail, AKT/PKB, AKT/mTOR, and TGF-β pathways have been recognized in CSC formation[88].

With regards to TCM treatments associated with CSC hindrance, investigations have revealed several notable findings. Antrodia cinnamomea, a fungus species, has well-documented anti-HCC effects, and has been found to hinder CD133+ CSC and downregulating onco-miRNAs in glioblastoma multiforme 8401 and breast adenocarcinoma (MDA-MB-231) cell lines[89]. Tiliroside, a compound isolated from Tribulus terrestris L., decreased CD133+ and carbonic anhydrases XII expressing while concurrently activated E2F1/E2F3/Caspase-3 axis in Hep3B and SNU-449 cell lines[90]. In addition, 8-bromo-7-methoxychrysin decreased expressions of CD133, CD44 and IL-6, and inhibited self-renewal of SMMC-7721- and MHCC97H-derived liver cancer stem-like cells[91]. Curcumin decreased expressions of several CSC markers (c-KIT, EpCAM, CD133, RING finger protein 51, and NANOG) and inhibited the oncogenic NF-κB signaling pathway[92]. Sophocarpine decreased expressions of CD133, CD90, and EpCAM as well as TGF-β to inhibit both EMT and CSC[93]. Matrine, extracted from Sophora flavescens, reduced the EpCAM+/CD133+ in HCC cells by inactivating the PI3K/AKT/mTOR and AKT/GSK-3β/β-catenin signaling pathways[94]. In addition, Brucea javanica has been found to decrease expressions of CD133, NANOG and EpCAM, subsequently inducing apoptosis and suppressing CSCs[95]. 2-Ethoxystypandrone, extracted from Polygonum cuspidatum, blocked STAT3 activation to decrease cancer stemness[96]. Meanwhile, the formula BRM270 decreased CD113+ cells and inhibited liver CSCs both in vivo and in vitro[97]. Songyou Yin (consisting of Salvia miltiorrhiza, Astragalus membranaceus, Lycium barbarum, Crataegus pinnatifida, and Trionyx sinensis) prevented CSCs by decreasing the expressions of CSC markers including CD90, CD24 and EpCAM, and increased chemosensitivity to oxaliplatin[98]. Moreover, differentiation therapy has revealed further opportunities for controlling CSCs. The combination of Astragalus membranaceus and Salvia miltiorrhiza extract has been found to increase the differentiation of HCC cells by modulating TGF-β/TβR and Imp7/8 protein expression[99]. Additionally, Rui-Chuan et al[100] reported that isoverbascoside induced SMMC-7721 differentiation, thereby acting as a potential anti-tumor target[100]. The compounds and formulas applied for preventing HCC recurrence via inhibition of cancer stemness are presented in Table 3.

In summary, CSC plays an important role in HCC recurrence. In this regard, TCM and its derivative compounds could suppress CSC markers, particularly in CD113, CD44 and EpCAM, reduce TGF-β which promotes CSC properties, and suppress the PI3K/AKT/mTOR and AKT/GSK-3β/β-catenin signaling pathways (Figure 1).

In the advanced stages of HCC and in patients presenting with recurrence, molecular target therapy has become a viable alternative treatment. Target therapy agents such as sorafenib [targeting VEGFR and platelet-derived growth factor receptors (PDGFR)], ramucirumab (targeting VEGFR), regorafenib (targeting VEGFR), gefitinib (targeting EGFR), erlotinib (targeting EGFR), lenvatinib (targeting VEGFR, PDGFR, FGF receptor), and everolimus (targeting mTOR) are commonly prescribed. However, patients having received target therapy have not exhibited significant beneficial effects in terms of overall survival, while drug resistance has further limited the anticancer effect. Previous studies have shown that inflammation and fibrosis have caused sorafenib-resistance and HCC progression. TNF-α and IL-6 are key cytokines which promote intrahepatic HCC progression via STAT3 activation[101]. The combination of two or three drugs which impact multiple targets may improve treatment to control the complex cancer metabolic system, whereby TCM may serve as a multi-target adjuvant therapy in preventing HCC recurrence.

Investigations have revealed that cisplatin and oxaliplatin, platinum-based chemotherapeutic agents, cause cytotoxic effects through DNA damage. The resistance to oxaliplatin in HCC has been associated with the lysine-specific demethylase 1/long intergenic non-protein-coding RNA 1134 (LINC01134)/SP1/p62 axis or the miR-129-5p/ETS translocation variant 1 axis[102,103]. It has been reported that trametes robiniophila extract repressed the expression of Yes-associated protein and apoptosis-related proteins (Bcl-2) to sensitize the oxaliplatin effect[104]. In as separate study, falcarindiol sensitized the cisplatin anti-Huh7 and LM3 effects by downregulation of the STAT3/pituitary tumor transforming gene 1 (PTTG1) pathway expression[105]. As applied in advanced or recurrent HCC, doxorubicin has been shown to intercalate the DNA, stabilize the topoisomerase II complex and halt the DNA replication process. In addition, dihydroartemisinin has been found to decrease P-gp expression through downregulating the p53 (R248Q)-ERK1/2-NF-κB signaling pathway to augment anticancer effects in mutant p53 (R248Q)-expressing Hep3B cells (doxorubicin resistant cell line)[106]. Of note, it has been reported that Solanum nigrum enhanced cisplatin and doxorubicin’s anti-HCC effect through apoptosis and autophagy by cleavage of caspase-7 and accumulation of microtubule-associated protein-1 Light chain-3 A/1B II[107]. Meanwhile, 5-fluorouracil (5-FU) is a thymidylate synthase inhibitor which interferes with DNA replication and leads to cytotoxicity. As reported, H1 (a derivative of tetrandrine, molecular formula: C₂₇H₄₀N₂O₆Br) and bufalin increased 5-FU sensitivity in 5-FU-resistant HCC cells (BEL-7402/5-FU)[108,109]. Additionally, bufalin induced apoptosis by increasing in the Bax/Bcl-xL ratio, inhibited drug efflux pump activity via downregulation of multidrug resistance protein 1 and reduced the expression of thymidylate synthase[108]. Furthermore, H1 downregulated the STAT3/ myeloid cell leukemia 1 (MCL-1) pathway to sensitize 5-FU treatment[109]. Sorafenib is a protein kinase inhibitor which acts against VEGFR and PDGFR, and rapidly accelerates fibrosarcoma (RAF) kinases. In separate studies, artesunate and tetrandrine increased the effectiveness of sorafenib on HCC apoptosis by inhibiting the PI3K/AKT/mTOR pathway[110,111]. Artesunate has also been shown to inhibit the RAF/MAPK pathway[110]. Meanwhile, Zhai et al[112] reported that bufalin reversed sorafenib resistance via the inositol-requiring enzyme 1 pathway in HepG2 and Huh7 cell lines[112]. Furthermore, solamargine has been shown to provide a synergistic anticancer effect with sorafenib by regulating HOXA distal transcript antisense RNA (HOTTIP)–the taurine upregulated 1 (TUG1)/miR-4726-5p/mucin 1 signaling pathway[113]. The combination of 8-bromo-7-methoxychrysin and sorafenib has been reported to decrease expressions of HIF-1α and the EMT regulator Twist1 to inhibit CSC[114]. To be applied in cases of HCC recurrence or in advanced cases, icaritin has been found to enhance the effects of doxorubicin and lenvatinib in Hepa1-6 and Huh7 cells[115]. The compounds involved in reversing drug resistance are listed in Table 4.

With regards to the benefits of acupuncture in the amelioration of drug resistance, a limited number of studies have focused directly on the antitumor and synergistic effects associated with acupuncture, electroacupuncture and moxibustion. Although, Yang et al[116] reported that electroacupuncture around a breast cancer tumor increased the local concentration of paclitaxel and decreased the tumor volume[116].

Drug resistance indeed limits the therapeutic effectiveness of drug treatments for recurrent HCC. However, investigations have demonstrated that the combination of two or three drugs impacting multiple targets may offer promising anti-HCC treatment strategies. As such, TCM has been found to provide a wide range of synergistic effects associated with platinum, doxorubicin, 5-FU, and protein kinase inhibitors. The mechanisms underlying these effects are associated with the RAF/MAPK, PI3K/AKT/mTOR, STAT3/PTTG1, STAT3/MCL-1 and HOTTIP-TUG1/miR-4726-5p/MUC1 pathways (Figure 1).