Emerging Potential of Naturally Occurring Molecules against Gastric Cancer: Insights Molecular Mechanism and Therapeutic Targets

Gastric cancer, also known as stomach cancer, is a cancer which develops from the lining of the stomach. Accumulated evidences and epidemiological studies have been indicated that natural products play an important role in gastric cancer prevention and treatment, although its mechanism of action did not elucidate yet. Particularly, experimental studies have been showed that natural products displayed a protective effect against gastric cancer via numerous molecular mechanisms such as suppression of cell metastasis, anti-angiogenesis, inhibition of cell proliferation, induction of apoptosis, and modulation of autophagy. Although chemotherapy remains the standard treatment for advanced gastric cancer along with surgery, radiation therapy, hormone therapy and immunotherapy, but its adverse side effects including neutropenia, stomatitis, mucositis, diarrhea, nausea, and emesis are well documented. Additionally, intake of naturally occurring phytochemicals could increase the efficacy of gastric chemotherapy and chemotherapeutics resistance. However, natural product structural stability and powerful bioactivity are important to develop novel treatments for gastric cancer that may minimize such adverse effects. Therefore, the purpose of this review is to summarize the potential therapeutic effects of natural products on prevention and treatment of gastric cancer with intensive molecular mechanisms of action, bioavailability, and safety


Natural plant extracts inducing apoptosis in gastric cancer
Several have been studies demonstrated that natural product extracts of plant or animal sources induced apoptosis against gastric cancer cells and inhibited tumor growth of xenograft animals (Table 3, 4). Acetonic extracts of the endolichenic fungus EL002332 isolated from Ribes nigrum L. (Saxifragaceae) showed selective cytotoxicity against AGS human gastric cancer cells [74]. Liu et al. reported that the polyphenol-rich extract of Ribes nigrum L. (Saxifragaceae), or black currant, induced apoptosis of MKN-45 cells via MAPKand PI3K/Akt-mediated mitochondrial pathways [75]. Euphorbia lunulata Bunge. (Euphorbiaceae) ethanol and n-hexane extract was processed to 10, 20, and 40 μg/mL solutions and applied to SGC-7901 and ADR cells for 24 h [76]. The solution increased the expression of Bax, caspase-3, caspase-8, and caspase-9, and suppressed the expression of Bcl-2, thereby inducing apoptosis.  [85]. The expression levels of pro-apoptotic proteins such as Bak, BH3 interacting-domain death agonist (Bid), Bad, AIF, cytochrome c, cleaved caspases and cleaved PARP were significantly upregulated. Levels of anti-apoptotic proteins such as FLIP, Bcl-2 and Bcl-xL were lowered. Kustiawan et al. discovered that the methanol extract of Trigona incisa propolis represented cytotoxic activity to the human gastric cancer cell line, KATO-III [83]. Methanol extract of Schizandra chinensis Baill Fructus (Schisandraceae) was treated to AGS cells at the dose of 100, 200, 300, or 400 μg/mL for 24 h [84]. The extract induced apoptotic cell death and generated ROS via mitochondria-and caspase-dependent pathways, elevating Bax, caspase-3, and caspase-9 expressions and inhibiting Bcl-2 expression. It is suggested that c-Jun N-terminal kinase and p38 MAPK pathway is also involved. Study by Zhang et al. indicated that trichloromethane fractions of Incarvillea compacta Maxim. Radix (Bignoniaceae) inhibited the proliferation of EBV-positive AGS cells (AGS-EBV) by inducing EBV lytic replication and apoptosis [85]. Aqueous extract of Euphorbia esula Latex (Euphorbiaceae) promoted caspase dependent apoptosis regulated by Bax and Bcl-2 family [86]. 5, 10, 20, 40, 80, 160 mg/L extract was treated on SGC-7901 human gastric cancer cells for 24, 48 h. The activity of caspase-8 and caspase-3 increased 1.73fold and 1.98-fold, respectively. Additionally, efficicicy of Astragalus membranceus Fisch. Radix (eguminosae/Fabaceae) was also studied in vivo model (Table 4) [79]. Xenograft nude mice injected SGC-7901 cells developed tumors of lesser size compared to mice only injected the vehicle.

Apoptosis-inducing mixtures in gastric cancer
Four studies discussing decoctions or pills of TCM focused on the apoptosis-inducing abilities of the mixtures (Table 5, 6). Jinlong capsule (JLC) is an effective Traditional Chinese Medicine widely used to treat gastric cancer patients [88]. It was discovered that JLC induces mitochondrial apoptosis of MGC-803 and BGC-823 cells through a cascadedependent pathway [89]. Xu et al. demonstrated that the TCM decoction Xiao Tan He Wei induces apoptosis against gastric tumors [90]. 1-Methyl-3-nitro-1-nitrosoguanidine (MNNG) was treated to wistar rats and GES-1 cells to induce precancerous lesions of gastric carcinoma in vivo and in vitro. The decoction induced apoptosis of GES-1 cells by inhibiting the NF-κB pathway and showed anti-metastatic efficacy to wistar rats via cell cycle arrest. Yangzhen Sanjie decoction is made from the medicinal herbs Astragalus membranceus Fisch. Radix  [92]. In an in vivo approach, tumor weight of the athymic nu/nu mice were subcutaneously injected HCT116 cells was smallest in the group treated with the fermented decoction (Table  6).

Emergeing role of autophagy in gastric cancer treatment mediated by natural products
Autophagy is a cellular process in which cytoplasmic contents are degraded within the lysosome/vacuole, and the resulting constituents are recycled [93,94]. Autophagy can be classified into macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA) [95]. Among these, macroautophagy, which has been studied the most, is the process of forming autophagosomes that surround organelles and fuses with lysosomes and natural products modulate autophagy [96]. In addition, autophagy can also be categorized into selective and nonselective autophagy. Based on the isolation target, separate kinds of selective autophagy such as mitophagy, pexophagy, and xenophagy can be distinguished [97]. Macroautophagy consists of several sequential steps: initiation, nucleation, elongation, maturation and fusion with the lysosome [98]. Phagosome originates from omegasome, a subdomain of ER, and associates with other organelles such as mitochondria, golgi complex, plasma membrane, recycling endosome, etc. during its development. Four molecules, Unc-51-like kinase 1/2 (ULK1/2), autophagy-related gene 13 (ATG13), family 200-kD interacting protein (FIP200) and AtG101 form the ULK1/2 complex and initiates the process. The mechanistic target of rapamycin complex 1 (mTORC1) is a major inhibitor of the ULK1/2 complex [93]. AMP-activated protein kinase (AMPK) inhibits mTORC1 and leads to the activation of ULK1/2 complex [98]. The ULK1/2 complex phosphorylates the class III phosphatidylinositol-3-kinase (PI3K) vacuole protein sorting 34 (VPS34) complex consisting of VPS15, Beclin-1 and AtG14 complex, which promotes the formation of phosphatidylinositol-3-phosphate (PI3P), which is an essential lipid molecule required for the nucleation step of the phagophore [99]. PI3P recruits PI3P-binding proteins such as WIPI2B, a protein known to facilitate LC3-lipidation [100]. Formation and expansion of the phagophore is related to the generation of Atg12-5-16L1 complex. Atg12 binds with Atg5 and composes a complex with Atg16L. The Atg12-5-16L1 complex lipidates LC3-I into LC3-II [101,102]. LC3-II, considered a marker of autophagy, is essential for phagosome elongation and fusion [103,104]. When the phagosome encloses and becomes a mature autophagosome, it fuses with a lysosome and degradation and recycling process follows ( Figure 2).

Figure 2.
Natural products regulates molecular mechanism of autophagy. Natural products initiate autophagy by the formation of a pre-autophagosomal structure via association of PI3K-AMPK, mammalian target of rapamycin (mTOR), ULK1, Vps34, and the Beclin-1 complex which contribute to the formation of the pre-autophagosomal structure in addition to activate phagophore formation. Fusion of mature autophagosome as well as lysosome causes autolysosome formation. Lastly, elimination of molecule is happened by acid hydrolases which has been produced nutrients as well as recycling metabolites.
Two single compounds and two plant extracts were reported to induce autophagy along with apoptosis against gastric cancer cells (Table 7). Rottlerin, extracted from Mallotus philipensis Muell (Euphorbiaceae), induced autophagy and caspase independent apoptosis against SGC-7901 and MGC-803 cells [105]. Autophagy is induced by downregulating mTOR and S-phase kinase-associated protein 2 (Skp2).  [110]. Proteins that mediate ER stress mediated apoptosis including glucose-regulated protein 78 (GRP78), C/EBP-homologous protein (CHOP) and caspase-12 has been greatly upregulated in the group treated kangfuxin. Transmission electron microscopy (TEM) image results has been shown much more autophagosomes compared with the control. In addition, the LC3-I/LC3-II ratio and expression levels of Beclin-1 were also higher in the kanfuxin group. Additionally, Natural plant extracts of Saussurea lappa Clarke, Dioscorea nipponica Makino, and Melandrium firmum has been found to induce anti-proliferative as well as apoptotic function [111][112][113]. Therefore, autophagy induction by natural products might possibly be targeted as a potential therapeutic approach to control gastric cancer. Table 7. Autophagy-Inducing Natural Products in vitro.

Classification
Compound

Anti-metastasis effects of natural products in gastric cancer
Metastasis is a major contributor of death in cancer patients, arising from a growing tumor from which cells escape to distant organs of body [121]. Targeting metastasis is an attractive strategy in cancer treatment. Sixteen studies highlighted the anti-metastatic ability of diverse natural products in vitro and in vivo models. Epithelial-mesenchymal transition (EMT) was inhibited in many cases while cell cycle arrest and other blockages of tumor proliferations were also observed ( Table 11, 12). Evodiamine is a chemical compound extracted from Evoida rutaecarpa (Rutaceae) [122]. The compound suppressed epithelialmesenchymal transition of AGS and SGC-7901 gastric cancer cells via inhibition of Wnt/β-catenin signaling pathway (Figure 4). Sulforaphane is an organosulfur compound extracted from Brassica oleracea var. italica Plenk (Brassicaceae) [123]. The compound ex-  [67]. The compound inhibited epithelial-mesenchymal transition of gastric cancer by upregulating miR-200a, E-cadherin and suppressing β-catenin when treated at dose of 0.5, 1 μM for 48 h. Tangerines, grapefruits, lemons and oranges contain low-molecular-weight citrus pectin (LCP) [128]. Wang et al. demonstrated that anti-metastatic effect of LCP by treating it on AGS cells in vitro at dose of 0.625, 1.25, 2.5, 5, 10 mg/mL for 24 h. Epithelial-mesenchymal transition was inhibited followed by downregulation of Cyclin B1, galectin-3 (GAL-3) and Bcl-xL. LCP had both apoptotic and anti-metastatic effects. N-butylidenephthalide is a compound extracted from the roots of Angelica sinensis (Oliv.) Diels Radix (Apiaceae) [63]. When treated at dose of 50 μg/mL for 24, 48 h on AGS, 75 μg/mL for 24, 48 h on NCI-N87 and 25, 50, 75 μg/mL for 48 h on TSGH-9201, inhibition of tumor metastasis was observed. The compound promoted E-cadherin expression while downregulating N-cadherin and vimentin slug. It is unclear whether natural products exert anti-metastatic effect in a multi-target manner. Further study is required to distinguish the specific mechanism. The primary mechanism observed was through the inhibition of cadherin-catenin adhesion. Compounds and extracts including baicalein, Celastrus orbiculatus extract, dehydroeffusol, etc., down-regulated N-cadherin, VE-cadherin, β-catenin and other related factors [125,126,129,130]. Activity of E-cadherin was repressed on the other hand, which inhibits EGFR kinase activity. The mechanism leads to downstream regulation of multiple growth factor related activities, which is associated with anti-metastatic activities of such natural products. Notably, Shi J et al. demonstrated the Xiaotan Sanjie decoction down-regulated VEGF-A, VEGFR-1 and VEGFR-2 [119]. In other aspect, the Bcl-2 family proteins were also found to play role in anti-metastatic effects of natural products. For example, products such as andrographolide and Xian Tan He Wei elevated Bax protein activity while inhibiting Bcl-2 protein [127,131]. Many other factors including PI3K, Akt, Rac1, CDX1/2 play a role in anti-metastatic activity of natural products, some of which are also related to apoptosis of tumor cells. For example, Lyu et al. reported the paeonol extract suppressed the expression of MMP-2 and MMP-9, which lead to inhibition of proliferation and so inhibition of metastasis (Figure 4) [64]. It is unclear whether natural products exert anti-metastatic effect in a multi-target manner. Further study is required to distinguish the specific mechanism.

Chemotherapy resistance and natural products in gastric cancer
Drug resistance has been an important issue in cancer treatment. It is known as a primary cause limiting cancer treatment [134]. Several studies have indicated that natural products could be used along with the primary drug to overcome drug resistance and reinforce its efficacy. Previous studies focused on how natural products can support conventional antibiotics by suppressing resistance [135]. This review highlighted natural products' potential in handling cancer chemotherapy resistance (Table 13, 14). Isorhamnetin, a flavonoid metabolite of quercetin commonly found in onions, minimized the apoptotic effects of capecitabine via inhibition of NF-κB and various NF-κB regulated gene products in tumor cells [136]. Attenuation of capecitabine resistance was also observed in an animal model in which athymic nu/nu female mice grafted with SNU-5 were treated with isorhamnetin (1 mg/kg) three times a week for 4 weeks. According to Wei et al., liquiritin isolated from Glycyrrhiza uralensis Fischer. Radix (Leguminosae/Fabaceae/Fabaceae) could circumvent the resistance of cisplatin-based chemotherapy [137]. SGC7901/DDP cells were treated with DDP (2 µg/ml) or liquiritin (80 µM) or both for 24 h. The combination greatly suppressed cell proliferation and induced apoptosis, autophagy and G0/G1 phase cell cycle arrest against DDP resistant gastric cancer cells compared to DDP single treatment. Cleavage of caspase-8, caspase-9, caspase-3 and PARP were activated and increased expression levels of LC-3B and Beclin 1 were observed. Abdelfattah et al. isolated a new alkyl sulfonic acid derivative from the methanolic extract of mycelium of Streptomyces sp. IFM 11694 and named it sulfotanone [138]. Its bioactivity was evaluated for effects on TRAIL-resistance of AGS cells by comparing cell viability in the presence and absence of TRAIL (100 ng/mL). Sulfotanone showed TRAIL-resistance overcoming activity in AGS cells at concentrations of 40 μM. Bufalin, a traditional Chinese medicine extracted from Venenum bufonis, showed synergetic effects with cisplatin to inhibit proliferation and promote apoptosis of gastric cancer cells SGC-7901, MKN-45, and BGC-823 by diminishing the activation of cisplatin-induced Akt and its downstream molecules under normoxic and hypoxic conditions [139]. Astragalus polysaccharide and apatinib cotreatment were reported to enhance apoptosis compared to apatinib monotherapy [140].
The efficacy of astragalus polysaccharide, an active component extracted from Astragalus mambranaceus Bunge Radix (Leguminosae/Fabaceae/Fabaceae), arises mainly from its ability to inhibit autophagy of apatinib-resistant cells which serves as a survival mechanism. Abdelfattah et al. isolated a new ana-quinonoid tetracene metabolite from the ethyl acetate extract of the culture of marine bacteria Streptomyces sp. EGY1 and named it sharkquinone [141]. Sharkquinone sensitized TRAIL-resistant AGS cells and suggested their potential use in combination with TRAIL against AGS cells. Tanshinone IIA is identified as an interesting agent with potential to treat doxorubicin-resistant gastric cancer cells. [142]. 5 µM Tanshinone IIA solution combined with 0.05 µg/mL of doxorubicin showed anticancer effect against doxorubicin-resistant cell lines including SNU-638, SNU-668 and SNU-216 and SNU-620. Apoptosis was mainly induced by inhibition of multidrug resistance-associated protein 1 (MRP1). Zuo Jin Wan Formula is a traditional Chinese medicine pill that consists of Coptidis Rhizoma (Ranunculaceae), and Evodiae rutaecarpa Bentham. Fructus (Rutaceae) in the ratio of 6:1. Sun et al. reported that Zuo Jin Wan Formula induced apoptosis in primary DDP-resistant gastric carcinoma cells by stimulating cofilin-1 mitochondrial translocation [143]. Translocation of cofilin-1 was mediated by Akt. Accumulation of Bax was observed while expression level of Bcl-2 was decreased. Natural protucts and their target signal is presented in figure 5. Although specific targets vary, most natural products aimed to prevent drug resistance by downregulating Akt and NF-κB and following pathways (Figure 4). Bufalin from Venenum bufonis and the Zuo Jin Wan formula effectively down-regulated Akt [139,143]. Similarly, the mineral isorhamnetin from Quercetin inhibited cell viability and prevented drug resistance by downregulating NF-κB [130]. Reduction of drug resistance by natural compounds was demonstrated in other aspects. Pseudolaric acid B from Pseudolarix kaempferi inhibited COX-2, PKC-α and P-gp, factors that promote angiogenesis by cancer cells. Liquirtin from the Glycyrrhiza genus promoted p53, p21 and caspase cleavages while inhibiting cyclin activities. The compound's anti-resistant ability may be focused on apoptotic effects. Other factors such as Bax/Bcl-2 in mitochondria, ERK1/2, MMP2, and PARP are broadly affected by many natural products covered in the review. Each compound, extract or decoction had anti-resistant effects over various aspects of cancer cells when tested in vitro or on animal models.

Limitation and future perspectives of natural products in gastric cancer treatments
Gastric cancer is known to account for the fifth highest incidence and the fourth highest mortality among all cancers worldwide [1]. Chemotherapy is one of the methods typically used in advanced gastric cancer treatment, but it exerts severe side effects that limit the efficacies and decrease quality of life. Development of therapeutic remedies with less adverse effects and lower chemo-resistance is required. Natural products are emerging as alternative resources to combat gastric carcinoma. While few natural products such as aflatoxins may be noxious or even carcinogenic [144], many phytochemicals with beneficial bioactivity can be successfully selected through deliberate investigations. Therefore, several natural resources obtained from dietary fruits and vegetables were discussed is this review. Curcumin, black currant extract, oligosaccharide isolated from tomato, sulforaphane derived from broccoli, citrus pectin originated from tangerine, grapefruit, lemon and orange are good examples. These medicinal resources are still being extensively used in traditional Korean and Chinese medicine. Many natural substances were shown to exhibit multiple effects. The variety is attributed to the structural diversity and multi-target characteristic of natural compounds [145]. Several decoctions were also reported to suppress cancerous cells and tumors comprehensively. In this light, decoctions have the potential to develop as effective therapeutic agents against gastric cancer based on their multi-efficacy and enhancement of efficacy through various methods of post-processing. NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; VEGF, vascular endothelial growth factor; COX-2, cyclooxygenase 2; MMP-9, matrix metallopeptidase 9. 7 While there have been similar reviews highlighting the anti-neoplastic efficacies of natural products or phytochemicals, few of them are written with regards to the chemical classification of each bioactive compound [146]. This review is not only a simple compilation of previous in vitro studies testing natural products on gastric cancer but goes as far as to systematically organizing previous works depending on each cancer related pathways, namely, apoptosis, autophagy, metastasis, drug-resistant capability and more. Studies grouped into each pathway were then organized according to the phytochemical classification of the active compounds. As there is currently no golden standard for classifying phytochemicals, we adopted a comprehensive and clear method previously demonstrated in a literature highlighting the efficacies of natural products on gastrointestinal diseases [147]. This will help researchers rule out or select appropriate candidate species of natural products for further studies. One limitation of this review is that it only included studies published from 2014 to 2020. Also, clinical trials were excluded to focus on laboratory experiments highlighting specific biological pathways. Several investigations were insufficient to elucidate anti-cancer mechanisms at molecular levels. They were generally focusing on the cytotoxicity of the chemicals or reporting newly discovered compounds, which makes incisive research burdensome. By and large, more than half of the studies only carried out experiments in vitro. More in vivo studies are recommended to bridge the advance to clinical trials and therapeutic use. Decoctions and extracts proven safe for human should go under well designed clinical trials to prove their efficacy in the evidence-based medicine perspective.
Natural products are indeed effective in the single compound to single target mechanistic perspective; however, it is worth highlighting the complex interactions between many compounds. While the importance of studying the interactions between multi-compound natural products and other drugs was previously highlighted in many literatures, it is also important to further investigate the interactions between different natural products, including herbal medicines in a biochemical manner [148]. A systemic approach with focus on structural similarities of several phytochemical compounds and human metabolites is a potential way of clearly highlighting the efficacies of multi compound drugs. Furthermore, the fact that current drug interaction analytical methodology for novel multi-targeted agents closely resembles the Kun-Shin-Choa-Sa in many perspectives makes it more intriguing to apply this principle in modern drug studies [149,150].

Conclusions
In this review, we summed up seventy-six studies considering natural products that have anti-cancer efficacy against gastric cancer. Natural products mainly induced cell death by apoptosis and autophagy, cell cycle arrest, inhibit angiogenesis and metastasis, and circumvent chemo-resistance against stomach cancer cells in vitro or in vivo through various molecular mechanisms. Several compounds, extracts and decoctions showed multiple efficacies, attributed to structural complexity and multiple target pathways and proteins of natural products. Thus, natural substances implicate possibilities of being used in nutrition or medications which may lead to novel discoveries in alternative medicine in cancer treatment. This review would provide data for future research and clinical trials to develop novel drugs from natural products for gastric cancer treatment.