Plant-derived natural products and combination therapy in liver cancer

Liver cancer is one of the malignant cancers globally and seriously endangers human health because of its high morbidity and mortality. Plant-derived natural products have been evaluated as potential anticancer drugs due to low side effects and high anti-tumor efficacy. However, plant-derived natural products also have defects of poor solubility and cumbersome extraction process. In recent years, a growing numbers of plant derived natural products have been used in combination therapy of liver cancer with conventional chemotherapeutic agents, which has improved clinical efficacy through multiple mechanisms, including inhibition of tumor growth, induction of apoptosis, suppression of angiogenesis, enhancement of immunity, reversal of multiple drug resistance and reduction of side effects. The therapeutic effects and mechanisms of plant-derived natural products and combination therapy on liver cancer are reviewed to provide references for developing anti-liver-cancer strategies with high efficacy and low side effects.


Introduction
Liver cancer is one of the most common malignant cancers with more than 906,000 new cases and 830,000 deaths annually worldwide (1). Liver cancer has brought significant threats and challenges to human's physical and mental health as well as social and economic development (2). Currently, the main treatments for liver cancer include surgical resection (3), radiotherapy (4), chemotherapy (5), local ablation therapy (6) and liver transplantation (7). Although traditional therapies are effective for liver cancer at early stage, they have limited efficacy for advanced liver cancer due to severe side effect, drug resistance, multiple recurrences and metastases (8,9). For the past few years, plant-derived natural products have been evaluated as potential anticancer drugs that preferentially kill tumor cells with low toxic effect on normal cells. A variety of plant-derived natural products with antitumor activities have been identified, such as alkaloids, terpenoids, phenols, flavonoids, which can inhibit tumor-cell invasion and migration (10), induce apoptosis (11), suppress angiogenesis (12) and proliferation (13). Although these plant-derived natural products have many advantages, such as abundant resources, low toxic effect and diversity in targets and molecular mechanisms, they have limited efficacy for clinical application due to low solubility and poor bioavailability (14). Therefore, there is an urgent need to develop new therapy with high efficacy and low side effects to prevent and treat liver cancer.
Numerous studies have shown that combined therapy has great potential value in treating of liver cancer. The combination of chemotherapy with natural compounds is likely to increase the efficacy of drug treatment as well as reduce the adverse responses (15). Some natural products may sensitize to conventional cytotoxic therapy, reinforce the drug effective concentration, reverse drug resistance, intensify the combined effect of both administered therapeutics or exert cytotoxic effects specifically on tumor cells (16). Moreover, natural products combined with immunotherapy, can reduce the development of cancer and enhance the immune function by targeting multiple signaling pathways (17). The desired effect could be to diminish burden on the patient's organ by replacing part of the dose of a conventional chemotherapeutic with a natural substance with a defined effect. Many natural compounds are well tolerated by the patients and do not cause toxic effects even at high doses. Interaction of conventional chemotherapeutics with natural compounds introduces a new aspect in the research and therapy of cancer. It could be a complementing approach to potentially achieve improvements, while minimizing side effects associated with conventional chemotherapy. In this paper, we review the recent studies on the functions and mechanisms of plant-derived natural products and combination therapy in the treatment of liver cancer.

Search strategy
This review followed the PRISMA guidelines. The primary search for article screening used in this review was conducted using PubMed (387), CNKI (277), Web of science (236), X-MOL (193) and the Wanfang database (178), and the medical subject headings (natural product, liver cancer, combination). After completing the initial search, therefore to avoid duplication, we removed 550 articles from the literature, and selected 721 articles. After screening for eligibility of the articles, 128 articles were included in the study. PRISMA flow chart indicates only the identification and screening processes of the anti-liver-cancer activity of plant-derived plant products and combination therapy ( Figure 1).

Plant-derived natural products in the treatment of liver cancer Alkaloids
Alkaloid is a kind of basic organic compounds containing nitrogen in plants. The representative anti-tumor compounds include matrine, berberine, evodiamine, camptothecin, vincristine and caffeine. Matrine exists in dried roots of Sophora flavescent and induces HepG2 cell apoptosis through increasing Bax/Bcl-2 ratio and inhibiting the ERK1/2 signaling pathway (18). Dai et al. reported that matrine inhibits BEL-7402 cell and SMMC-7721 cell invasion and metastasis by upregulating the expression of microRNA-199a-5p (miR-199a-5p) and reduces the levels of hypoxia-inducible factor-1a (HIF-1a) (19). Moreover, matrine also inhibits liver cancer migration and induces apoptosis through increasing the expression of microRNA-345-5p (miR-345-5p) and reduces the levels of circular RNA_0027345 (circ_0027345) and homeobox-containingD3 (HOXD3) (20). In addition, Zhang et al. researched that matrine at non-toxic dose could significantly suppress PLC/PRF/5 and MHCC97L cells migration and invasion. And, it also significantly decreased lung metastasis in orthotopic HCC mouse models (21). Berberine, an isoquinoline alkaloid, has been extracted from the rhizome of Coptis and Phellodendri and considered as anticancer drug in China because of its wide range of pharmacological effects. Berberine induced autophagy of HepG2 and MHCC97-L cells through activating Beclin-1 via enhancing p38 mitogen-activated protein kinase (MAPK) signaling pathway and inhibiting protein kinase B (Akt) activity (22). Hou et al. researched that berberine decreased the expression of CD147, thereby induced apoptosis of HepG2 and SMMC7721 cells in a dose-and time-dependent manner (23). In addition, berberine inhibits transforming growth factor-b/ Smad (TGF-b/Smad) signaling pathway and interferes epithelialmesenchymal transition (EMT), thereby inhibiting migration and invasion of HepG2 cells (24). A study showed that evodiamine induced apoptosis of HepG2 and PLHC-1 cells in a dose-dependent manner by inactivating phosphatidylinositol 3-kinase (PI3K) signaling pathway, downregulating B-cell lymphoma 2 (Bcl-2) expression and upregulating Bcl-2-associated X protein (Bax) (25). Evodiamine can also inhibit the proliferation of hepatocellular carcinoma cells by activating Hippo-Yes-associated protein signaling pathway in vitro and in vivo (26). Camptothecin has been used clinically to treat liver cancer, stomach and colorectal cancer for a long time, which can inhibit Huh7 and H22 cell proliferation, FIGURE 1 The flowchart provides an overview of the study search and selection process. A search of the databases PubMed, CNKI, Web of Science, X-MOL, and the Wanfang database was undertaken, and then duplicate articles and any which did not meet the criteria of our study were removed before the relevant articles were selected for further study. metastasis and angiogenesis by upregulating of reactive oxygen species (ROS) and nuclear factor E2-related factor 2 (Nrf2) expression, reducing the expression of N-cadherin, matrix metalloproteinase 9 (MMP9), Snail and Twist, and increasing Ecadherin (27). Notably, 10-hydroxycamptothecin (0.5 mg/L) inhibited the proliferation of BEL-7402 cells through induction of apoptosis, and showed synergistic effect with human cytokineinduced killer cells (optimal target ratio of 20:1) (28). According to reports, vincristine could decrease the expression of Ki-67, MMP2 and MMP9, and increase the expression of cleaved caspase 3, thereby inhibiting proliferation and reducing metastasis by PI3K/Akt signaling pathway in Hep3B cells (29). Caffeine is an alkaloids compound extracted from tea, cocoa bean and coffee fruit. Caffeine and X-ray irradiation have synergistic effect on the inhibition of cell proliferation and induction of apoptosis, and cell cycle arrest at G2/M phase (30). Cinchonine is a natural compound present in Cinchona bark. It exerts multidrug resistance reversal activity and synergistic apoptotic effect with paclitaxel in uterine sarcoma cells. Jin et al. demonstrated that cinchonine inhibited liver cancer cell proliferation and promoted apoptosis in a dose-dependent manner. It can activate caspase-3 and PARP1 cleavage, increased the expression of GRP78 and PERK, and suppressed HepG2 xenograft tumor growth in mice (31). As a transcription factor, the tumor suppressor p53 can participate in many important biological processes, such as cell cycle arrest and apoptosis. It can activate the expression of antioxidant proteins such as catalase, glutathione peroxidase 1 (GPX 1), manganese superoxide dismutase (MnSOD), which leads to the imbalance of ROS, and finally leads to apoptotic (32). Moreover, Sanguinarine significantly inhibited liver cancer cell proliferation in a p53-dependent manner by inducing cell cycle arrest and ROS-associated apoptosis. Importantly, it dramatically suppressed tumor growth in an HCC xenograft model, with little cytotoxicity (33).

Terpenoids
Terpenoids are hydrocarbons and oxygenic derivatives in natural plants, which contain isoprene structure. It has been found that terpenoids have antitumor activities, such as saikosaponin D, triptolide, ginsenoside, artemisinin, celastrol and norcantharidin. Saikosaponin D is the most important active component of the Chinese medicinal herb Bupleurum, which can selectively upregulate the protein expression of light chain 3II (LC3II) and Beclin 1, down-regulate the protein expression of P-S6K1, and induce autophagy by negatively regulating the mammalian target of rapamycin complex (mTORC) signaling pathway to reduce liver cancer cell growth (34). Moreover, it can increase radiation-induced apoptosis of liver cells via inhibiting mTOR phosphorylation to provide a possible potential therapeutic method for radio sensitization of liver cancer (35). In addition, Wu et al. discovered that saikosaponin D inhibited HepG2 cell proliferation by induction of apoptosis, improved the survival rate and suppressed the growth of liver tumors (36). Triptolide, a natural diterpenoid compound, is the main active ingredient of Tripterygium wilfordii, which inhibits the proliferation of HepG2 cells through induction of autophagy and apoptosis via up-regulating the expression of microRNA -194 (miR-194), LC3 and Beclin-1, and Bax/Bcl-2 ratio (37). Another study showed that it induced HepG2 cell apoptosis by inhibiting the expression of Ras (38). Gan et al. demonstrated that triptolide significantly suppressed the proliferation of H22 cells with IC 50 value of 266.8 ng/mL at 24 h and induced apoptosis in H22 cells through decreasing the expression of cycloxygenase-2 (COX-2) (39). Ginsenoside-Rg3 inhibited cell proliferation and invasion of SMMC-7721 cells through PI3K/AKT pathway, which effectively decreased the levels of phosphorylated (p)-AKT, p-PI3K, MMP2, MMP9 and long non-coding RNA HOX antisense intergenic (lncRNA HOTAIR) (40). Recently, Zhang et al. showed that artemisinin enhanced the effect of anti-programmed cell death-ligand 1 (anti-PD-L1) immunotherapy by blocking the accumulation and function of myeloid-derived suppressor cells (MDSCs) via regulating PI3K/ AKT/mTOR and MAPK signaling pathways and polarizing M2-like pro-tumor phenotype to M1-like anti-tumor phenotype (41). Dihydroartemisinine inhibits proliferation and induces apoptosis of HepG2 cells through regulating Bcl-2/Bax/caspase-3 apoptosis signal pathway (42). It can also induce liver cancer ferroptosis by promoting the formation of phosphatidylethanolamine-binding protein 1/15-Lipoxygenases (PEBP1/15-LO) and lipid peroxidation in cell membrane (43). Celastrol is a quinone methyl triterpene monomer compound of T. wilfordii Hook. f. and inhibits HepG2 cell proliferation by activating AMPK signaling pathway to suppress lipid metabolism and proliferation signal (44). Moreover, it can reduce the expression of nuclear factor kappa-B (NF-kB) and tumor necrosis factor alpha (TNF-a) in HepG2 and SMMC-7721 cells in a dose-and time-dependent manner (45). Recent studies have found that p53 can downregulate the expression of asparagine synthetase (ASNS) at the transcriptional level, thereby inhibiting asparagine synthesis and disrupting aspartate homeostasis, which can suppress the growth of lymphoma and colon tumors in vivo and in vitro. And depleting the levels of asparagine in cancer cells could release liver kinase B1 (LKB 1), and activated monophosphateactivated protein kinase (AMPK) -p53 signaling, triggered p21dependent cell cycle arrest and promoted survival of cancer cells in asparagine-deficient conditions (46). Norcantharidin inhibits Huh7 cell proliferation and induces apoptosis in a time-and dose-dependent manner by activating p53 and p21 proteins and arresting cell cycle at G2/M phase (47). Tan et al. reported that genipin suppressed orthotopic HCC tumour growth through inhibiting IRE1a-mediated infiltration and priming of tumour associated macrophages (TAMs). It could reduce infiltration of inflammatory monocytes into liver and tumour, and inhibited the TAMs migration (48). Natural compound andrographolide (Andro), isolated from medicinal herb Andrographis paniculata, was reported to inhibit hepatoma tumor growth. It can inhibit the hepatoma tumor growth and alters the expression of miRNAs profile and downstream signals (49). the rhizome of the perennial herb turmeric in Zingiberaceae. In vitro and in vivo results showed that curcumin downregulated the expression of Bcl2-associated transcription factor (BCLAF1), inhibited the activation of PI3K/AKT/glycogen synthase kinase-3b (GSK-3b) pathway, and triggered mitochondria-mediated apoptosis in liver cancer (50). Sun et al. reported that curcumin inhibited SMMC-7721 cell proliferation by induction of apoptosis via activating C/EBP homologous protein (CHOP) signaling pathway, reducing the expression of Bcl-2 and increasing cleaved caspase-3 and suppressed SMMC-7721 cell growth in xenograft tumor nude mice by activating endoplasmic reticulum (ER) stress pathway (51). Curcumin can induce liver cancer cell death by induction of apoptosis and pyroptosis. In addition, curcumin activates the gasdermin E (GSDME)-related scorch death and mitochondria-dependent apoptosis signaling pathways (52). Resveratrol is a phytoestrogen derived from grapes and berries and has several potential biological effects, including antioxidant, anti-inflammatory, cardioprotective and anticancer properties (53). Resveratrol inhibited the wound healing and invasion of HepG2 and Huh7 cells, increased the expression of E-cadherin, and decreased the expression of vimentin and Twist1. It inhibited metastasis and EMT through up-regulating the expression of microRNA-186-5p (miR-186-5p) (54). Furthermore, resveratrol can decrease the expression of membraneassociated RING-CH protein 1 (MARCH1) and p-Akt, increase the expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) dose-dependently, hence inducing apoptosis and inhibiting the proliferation and invasion of HepG2 and Hep3B cells (55). 6-gingerol is an active polyphenolic compound extracted from ginger and can regulate the AKT/extracellular signalregulated kinases 1/2 (ERK1/2) signaling pathway, increase P27/Kip1 and p21/Cip1 expression and inhibit cyclins D1, c-myc and cyclindependent kinase 4 (CDK4) protein expression, thereby arresting cell cycle at G2/M phase in Huh7 cells (56). Notably, a study showed that epigallocatechin gallate (EGCG) and EGCG derivative Y6 could significantly inhibit tumor growth and angiogenesis through MAPK/ERK1/2 and PI3K/AKT/HIF-1a/vascular endothelial factor (VEGF) pathways (57). Guo et al. demonstrated that EGCG combined with paclitaxel effectively inhibited the proliferation of HepG2 cells and the tumor growth in bearing cancer nude mice (58). In addition, it combined with adriamycin to exert synergistic antiliver cancer effect, which induced apoptosis of BEL-7404/ADR cells and arrested cells in S/G2 phase of the cell cycle in a dose-dependent manner (59). Tannic acid is a natural polyphenol compound primarily found in fruits and nuts. Tannic acid can induce apoptosis by DNA disruption via caspase-dependent and caspaseindependent mechanisms. It can also induce oxidative stress to promote liver cancer cell death (60). The combination of tannic acid with cisplatin induced HepG2 cell apoptosis through enhancing the activation of ER stress ATF6-CHOP pathway (61). Magnolol, a natural compound extracted from herbal plant Magnolia officinalis, has been recognized as a liver protection and antitumor drugs. It can significantly suppress tumor size and tumor growth rate, decreased the expression of p-ERK, NF-kB p65 (Ser536), MMP-9, VEGF, Xlinked inhibitor of apoptosis protein (XIAP), and cyclinD1, and increased the expression of caspase-8 and caspase-9, which in turn induced apoptosis in vivo (62). Rosmarinic acid is a natural polyphenolic compound that exists in many medicinal species of Boraginaceae and Lamiaceae. Rosmarinic acid can regulated immune response and induced apoptosis of H22 cells. It could effectively inhibit the tumor growth through decreasing the expressions of IL-6, IL-10 and signal transducer and activator of transcription 3, upregulating Bax, caspase-3 and down-regulating Bcl-2 (63).

Flavonoids
Flavonoids are phenolic phytochemicals and are commonly found in fruits, vegetables and plant-based beverages (e.g., green tea and wine), which have a polyphenolic structure and are usually composed of a 15-carbon skeleton (including two benzene rings) and a heterocyclic nucleus containing an oxygen atom. Many studies have reported that flavonoids have a significant role in tumor treatment, such as kaempferol, silymarin, quercetin, chrysin and diosmetin. Kaempferol is a natural flavonol and widely distributes in many vegetables and fruits. Kaempferol remarkably decreased the expression of miR-21 and increased the expression of PTEN, thereby inhibiting HepG2 cell proliferation and metastasis through inactivating PI3K/AKT/mTOR signaling pathway (64). Moreover, it can promote apoptosis of HepG2 cells in a dose-dependent manner through decreasing the expression of CDK1 and increasing the expression of Bax and JUN (65). Another study showed that kaempferol was able to increase the protein expression of Bax, glucose-regulated protein of 78 kDa (GRP78), GRP94, PERK, cleaved ATF6, IRE1a, CHOP, caspase-3 and caspase-4, thereby inducing HepG2 cell apoptosis by mitochondrial pathway and ER stress pathway (66). Silymarin is a lipid compound extracted from the fruit of Silybum marianum and inhibits MHCC97 cell invasion and metastasis through reducing Akt phosphorylation and integrin b1, VEGF and MMP-9 expression. Moreover, it can restore the cell adhesion ability by upregulating E-cadherin, thereby inhibiting invasion and metastasis of liver cancer cells (67). Silibinin is another bioactive polyphenolic flavonoid isolated from the fruits and seeds of S. marianum (68), which can achieve anti-liver-cancer effects through multiple mechanisms, including induction of apoptosis, suppression of angiogenesis and block of cell cycle (69, 70). Quercetin is a dietary polyphenolic compound with wide distribution in onions, grapes, berries, apples, broccoli and cereals (71), and has the capability to reduce the expression of markers in cancer stem cells and myofibroblasts, as well as the expression of ABCC3 to reduce the acquisition of drug resistance in the early liver cancer stages (72). Quercetin inhibited the growth of HepG2 cells in a dose-and time-dependent manner, with IC 50 values of 110.50, 63.73 and 46.38 µmol/L at 24 h, 48 h and 72 h, respectively, and suppressed the growth of subcutaneous xenografts in BALB/c mice (73). Another study showed that quercetin inhibited HepG2 cell proliferation by NF-kB pathway and activator protein-1 (AP-1)/c-Jun N-terminal kinase (JNK) pathway, which increased the expression of p-JNK, p-AKT, p-p38, caspase-3 and decreased the expression of Bcl-xL, p-ERK and NF-kB (74). Chrysin, also known as aspicin, is a natural flavonoid compound extracted from Pterophylla globulina, with extensive pharmacological activities (75). Chrysin significantly inhibited SMMC-7721 cell proliferation and induced apoptosis by activating MAPK signaling pathway (76). Moreover, chrysin could effectively inhibit the progression of tumor and promote the antitumor immunity of mice concomitant with increased CD4 + and CD8 + T cell proportions in tumor tissues of H22 xenograft mice model. Additionally, chrysin reduced the expression of PD-L1 through blocking of STAT3 and NF-kB pathways in vitro and in vivo (77). Diosmetin is an effective component of medicinal plants in southern China. Liu et al. found that diosmetin inhibited liver cancer cell metastasis, invasion and adhesion by regulating the PKCd/MAPK/ MMP pathway (78). It also can significantly downregulate the levels of Bcl-2, CDC2 and cyclinB1, and upregulate the levels of Bax, cleaved-caspase-3, cleaved-caspase-8, cleaved-PARP, Bak, p53 and p21, thereby inhibiting HepG2 cell proliferation and inducing cell apoptosis and arresting cell cycle at G2/M phase (79). Similarly, another study showed that diosmetin reduced the expression of Bcl-2, CDK1 and CCND1 in a dose-dependent manner in vitro and the expression of Ki67 in vivo, thereby inhibiting the proliferation and migration and arresting cell cycle at G1 phase of liver cells (80). Tan et al. observed that oral administration of baicalin completely blocked orthotopic growth of implanted HCC. And it induced repolarisation of TAM and M2-like macrophages, which is associated with elevated autophagy, and transcriptional activation of RelB/p52 pathway (81).

Others
Emodin is an anthraquinone compound extracted from Palmar rhubarb and Amomum amomatum and inhibits liver cancer cell proliferation and metastasis by regulating VEGFR2-AKT-ERK1/2 signaling pathway and miR-34a signaling pathway (82). Aloin is a natural anthracycline compound, which can inhibit MHCC97H cell proliferation, migration and invasion and induce apoptosis through regulating Bcl-2/Bax ratio (83). Tanshinone is a phenoquinone derivative extracted from Salvia miltiorrhiza and inhibits HepG2 and Huh7 cell proliferation by blocking G0/G1 phase cell cycle via decreasing the expression of cyclin D1 and increasing p21, as well as inhibiting p53/damage-regulated autophagy and inducing apoptosis (84). As the most frequently mutated gene in human tumors, p53 can induce apoptosis, cell cycle arrest and cell senescence. And it plays an important role in suppressing proliferation of tumor, and is a major target for developing new drugs to reactivate its tumor suppressing activity for anticancer therapies. Taking p53 mutant Y220C as the core, Joseph et al. studied the structure-guided development of highaffinity small molecules stabilizing p53-Y220C in vitro and the synthetic route developed in the process. They found two new chemical probes with submicromolar binding affinity in vitro, which represents an important step toward the novel and effective Y220C ligand in the clinical evaluation of tumors (85). Cryptotanshinone significantly inhibited the cell viability of HepG2 cells with an IC 50 of 93.73 mmol/L and induced ferroptosis by ROS accumulation via reducing glutathione (GSH) level and the expression of cystine/glutamate antiporter system light chain (xCT) and glutathione peroxidase 4 (GPX4) (86).
Natural polysaccharides are rich dietary components with significant roles in tumor prevention and treatment. Astragalus polysaccharides are the active component of the dried root of Radix astragali and can inhibit HepG2 cell proliferation (87), reverse the drug resistance of 5-fluorouracil (88), suppress EMT (89) and induce apoptosis (90). Astragalus polysaccharides can inhibit the growth of HepG2.215 cells by blocking HepG2.215 cells from entering the G2/ M phase and inducing apoptosis (91). Poria cocos polysaccharides induced HepG2 cell apoptosis by inhibiting the expression of NODlike receptor thermal protein domain associated protein 3 (NLRP3)/ caspase-1/gasdermin D (GSDMD) in the classical pyroptosis pathway (92). Another study showed that Lycium barbarum polysaccharides inhibited SMMC-7721 cell proliferation and metastasis and induced apoptosis by reducing the expression of MMP-2, MMP-9 and VEGF (93). Saponins can significantly inhibit tumor cell proliferation. Astragaloside IV, also known as astragalus saponins, was able to upregulate the expression of caspase-3 and downregulate the expression of interleukin (IL)-6 by inhibiting the STAT3 signaling pathway, thereby inducing the apoptosis of liver cancer cells (94). Astragaloside IV also inhibits HepG2 cell proliferation and induces apoptosis by regulating oxidative stress and NF-kB signaling pathway (95). In addition, astragalus saponins promoted the secretion of IL-2 to enhance the proliferation and transformation of lymphocytes, thereby improving the immune function of rats and inhibiting liver cancer cells growth (96). promote the improvement of liver function, regulate immune function, reduce AFP level, has the advantages of low recurrence rate, has significant curative effect, and is worthy of further promotion and application (101). Furthermore, Zhao et al. founded that the therapy of Fuzheng Huayu Jiedu prescription can effectively improve the clinical effect on advanced liver cancer with less adverse reactions and high safety, and its mechanism may be related to its remarkable reduction of levels of inflammatory factors, inhibition of inflammatory responses, improvement of immune function and enhancement of resistance (102). Hedyotis diffusa injection is a Chinese patent medicine, which has the effect of clearing heat and detoxifying, benefiting dampness and swelling. Hedyotis diffusa injection combined with transarterial chemoembolization in the treatment of the patients with primary liver cancer can improve the disease control rate and improve the levels of T cell subsets. Moreover, it is superior to single transarterial chemoembolization (103).
Supplementary Table 1 summarized the effects and mechanisms of plant-derived natural products on liver cancer. Although many plant-derived natural products are effective, they have limited efficacy for clinical application. Conventional mono-therapeutic methods target rapidly proliferating cells and destroy their growth, which it does not differentiate between healthy and cancerous cells. Chemotherapy can be toxic to the patient with multiple side effects and risks, such as nausea and vomiting and tumor metastasis (104). And it can also strongly reduce their immune system by affecting peripheral blood lymphocyte subsets and increasing susceptibility to host diseases (105). Although combination therapy can be toxic if one of the technologies is chemotherapeutic, the toxicity is remarkably less because a lower therapeutic dosage of each individual drug is demanded (106). It may be able to prevent the toxic effects on normal cells while concurrently producing cytotoxic effects on cancer cells. Further, monotherapy treatment is more develop drug resistance because continuous treatment with a single compound induces cancer cells to create alternative salvage approach (107). The most common mechanism of multidrug resistance is the elimination of drugs from the cell by ATP-dependent efflux pumps, thereby reducing intracellular drug concentrations and giving rise to resistance. As an example, cells in adenocarcinoma, when treated with adriamycin, upregulate the expression of ATP-binding cassette (ABC) transporter family, P-glycoprotein, to eliminate the drug, leading to a state of drug resistance (108). However, combination therapy also produced a more effective treatment response which target different pathways, and therefore this treatment modality reverses the drug resistance (109). Generally, conventional chemotherapy only targets non-stem cell tumor cells, resulting in an increasing proportion of stem cells in tumors after chemotherapy. And it is also a key mechanism for induction of drug resistance, regulation of metastasis and recurrence of malignant (110). However, combination therapy (for example quercetin in combination with chemotherapy drug doxorubicin) could reduce drug resistance and attenuated the likelihood of relapse (111). Therefore, combination therapy of plant-derived natural products with chemotherapeutics or immunotherapy is drawing researcher's attention on liver cancer to induce apoptosis and inhibit cell proliferation and angiogenesis. It has the potential to develop therapeutic or adjuvant agents for human liver cancers.

Anti-liver cancer mechanism by combination of plant-derived natural product ingredients
Mutations and epigenetic changes allow cancer cells to grow and replicate uncontrollably and invade normal tissues. This uncontrolled growth and proliferation arise due to loss of function mutations in tumour organelles such as lysosome (112), mitochondria (113), as well as endoplasmic reticulum (114). The limited efficiency and serious side effects associated with the use of conventional anticancer therapies encouraged scientists to focus on the discovery and development of new anticancer agents derived from natural products. For much of history, people have used plants and plant extracts to treat their ailments. Although the established natural plants as monotherapy of cancer had many advantages, they had limited efficacy for clinical application. If they are used in combination with other natural products, their anti-cancer effect is more effective. Secondary metabolites from plant sources like flavonoids, alkaloids, terpenoids, saponins, and others had been reported as important sources for potent anticancer agents. It was demonstrated that combination treatment acts by reducing the concentration of the compound required for drug efficacy through enhancing the effect on the same target or mechanism of action. The anticancer effect of these natural products in combination with others is mediated by different mechanisms, including inhibiting proliferation, blocking cell cycle and reversing multidrug resistance.

Inhibit liver cancer cells proliferation
Proliferation and invasion of liver cancer cells were inhibited through decreasing the expression of the tumor suppressor gene deleted in liver cancer 1 (DLC1), translationally controlled tumor protein (TCTP) and Cdc42 (115). Resveratrol in combination with dihydroartemisinine acts by regulating DLC1/TCTP protein expression and inhibiting migration-related signaling pathways in HepG2 and MDA-AB231 cells through inducing Cdc42 regulation of JNK/NF-kB and N-WASP signaling pathways (116). PI3K-AKT signaling pathway is closely related to the proliferation of liver cancer cells (117). The combination of natural products acts by inducing anti-tumor effect through inhibiting the invasion and proliferation of tumor cells. Sodium arsenite and astrageoside IV inhibit HepG2 cell proliferation, block cell cycle, promote apoptosis, and suppress tumor growth. It can reduce the expression of PI3K, AKT, mTOR through PI3K/AKT/mTOR signaling effect, and the synergistic effect is more significant (118). Apoptosis an active and ordered multistep cell death process that controlled by genes, and involves two major pathways, including intrinsic and extrinsic apoptosis pathways in human cancer cells. The intrinsic pathway of apoptosis is mediated mainly by mitochondria within the cell, and this pathway is regulated by pro-apoptosis Bcl-2 family proteins (119). Natural products principally induced liver cancer cells apoptosis and inhibited proliferation through caspase cascade reaction and NF-kB signaling pathway.
Wen et al. developed citrus net skin black tea (CRPBT) by combining citrus reticulate peel and black tea. CRPBT can significantly down-regulate the phosphorylation of PI3K and AKT in liver cancer cells, up-regulate the Bax/Bcl-2 ratio, and inhibit the expression of MMP2/3, N-cadherin and Vimetin proteins. It can inhibit invasion, proliferation and induce apoptosis of liver cancer cells (120). A combination of curcuma zedoary and kelp could inhibit liver cancer cell proliferation and metastasis by inhibiting endogenous H2S production and down-regulating the transcription activator 3 (STAT3)/BCL-2 and VEGF pathway in vitro and in vivo (121). Bufalin (BFL) and cinobufagin (CBF) have a synergistic effect. Metabolic pathways, including methionine metabolism, energy metabolism, lipid metabolism, and amino acid metabolism, were modulated and subsequently led to apoptosis and cell cycle arrest of HepG2 cells. Zhang et al. researched that the cotreatment group can significantly decrease mitochondrial membrane potential (MMP) and oxygen consumption rate (OCR), which associated with maximal respiration (122). Triptolide and sodium cantharidinate exert a synergistic toxic effect on hepatoma cell line 7721, which is related to increasing capase-3 activity and suppression of NF-kB (123). Quercetin and rosmarinic acid had a more significant synergistic effect on inhibiting HepG2 cells proliferation and migration in a dosedependent manner by up-regulating the expression of E-cadherin and down-regulating N-cadherin gene expression (124). Manikandan et al. showed that curcumin in combination with catechin which is polyphenolic compound have a synergistic effect on inhibiting the proliferation and inducing cell apoptosis. It treated HepG2 cells showed small morphological changes which significantly destruction of monolayer, and change the DNA fragmentation (125). Wang et al. evaluated the effects of co-treatment of vincristine and berberine on hepatic carcinoma cell lines. They find that combinational usage of these two drugs can significantly induce cell growth inhibition and apoptosis even under a concentration of vincristine barely showing cytotoxicity in the same cells when used alone (126). The combination of arsenic trioxide and tanshinone have synergistic effect, attenuation and antihepatocarcinoma effect. Tanshinone can reduce the blood routine changes and liver and kidney function damage caused by arsenic trioxide (127). Cryptotanshinone induced liver cancer cells apoptosis and enhanced the effect of Arsenic trioxide by downregulating phosphorylated STAT3, Bcl-2 in vitro and in vivo (128).

Block liver cancer cells cycle
The cell cycle system plays a critical role in regulating activities such as cancer cell proliferation and apoptosis. The pathogenesis of neoplasms because of lost control in cycle progression is common. Uncontrol cell division is an essential factor in the development of cancer. The cell cycle consists of G1, the S-phase, G2, and the M (mitotic) phase (129). Cells are preparing for DNA synthesis during G1 and they perform surveillance to establish the integrity of newly synthesized DNA during G2 before initiating mitosis. Chromosomal DNA is replicated during the S-phase. If this phase arrest, the cells will undergo variation or abnormal division, which will then terminate the proliferative (130). In the G2 phase, after the nucleic acid is amplified, the other cellular components are partitioned between two daughter cells during the M-phase. If the stagnation occurs, the normal growth and gathering of the cells is hindered, which inhibit proliferation (131). Cyclins and their cognate cyclin-dependent protein kinases (CDKs) are necessary components required for traversing the cell division cycle, with controlling balance of all stages in cell cycle (132). CDK2/4/6, Cyclin D1, and Cyclin E are the major regulatory proteins in the G1 phase; and the reduction of Cyclin B1 and CDK1/2 activity is a key marker of cycle arrest in the G2/M phase (133, 134).
Zhang Di demonstrated that chrysin in combination with diosmetin or triptolide acted by the induction of apoptosis through inhibiting HepG2 cells migration as well as arresting the cell cycle in G1 and G2 phases in vitro. Moreover, it had a stronger inhibitory effect on H22 solid tumors than single drug application (135). Xiao et al. revealed that a combination of biochanin A with BRAF inhibitor SB590885 synergistically inhibiting proliferation by disrupting of the ERK MAPK and the PI3K/AKT pathways in vitro, and promoting cell cycle arrest and apoptosis (136). Quercetin in combination with maleic anhydride derivatives acts by inducing apoptosis of liver cancer cells through arresting cells in S-phase by oxidative stress response, causing DNA damageactivating the intrinsic apoptosis pathway (137). Curcumin (20 µmol/L) combined with vincristine (1 pg/L) inhibits proliferation and clone formation of HepG2 cells, reduces mitochondrial membrane potential and MDR1 and LRP protein expression, increases P21 expression, thereby blocking G2/ M phase cell cycle and inducing apoptosis (138). Curcumin (5 pg/L) and glycyrrhetinic acid (10 pg/L) obviously inhibited HepG2 cells proliferation in a concentration-and time-dependent manner, arrested the G2 phase, and the synergistic effect is more remarkably (139). The combination of Curcumin (40 µmol/L) and artemisinin (80 µmol/L) could effectively inhibit HepG2 cells proliferation and arrest cell cycle and promote apoptosis. In addition, it can inhibit telomerase activity in liver cancer cells (140). Tanshinone IIA block subG1 phase cell cycle, while resveratrol arrest S phase and G2/M phase of HepG2 cells. The combination of them induce cell apoptosis and arrest subG1 cell cycle. Moreover, it exerted synergistic cytotoxicity and robustly induces apoptosis, and DNA fragmentation (141). Berberine (15 µmol/L) combined with tarpinine derivative HMQ1611 (15 µmol/L) inhibits cyclin D1, cyclin E, CDK1 expression and arrests the cell cycle at G1 phase through PI3K/AKT/mTOR signaling pathways to induce apoptosis of HepG2 cells. Furthermore, the combination therapy had strong suppressive effect on proliferation by inhibiting the phosphorylation of LDL receptor-associated protein, which was related to Wnt/bcatenin signaling pathway (142)

Reverse liver cancer cells multidrug resistance
Liver cancer cells will be exposed to a certain chemotherapy drug for a long time under chemotherapy. And it can develop multidrug resistance (MDR) to this drug and cross-resistance to other chemotherapy drugs with different functional structures, leading to chemotherapy failure (145). This mechanism of multidrug resistance arises due to activity of the superfamily members of adenosine triphosphate binding cassette (ABC) such as P-glycoprotein (P-gP), multidrug resistance associated protein (MRP) and breast tumor resistance protein (BCRP) (146). Plant-derived natural products act by enhancing the tumor killing effect of chemotherapeutic drugs and inducing their anti-tumor activity through reversing the multidrug resistance of liver cancer cells.
Sun et al. demonstrated that curcumin reversed the multi-drug resistance caused by cucurbitacin B through inhibiting the expression of P-gP, thereby inducing liver cancer cell apoptosis (147) (152). The combination of emodin (10 mg/L) and curcumin (10 mg/L) enhances sensitivity of BEL-7402 hepatoma cells and reduces the dosage of emodin and induces apoptosis (153). Curcumin (5-20 µmol/L) and paclitaxel (0.05-0.2 µmol/L) have synergistic anti-proliferation and pro-apoptosis effects on Hep3B cells. It can decrease paclitaxel-induced NF-kB activation, mediate Lin28 level, thereby enhancing sensitivity of liver cancer cells to paclitaxel (154). Matrine also reverses resistance of K562/ADM cells to doxorubicin and vincristine by arresting cell cycle and inducing autophagy (155). Table 1 summarizes their effects and mechanisms on liver cancer.

The synergistic effect of plant-derived natural products in combination with chemotherapy
Clinical chemotherapy drugs mainly include cisplatin which is platinum compounds, and doxorubicin which is antibiotics, and hormones. Cisplatin and adriamycin mainly affect cell division and induce apoptosis by interfering with DNA replication (156). However, chemotherapeutic drugs significantly reduce the immune function of the body, and induce multiple organ damage (157,158).
Radiation and chemotherapeutic agents as the backbone for cancer are not very effective and toxic not only to tumor cells but also to normal cells, and are not affordable for most. Therefore, plant-derived natural products in combination with chemotherapeutic drugs are generally free of deleterious side effects and usually inexpensive, which have played a significant role in the development of anticancer drugs over the years. It remains to mention that extraction from natural plants is also contributed to the synergistic effects with other anticancer drugs such as cisplatin, doxorubicin, and hormones. Plant-derived natural products in combination with chemotherapeutic agents act as an anticancer agent by inhibition of tumor cell angiogenesis, invasion and migration, induction of autophagy.

Suppress liver cancer cells angiogenesis
Liver cancer is a kind of tumor rich in blood vessels. And blood vessels providing essential nutrients and oxygen for the occurrence and development of liver cancer cells, and are closely related to the growth and metastasis of liver cancer cells (159). Vascular endothelial growth factor (VEGF) is the most important type of angiogenesis regulatory factor, which promotes the division and proliferation of vascular endothelial cells through paracrine and promoting formation of blood vessels (160). Angiopoietin (Ang)/Tie2 pathway regulates vascular and lymphatic development, vascular permeability, angiogenesis remodeling, and tumor vascularization (161,162). Tumor cells act by up-regulating VEGF through inducing miR-21 targeting AKT and ERK signaling pathways, thereby promoting tumor angiogenesis (163). The mTOR pathway can also promote tumor angiogenesis through inducing the transformation of tumorassociated macrophages (TAM) into M2-type. Therefore, it can reduce proliferation and metastasis of liver cancer cells through preventing the formation of new blood vessels (164,165).
Ao et al. found that the main component of kanglite injections is the extract from coix seed in traditional Chinese medicine, this active compound in combination with thalidomide can significantly inhibit human liver cancer blood vessels through inhibiting VEGF and B-FGF, thereby inducing angiogenesis. At the same time, it can regulate cellular immunity of the body, which has synergistic effect with chemotherapy (166).

Inhibit invasion and migration of liver cancer cells
A critical event during tumorigenesis is the transformation from a static primary tumor to an aggressive and invasive oner. During the processes, tumor cells show an increased capacity to migrate, and multitudinous intracellular molecules participate in migration. Metastasis is significantly pathological characterized by malignant tumors, while the invasion and migration of liver cancer cells lead to poor prognosis of liver cancer patients, prone to recurrence and short median survival (173). The invasion and migration of tumor cells is a complex process controlled by multiple factors (174). It mainly included EMT, inhibition of matrix metalloproteinases (MMPs) hydrolysis and cell adhesion to basement membrane and extracellular matrix (175). And it is affected by the regulation of tumor metastasis genes and tumor metastasis suppressor genes (176). Combining natural products with chemotherapy drugs can prevent and cure cancer metastasis safely and effectively.
Shan et al. confirmed that mannose and cisplatin had a synergistic effect, which inhibited proliferation and migration of HepG2 cells, furthermore, it can change morphology with separation, shedding and aggregation (177) (154) generation of ROS and influencing MAPK signaling pathway, thereby inhibiting the proliferation and metastasis and inducing apoptosis of SMMC-7721 and Hep3B cells (181). Data from a study in vitro revealed that combined metformin (10 mmol/L) and curcumin (5 and 10 µmol/L) could induce apoptosis and inhibit metastasis in HepG2 and PLC/PRF/5 cells. The anticancer effects could be attributed to inhibition of VE GF, MMP2/ 9, and VEGFR-2 prote in expression (182).

Induce liver cancer cells autophagy
Autophagy is a genetically controlled and evolutionarily conserved form of cell death, which is also related to occurrence and development of tumor, thereby has becoming a new tumor therapy method. Several cellular classical signaling pathways of natural plants target intracellular autophagy including Beclin-1, phosphoinositide 3-kinase/Akt/mechanistic target of rapamycin pathway (PI3K/AKT/mTOR) and p53, have been known to control cell proliferation and apoptosis (183,184). Beclin-1 and LC3 are two key markers of autophagy. Beclin-1 can enhance autophagy and induce apoptosis of tumor cells (185). p53 can induce autophagy by activating cathepsin D (CTSD), and further enhance the p53mediated tumor suppression effect (186). In addition, p53 can activate the expression of transglutaminase 2 (TGM 2) to promote autophagy and tumor suppression functions, and TGM 2 contributes to the development of p53-induced autophagy program and the function of tumor suppressor (187). Moreover, autophagy is also regulated by p53 in the elimination of aberrant mitochondria. In radiation-resistant cancer cells, p53 induces mitophagy by increasing the levels of BCL 2 interacting protein 3 (BNIP 3) to remove abnormal mitochondria to maintain mitochondrial oxidative phosphorylation (OXPHOS) and inhibit the glycolytic pathway (188). Importantly, PI3K-AkT-mTOR signaling pathway is a well-characterized antiapoptotic cascade in human cancers, since its activation leads to cell proliferation and cell survival. The inducing cell survival of this pathway was mediated by activating anti-apoptotic factors and inhibiting pro-apoptotic factors. AKT can induce anti-apoptotic effect through phosphorylating target proteins by a variety of pathways, such as prevent the apoptosis cascade through inhibiting caspase-9 activity (189). Natural products act by inducing autophagy and apoptosis of cells through down-regulating the expression of mTOR, AKT, PI3K and other proteins (190).
Hu et al. suggested that a combination of matrine with cisplatin promoted apoptosis through activating the caspase apoptosis pathway and inhibiting expression of survivin-related caspase-9 protein (191). A combination of tannic acid with cisplatin acts by inducing apoptosis through activating caspase-3 signaling cascade (192). Xu et al. revealed that saikosaponin D in combination with doxorubibin significantly induced autophagy and apoptosis of HepG2 cells. It suppressed cell proliferation and invasion through up-regulating of ROS, Oatp1b1 as well as TGF-b1 (193). Wang et al. showed that ginsenosides in combination with anti-cancer drugs regorafenib acted by inhibiting growth of HepG2 cells through regulating the expression of Survivin and Caspase-3 genes (194). Ginkgol C17:1 inhibited cisplatin-induced autophagy via AMP-activated protein kinase/ULK1signaling and increased cisplatin-induced apoptosis in HepG2 cells via the PI3K/AKT/mTOR signaling pathway (195). Astragaloside IV effectively protected against cisplatin-induced injury by inducing autophagy and limiting the expression of NLRP3 (196). Aloin in combination with metformin synergistically inhibited HepG2 and Bel-7402 cell proliferation and invasion and induced apoptosis and autophagy through activating PI3K/AKT/ mTOR pathway in vitro. In addition, it has a synergistic effect by inhibiting growth and promoting apoptosis and autophagy in HepG2 xenograft mice model (197). Sinapic acid combined with cisplatin inhibits proliferation and migration of HepG2 and SMMC-7221 cells. At the same time, this combination therapy induces autophagy by upregulating the protein expressions of LC3II, Beclin-1 and Atg5, and downregulating the expression of p62 (198). The combination of 6shogaol and 5-FU inhibit AKT/mTOR/MRP1 signaling pathway and induce liver cancer cell apoptosis by decreasing the expression of AKT, mTOR, MRP1 and cyclin-related proteins (199).
With the development of nanotechnology, nano-drug delivery system can improve drug solubility improve drug stability, increase drug targeting, reduce drug toxicity and side effects. Ceria Nanoparticles can kill cancer cells through induce cancer cells to produce ROS, reduce the expression level of various antioxidant enzymes, cause mitochondrial damage and apoptosis (200). Xu et al. synthesized a composite material DCQ which dextran-coated cerium oxide nanoparticlesloaded quercetin, and discovered that the composite material was more toxic to HepG2 cells, and had no obvious toxic effect on normal cells, human umbilical vein endothelial cells (HUVEC). The composite material can induce the production of ROS, inhibit the fusion of autophagosomes and lysosomes in HepG2 cells and cause the accumulation of autophagosomes, block autophagy and promote apoptosis of HepG2 cells (201). Dong et al. synthesized a nano-layered double hydroxide (NLDH) co-loaded fluorouracil (Fu) and curcumin (Cur) mixed dosage form (LDH-Fu-Cur, LFC). Compared with pure fluorouracil, fluorouracil plus curcumin, and LDH-Fu, LDH-Fu-Cur more efficiently inhibited the growth and promoted the apoptosis of 7721, LM3 and Hep G2 cell lines. It also could significantly downregulate the Bcl-2 gene expression of 7721 cells and induce cell apoptosis by activating caspase 3 and caspase 9 (202). Moreover, han explored a unique amphiphilic PCL-AuNC/Fe (OH)3-PAA Janus nanoparticle (JNP) to simultaneously preserve the hydrophilic drug (doxorubicin) and hydrophobic drug (docetaxel) in their distinct domains. It realized the optional sequential drug release by a single inorganic JNP for the first time, and the simultaneous release of the two drugs can improve efficacy and reduce toxic side effects. Furthermore, the mice treated with dual drug loaded PCL-AuNC/ Fe (OH)3-PAA JNPs under near infrared (NIR) laser irradiation showed better tumor inhibition than solo drug, cocktail and dual drug treated groups, induced aopotosis and blocked the cell cycle of HepG2, and inhibited the growth of tumor in vivo, indicated the effectivity and significance of combined cancer therapy (203). To improve the utilization of curcumin (CUR) and 5-fluorouracil (5-FU) chemotherapeutic drugs in the treatment of liver tumors, increase the accumulation of drugs in tumor sites and reduce the side effects of drugs on the systemic system, Ni Wenfeng constructed a novel targeting nanocarrier to transport the drug to the liver tumor site. In their study, an amphiphilic triblock PEG-PLGA-PEG copolymer was coupled with two targeting ligands (BIO and LAC) to prepare dual-targeted nanoparticles (BLPPNPs), which encapsulated curcumin (CUR) and 5-FU. And the mechanism of synergistic antihepatocarcinogenesis of the two drugs may be that CUR downregulates the expression of DPYP protein by up-regulating the expression of p53 protein, increasing the cytotoxicity of 5-FU and enhancing the anti-tumor effect. At the same time, CUR may decrease the expression of Bcl-2 protein, increase the release of cyt c, and promote the apoptosis of hepatoma cells (204). Zhang successfully prepared a kind of multifunctional nanoparticles: Janus-magnetic mesoporous silica nanoparticles (Fe 3 O 4 -MSNs), with the surface modified by hyaluronic acid (HA) and nano fluorescent probe quantum dots (QD), and chemotherapy drug doxorubicin and berberine (ber), Fe 3 O 4 -MSN/Dox+Ber@HA-QD. The in vitro experiments showed that the nanoparticles could selectively enter HepG2 cells through the CD44 receptor mediated endocytosis pathway and release dox and rer rapidly, which had better cytotoxic effects at low concentrations. The in vivo results also suggested that the nanoparticles have a significantly lower recurrence of the hepatocellular carcinoma induced by dox and significantly reduced the toxic and side effect of doxorubicin while having a better antitumor effect than other groups. In addition, the nanoparticles can also achieve MRI and fluorescence imaging of the tumor site, providing the possibility of drug delivery and therapeutic effects (205). Table 2 summarizes their effects and mechanisms on liver cancer.
Anti-liver cancer mechanism of plantderived natural products in combination with immunotherapy The immune system of the body can recognize and eliminate foreign invading antigens, mutated cells and senescent cells of the body, and maintain the stability of the internal environment. However, tumor cells have the ability to escape immune surveillance, immune identification, and eradication, and immune system dysfunction lead to the development of malignant cells with clinical manifestationsb (206). According to the immune escape mechanism of tumor cells, who established a variety of immunotherapies of cancer (207). This kind of breakthrough therapy is defined that regulates immunological response through activating the organism's immune defense system or action of biological compounds to suppress and prevent tumor growth. At present, the commonly immunotherapy methods include immune checkpoint blockade therapy (208), antibody therapy (209), tumor vaccine therapy (210). Cancer immunotherapy has become an irreversible trend herald in the field of cancer therapy and is regarded as the fourth type of anti-tumor treatment after surgery, radiotherapy, and chemotherapy due to the obvious efficacy and low side effects. Although immunotherapy has made significant progress in the treatment of liver cancer, it needed to be further improved the clinical efficacy, find more specific immune targets, while avoid unnecessary targeting and off-target toxicity (211). Therefore, a combination of natural products and tumor immunotherapy can learn from each other and coordinate with each other to improve the effective rate of treatment.

Regulate immune function
It has been known that immune function of body is closely related to the occurrence and development of tumors. As host immune function is low or suppressed, the incidence of tumor will be increased. And in the progressive growth of tumor, the immune function will also be inhibited by the tumor. These two factors are mutual causation and the fluctuation directly affects the occurrence and development of tumor. Tumor microenvironment is an extremely complex system that involves several kinds of multifunctional immunizing cells and molecules, as well as tumor cells. It is a dynamic system composed of cancer cells, cytokines, extracellular matrix and immune cell subsets (212). Formation of Individual tumor is likely to develop because the tumor-associated microenvironment promotes tumor cell growth and protects tumor cells from elimination of systemic effector cells (213). Natural products in combination with immunotherapy act by inducing antitumor effects through restoring immune recognition and immune elimination by regulating the immune function.

Induce liver cancer cell apoptosis
Endoplasmic reticulum (ER) stress results from the accumulation of unfolded or misfolded proteins in the ER, is involved in regulating apoptotic process in tumor cells (219). It will trigger endogenous and exogenous apoptotic signals as ER dysfunction persists in eukaryotic cells, in addition, the heavy and continuing endoplasmic reticulum stress can lead to cell dysfunction and even cell death. When endoplasmic reticulum stress (ERS) occurs in cells, the expression   Reduced the toxic and side effect of doxorubicin while having a better antitumor effect than other groups (205) cells and induce apoptosis of liver cancer cells (221). Wang et al. demonstrated that paclitaxel in combination with anti-human stathmin1 antibody inhibits proliferation and induces apoptosis of liver cancer cells (222). Lin et al. found that the combination of taxol and immunosuppressant cyclosporin A increases the expression of caspase-9 and caspase-3, induces apoptosis of liver cancer cells through PI3K-mTOR pathway, and effectively reverses drug resistance of paclitaxel (223). Huang et al. confirmed that a combination of gambogic acid and proteasome inhibitors induces liver cancer cell apoptosis and inhibits growth through inducing cytotoxicity and enhancing proteasome inhibition and ER stress (224). A combination of n-glycosylation inhibitor tunicamycin and camptothecin acts by inducing resistance through up-regulating GRP78 and blocking G1 phase cell cycle (225). Nicotinamide and STAT3 inhibitor significantly induced apoptosis and inhibited proliferation by reducing phosphorylation of STAT3 Y705 in HepG2 cells, down-regulating the expression of SNAIL1, VEGFA and ZEB1, EMT-related gene and glucose metabolism. Moreover, the synergistic effect is more significant (226). Photodynamic therapy (PDT), with its unique advantages of minimally invasive, good selectivity, high safety and unique efficacy, has been used as a new therapeutic method for liver tumor treatment. As a natural product, hypocrellin B (HB) can be used as a photosensitive agent for PDT treatment of many diseases, especially for tumor treatment. In order to improve poor water solubility, fast metabolism in vivo and no specific tissue distribution of HB, zhang collected liver cell membrane (CCCM) and modified tumor-targeting ligand (transferrin, TF) into homologous liver cancer cell membrane (TF-CCCM), and prepared ethylene nanoparticles (HB NPs) by double emulsion method. Then TF-CCCM-HB NPs was prepared by extrusion method by mixing the two in a certain proportion. TF-CCCM-HB NPs has strong targeting ability and phagocytosis in homologous liver cells, and suppress the proliferation of tumor cells in a dose-dependent manner. Moreover, it has a synergistic effect with PDT, which can promote the production of ROS and reduce mitochondrial membrane potential. At the same time, TF-CCCM-HB NPs combined with PDT can inhibit the growth of tumor in mice model with liver cancer, prolong the survival time of tumor-bearing mice, and inhibit tumor metastasis and recurrence, which has good biosafety (227). Bai prepared a form drug/gene co-loaded nanoparticles (sCDP/DOX/miR-122), which a nanosystem based on b-cyclodextrin-cored star polymer (sCDP) and co-deliver antitumor drug doxorubicin (DOX) and miRNA-122. s CDP/DOX/miR-122 nanoparticles can remarkably inhibit HepG2 cell proliferation, promote cell apoptosis and increase the expression of p53 and cleaved caspase 3; it also can downregulate the expression of Bcl-w and CCNG1 leading to irreversible cell apoptosis, decrease the expression of MDR1, MRP and P-gp, and improve the sensitivity to DOX of HepG2 cells, thus realizing the synergistic antitumor effect in vitro. sCDP/DOX/miR-122 nanoparticles can slow down the weight loss of nude mice bearing tumor and significantly inhibit tumor growing, thus realizing a synergistic anti-tumor effect in vivo (228). Yuan synthesis iron oxide nanoparticles loaded polydopamine (PDA) and doxorubicin (Dox). Rats were treated with hepatic artery via interventional methods, and it was verified that Fe 2 O 3 -PDA-Dox nanoparticles can simultaneously have the ecceft of Transcatheter arterial chemoembolization (TACE) and photothermal ablation (PTA). It also confirmed that combretastatin A-4 phosphate disodium (CA4P) can increase the uptake of Fe 2 O 3 -PDA-Dox nanoparticles by rat hepatocellular carcinoma, which can enhance the combined treatment of transcatheter arterial chemoembolization and photothermal ablation without additional increase in liver and kidney toxicity (229).  (233). Compound kushen injection significantly enhanced the anticancer activity of sorafenib at a subclinical dose without obvious side effects, and prevented the postsurgical recurrence and rechallenged tumor growth. It activated proinflammatory responses and relieved immunosuppression of tumor-associated macrophages in the liver cancer cell microenvironment by triggering tumor necrosis factor receptor superfamily member 1 (TNFR1)mediated NF-kB and p38 MAPK signaling cascades, which subsequently resulted in apoptosis of liver cancer cells (234). Table 3 summarizes their effects and mechanisms on liver cancer.

Conclusion
The high mortality and morbidity of liver cancer remain a primary challenge for scientific research. Although traditional therapies, such as surgical resection, radiotherapy and chemotherapy, are effective for liver cancer at early stage, the fiveyear survival rate of liver cancer patients is meager and often ends in Increased the expression of caspase-9 and caspase-3, induced apoptosis of liver cancer cells through PI3K-mTOR pathway, and effectively reversed drug resistance of paclitaxel (223) Gambogic acid/ Bortezomib failure. In recent years, plant derived natural products and their secondary metabolites possess characteristics of abundant products, low toxicity and side effects, diverse biologic activities, and high content of active ingredients. It has been considered as the most promising candidates for oncology therapies. However, due to poor water solubility, difficult extraction, and easy to develop drug resistance, a single treatment may not be able to achieve satisfactory efficacy. Therapies that combine natural products with chemotherapy drugs or immunotherapy have been developed to target a variety of cancer pathways. The dose requirement of each agent in combination therapy can be reduced, which reduces the side-effects compared to monotherapy. Targeting various pathways via multiple drug combinations can control disease preferably and decrease the chance of cancer cells becoming increasingly malignant and incurable. It can also target the heterogeneity of the tumor by two or more pathways to toxicity of cancer cells and disruption of their homeostasis (235). This review mainly focused on the mechanisms of plant-derived natural products and combination therapy against liver cancer. We have listed the mainly compounds and combination therapy and individually summarized their antitumour effects and mechanisms ( Figure 2). Like many other cancers, liver cancer arises as a result of accumulation of multiple genetic mutations that cause   Mechanisms of natural products and combination therapy against liver cancer. Alkaloids could inhibits migration through up-regulating TGF-b/Smad and miR-199a-5p, and down-regulating HIF-a expression. Terpeniods promotes apoptosis by inhibiting the PI3K/AKT pathway and the expression of caspase3. It also induces autophagy through up-regulating the expression of LC3II and Beclin-1. Ployphenols inhibits Cyclin D1 and CDK1/4 expression, which in turn block the cell cycle. It also induces apoptosis by activating ATF6/CHOP signaling pathway. Flavonoids induces apoptosis through activation of the ATF6/CHOP pathway and upregulation the ratio of Bax/Bcl-2. Moreover, other natural products inhibit migration through down-regulating VEGFR2-AKT-ERK1/2 signaling pathway and suppressing EMT. Combination of Natural product ingredients inhibits Cyclin D1 and CDK1/4 expression through downregulation of the Wnt/b-catenin pathway, which in turn inhibits cell proliferation. It also reverses multidrug resistant by decreasing the expression of P-gP, MRP and BCRP. Natural product in combination with chemotherapy suppress angiogensis through down-regulating VEGF and Ang expression. It also induces autophagy by upregulating expression of LC3ii and Beclin-1. Natural product in combination with immunotherapy act by inducing antitumor effects through restoring immune recognition and immune elimination by regulating the immune function. It also induces apoptosis by inhibiting the PI3K/AKT and p38MAPK pathway, which in turn induces apoptosis. abnormal cellular proliferation. Our study reviewed natural products and different combination therapy that have antitumor effects on liver cancer, which showed possible benefits in treating patients with liver cancer through mechanisms, such as activation of continuing endoplasmic reticulum stress, suppression of migration, inhibition of cell proliferation, induction of apoptosis and autophagy ( Figure 3). Therefore, it could provide effectively alternative or adjuvant treatment strategies for cancer patients.
However, it is important to note that the study also revealed some disadvantages of combination therapy for cancer. Multiple drug combinations can synergistically increase efficacy, but may also produce unnecessary side effects (236). The target molecular mechanisms of most natural products have not been fully elucidated, especially those related to combinations. Researchers need to further systematically and comprehensively study the immunity or molecular pathology of liver cancer to clarify the relevant mechanisms. Meanwhile, there is still a considerable long way in developing the antitumor strategy of combining natural products with other methods.