Modulating Effects of Cancer-Derived Exosomal miRNAs and Exosomal Processing by Natural Products

Simple Summary Cancer cells generate exosomes (extracellular vesicles) to regulate many cell functions for tumor progression. Many exosome-modulating clinical drugs have been developed for effective cancer therapy, but the functions and exosome processing (secretion and assembly) modulation by natural products are not well understood. In this review, we fill the gaps between natural products-modulated miRNAs and exosome-processing by the target gene prediction of the bioinformatics database. The cancer-derived exosomal miRNAs and their exosome processing and modulated cell functions by natural products are well organized. Consequently, this review provides a comprehensive and potential modulating mechanism and targets for exosome processing and cancer cell functions for natural products. Abstract Cancer-derived exosomes exhibit sophisticated functions, such as proliferation, apoptosis, migration, resistance, and tumor microenvironment changes. Several clinical drugs modulate these exosome functions, but the impacts of natural products are not well understood. Exosome functions are regulated by exosome processing, such as secretion and assembly. The modulation of these exosome-processing genes can exert the anticancer and precancer effects of cancer-derived exosomes. This review focuses on the cancer-derived exosomal miRNAs that regulate exosome processing, acting on the natural-product-modulating cell functions of cancer cells. However, the role of exosomal processing has been overlooked in several studies of exosomal miRNAs and natural products. In this study, utilizing the bioinformatics database (miRDB), the exosome-processing genes of natural-product-modulated exosomal miRNAs were predicted. Consequently, several natural drugs that modulate exosome processing and exosomal miRNAs and regulate cancer cell functions are described here. This review sheds light on and improves our understanding of the modulating effects of exosomal miRNAs and their potential exosomal processing targets on anticancer treatments based on the use of natural products.


Introduction
Exosomes are extracellular vesicles of 30-100 nm in size that are secreted by both cancer and normal cells [1,2]. Cancer cells secrete more abundant and complex compositions in exosomes than normal cells. Cancer-derived exosomes exhibit diverse functions in regulating proliferation, migration, invasion, metastasis, drug resistance, inflammation, and immune responses [1,3,4].
Among the non-coding RNAs, this review focuses only on miRNAs, particularly exosomal miRNAs. miRNA uptake into exosomes is not a random but selective process involving secretion and transportation between exosome donors and receptors [15]. miRNAs are a group of small non-coding RNAs of 21-25 nucleotides in size. miRNAs can modulate gene expression by inhibiting mRNA translation or improving the mRNA degradation of target genes [14]. By binding to the 3 -untranslated regions (UTR) of target genes, miRNAs can knock down target gene expressions to assess their diverse functions.
As mentioned above, exosomes and miRNAs have a close relationship in regulating cell functions. Recently, anticancer studies using natural products have shown progression in research involving exosomes and miRNAs. However, the potential impacts of exosomes and miRNAs on natural-product-regulating cancer cell functions lack systemic organization. The modulating effects of natural products on exosome biogenesis and exosomal miRNAs are discussed later, particularly in regard to their capacity for regulating exosomal processing (secretion and assembly). Moreover, some natural products and exosomal miRNAs show anticancer effects but lack investigation regarding their impacts on exosomal processing. This gap can be filled by utilizing the miRDB database [31], a bioinformatic tool which can predict the target genes of exosome processing by inputting natural-product-modulated exosomal miRNAs.
In the following review, we first explore the relationship between exosome processing (secretion and assembly) and natural products (Section 2), because the impact of exosome processing is rarely discussed in detail in the literature. Next, the prediction of the targeting of exosome-processing and AKT-signaling genes of exosome miRNAs is assessed (Section 3), because the contribution of exosome processing is rarely emphasized in the literature. The modulating effects of exosome production by natural products and their exosome delivery potential for cancer treatment (Section 4) are explored. Finally, the regulation of the cancer cell functions of natural-product-modulating miRNAs and exosomes (Section 5) is summarized ( Figure 2). Consequently, this review sheds light on the organization of the relationship between exosomal processing and its related genes, exosomal miRNAs, cell functions, and natural products.
As mentioned above, exosomes and miRNAs have a close relationship in regulating cell functions. Recently, anticancer studies using natural products have shown progression in research involving exosomes and miRNAs. However, the potential impacts of exosomes and miRNAs on natural-product-regulating cancer cell functions lack systemic organization. The modulating effects of natural products on exosome biogenesis and exosomal miRNAs are discussed later, particularly in regard to their capacity for regulating exosomal processing (secretion and assembly). Moreover, some natural products and exosomal miRNAs show anticancer effects but lack investigation regarding their impacts on exosomal processing. This gap can be filled by utilizing the miRDB database [31], a bioinformatic tool which can predict the target genes of exosome processing by inputting natural-product-modulated exosomal miRNAs.
In the following review, we first explore the relationship between exosome processing (secretion and assembly) and natural products (Section 2), because the impact of exosome processing is rarely discussed in detail in the literature. Next, the prediction of the targeting of exosome-processing and AKT-signaling genes of exosome miRNAs is assessed (Section 3), because the contribution of exosome processing is rarely emphasized in the literature. The modulating effects of exosome production by natural products and their exosome delivery potential for cancer treatment (Section 4) are explored. Finally, the regulation of the cancer cell functions of natural-product-modulating miRNAs and exosomes (Section 5) is summarized ( Figure 2). Consequently, this review sheds light on the organization of the relationship between exosomal processing and its related genes, exosomal miRNAs, cell functions, and natural products.

Figure 2.
Connections between different sections of this review. Connections between natural products and exosomal processing are examined in Sections 2 and 4. The connection between exosomal processing and exosomal miRNAs is examined in Section 3. Finally, connections between cancer cell functions, exosomal miRNAs, and natural products are examined in Section 5.

Exosome Processing (Secretion and Assembly) Genes
Several extracellular biogenesis genes, including exosomal secretion and assembly, were summarized using the Mouse Genome Database in Gene Oncology (GO) functions http://www.informatics.jax.org/vocab/gene_ontology/GO:1990182 (retrieval date: 11 November 2022) [32]. Exosomal secretion begins with the fusion of the partial endosomal membrane of a multivesicular body (MVB) with the plasma membrane, and it ends with Connections between different sections of this review. Connections between natural products and exosomal processing are examined in Sections 2 and 4. The connection between exosomal processing and exosomal miRNAs is examined in Section 3. Finally, connections between cancer cell functions, exosomal miRNAs, and natural products are examined in Section 5.

Exosome Processing (Secretion and Assembly) Genes
Several extracellular biogenesis genes, including exosomal secretion and assembly, were summarized using the Mouse Genome Database in Gene Oncology (GO) functions http:// www.informatics.jax.org/vocab/gene_ontology/GO:1990182 (retrieval date: 11 November 2022) [32]. Exosomal secretion begins with the fusion of the partial endosomal membrane of a multivesicular body (MVB) with the plasma membrane, and it ends with the release of membrane-bounded vesicles into the extracellular space ( Figure 1). Three main functions of exosomal secretion are classified: multiple vesicular body fusion into the apical plasma membrane, the negative regulation of exosomal secretion, and the positive regulation of exosomal secretion. Furthermore, exosomal assembly is the process in which a set of components are incorporated, aggregated, and bonded to generate an extracellular vesicular exosome. Three main functions of exosomal assembly are classified: extracellular exosome assembly, the positive regulation of extracellular exosome assembly, and the negative regulation of extracellular exosome assembly.
For exosomal assembly, the CD34 antigen (CD34) gene is listed among the cells that affect "extracellular exosome assembly". Four genes are listed as modulators of "positive regulation of extracellular exosome assembly", including PDCD6IP, SDC1, SDC4, and SDCBP, which also belong to the group of genes with an exosomal secretion function. Three genes are listed among the cells that affect the "regulation of extracellular exosome assembly", including PDCD6IP, STAM, and TSG101, which also belong to the group of cells with an exosomal secretion function mentioned above.

Exosomal Secretion and Assembly Effects of Natural Products in Regulating Cell Functions
Some of the exosomal secretion (Section 2.2.1) and assembly (Section 2.2.2) genes have been reported to be regulated by natural products (Table 1).

Exosomal Secretion Effects of Natural Products in Regulating Cell Functions
Natural-product-derived exosomes exhibit a capacity for the sophisticated regulation of cell functions in non-cancer and cancer cells. The potential roles of exosome-processing genes in natural product treatments can be predicted by target gene retrieval using the miRDB database (Table 1).
For cancer cells, several natural products exhibit modulating effects on cell functions in regard to exosome processing. The methanolic extract of Moringa oleifera leaves suppresses the proliferation and causes the G1 arrest and apoptosis of cervical cancer cells by downregulating COPS5 (Table 1) [39]. Rutin, a bioflavonoid, induces the apoptosis of cervical cancer cells by downregulating COPS5 [40]. Heteronemin, a marine sesterterpenoid, induces ferroptosis by upregulating the protein expression of divalent metal transporter-1 (DMT1) and STEAP3 in pancreatic cancer cells [41]. Dihydroartemisinin, a metabolite of artemisinin, downregulates the DMT1 and STEAP3 genes controlling iron uptake in liver cancer HepG2 cells (Table 1) [42].
Exosomal proteins can activate AKT signaling in the regulation of metastasis. Annexin A5, one of the exosome proteins in prostate cancer tissues, activates AKT signaling to stimulate the epithelial-mesenchymal transition (EMT) and upregulate matrix metalloproteinase-2 (MMP2) and MMP9 expression [5]. The exosomal miRNAs involved in AKT signaling have been applied in animal [59] and preclinical experiments [60]. Bonemarrow-mesenchymal-stem-cell-derived exosomes, which are rich in miR-126-3p (miR-126), enhance the migration and angiogenesis of human umbilical vein endothelial cells (HUVECs) [59]. This exosomal miR-126-3p stimulates vascularization at wound sites and improves cutaneous wound healing in mice models. Plasma exosomes isolated from Graves ophthalmopathy with an effective response to intravenous glucocorticoid therapy contain a high level of miR-885-3p, showing AKT inhibition and improving glucocorticoid sensitivity [60]. Consequently, the exosomal miRNAs with AKT modulating ability are potential tools for preclinical applications. Table 2. Connecting some exosomal miRNAs to the predicted targets of exosome processing and AKT genes.

Natural Products Modulate Exosome Production and Their Exosome Delivery for the Purpose of Cancer Treatment
Many natural-product-derived exosomes have been demonstrated to exhibit theranostic effects in cancer therapy [63][64][65][66]. The modulating results of exosome biogenesis and delivery by natural products are discussed as follows below.
Some natural products improve exosome biogenesis. Sulforaphane suppresses the fusion of early and late endosomes (GFP-Rab5a and GFP-Rab7a) with the lysosome, blocks the autophagy flux, promotes exosome production, and triggers exosome-dependent paracrine senescence by downregulating mTOR and transcription factor binding to IGHM enhancer 3 (TFE3) [67]. Sulforaphane induces a high protein concentration of exosomes and causes the accumulation of exosome marker CD63 in esophageal cancer cells. Moreover, supernatants from sulforaphane-treated cancer cells show high CD63 expression [67]. Consequently, sulforaphane triggers exosome biogenesis and secretion in esophageal cancer cells.
In contrast, some natural products suppress exosome biogenesis. Autophagy and lysosome dysfunction enhance exosome secretion [68,69] and vice versa. Asteltoxin inhibits mitochondrial ATP synthase and exosome generation by upregulating AMPK-dependent mTORC1 inactivation and lysosome activation [70]. Transmission electron microscopy analysis shows that asteltoxin induces lysosome-MVB fusion, causing the downregulation of exosome generation. Berberine suppresses the proliferation of colon cancer cells by downregulating acetyl-CoA carboxylase (ACC) for fatty acid synthesis and reducing exosome biogenesis and the secretion of colon and cervical cancer cells [71], an observation which is supported by the finding that berberine downregulates syntenin and TSG101, as intracellular vesicle markers. Some natural-product-derived exosomes exhibit modulating effects on cell functions. Exosomes used in phytoagent deoxyelephantopin treatment, a plant deoxyelephantopin derivative, suppress the ROS-mediated proliferation of breast cancer cells, reversed by Nacetylcysteine [72]. Phytoagent deoxyelephantopin also enhances calcium-dependent exosome secretion from breast cancer cells. Momordica charantia-derived exosome-like nanovesicles suppress the proliferation and migration of glioma cells by downregulating phosphorylated PI3K/AKT [73]. Fusobacterium nucleatum is rich in colon cancer lesions associated with colon cancer carcinogenesis and metastases. Exosomes from Fusobacterium nucleatum enhance the invasion of colon cancer cells. This invasion is prevented by the bioactive compounds of Paris polyphylla, such as pennogenin 3-O-beta-chacotrioside and polyphyllin VI, which exhibit cell-killing effects on Fusobacterium nucleatum [74]. All this warrants an advanced examination exploring the impacts of exosomal biogenesis by natural products and natural-product-derived exosomes on cell functions in the future.
Moreover, exosomes are naturally generated, showing lower cytotoxicity and immunogenicity and higher biocompatibility than lipid-based nanoparticles [6,65,75]. Exosomes were reported to effectively deliver several natural products that can be exploited for preclinical anticancer therapy in vitro and in vivo [65]. The oral delivery of paclitaxel using milk-derived exosomes results in less side effects of immunologic toxicity and higher antitumor effects than i.v. in lung-tumor-xenograft nude mice [76]. Exosome-delivered curcumin exhibits a high in vitro stability and in vivo bioavailability [77]. Celastrol-loaded milk exosomes show a high degree of anti-lung tumor growth with in vivo biosafety [78]. This warrants the advanced testing of more natural products based on exosome delivery strategies in the future.

The Role of Natural-Product-Modulating miRNAs and Exosomes in Regulating Cancer Cell Functions
A mounting array of literature reports that natural products modulate many miRNAs that regulate their target genes to affect several of the cell functions of cancer cells [79]. However, most of these studies did not investigate the impacts of exosomal miRNAs on anticancer effects using natural products.
Recently, several natural-product-induced exosomal miRNA studies have been reported. Aurea helianthus extract inhibits the migration and induces the senescence and autophagy of endometrial cancer cells [80]. Several miRNAs derived from the induced exosomes in these extract-treated endometrial cancer cells were upregulated or downregulated. However, there is a lack of systemic information on the modulating effects of drug-induced exosomal miRNAs based on natural products. Most natural-product-modulating miRNA studies have focused on impacts on cancer cell functions without considering the contribution of exosomes. Consequently, there are gaps between exosomal miRNAs and natural products in terms of their anticancer effects.
Exosomal miR-1246 is reported to regulate cell migration. Exosomal miR-1246 from highly metastatic oral cancer cells promotes the migration and invasion of poorly metastatic oral cancer cells by downregulating the DENN/MADD-domain-containing 2D (DENND2D) [134]. In addition to antimigration, miR-1246 was reported to modulate proliferation (Table 3). Bladder cancer T24 cells highly express miR-1246. Curcumin inhibits the proliferation of bladder cancer cells by downregulating miR-1246 [98]. Combined treatment (curcumin and X-ray) synergistically suppresses its proliferation to a greater extent than individual treatments by decreasing miR-1246 expression [98].
Exosomal miR-30d-5p appears in higher levels in cervical cancer tissues than in normal controls [135]. However, the miR-30d-5p-modulating cell function has rarely been reported, particularly in regard to the antiproliferation of cancer cells. A recent study of natural products reported the antiproliferation effect achieved by the modulation of miR-30d-5p (Table 3). Piperlongumine, a long-pepper-derived amide alkaloid, suppresses the proliferation of osteosarcoma cells by downregulating miR-30d-5p and upregulating its target, the suppressor of cytokine signaling 3 (SOCS3) [130].
Exosomal miR-421 regulates the chemoresistance of cancer cells. Exosomes from cisplatin-resistant oral cancer patients enhance the proliferation and reduce the cisplatin sensitivity of cisplatin-resistant cells by downregulating miR-421 expression [141]. The hypermethylation of transcription-factor-activating-enhancer-binding protein 2e (TFAP2E) enhances 5-fluorouracil chemoresistance in gastric cancer cells by upregulating exosomal miR-421 [142]. However, the impact of apoptosis by exosomal miR-421 is unclear. A recent natural product study reported the apoptosis function through the modulation of miR-421 (Table 3). Isoliquiritigenin induces the apoptosis and DNA damage of oral cancer cells by downregulating miR-421 expression [116].
miR-23b-3p suppresses the proliferation and migration of prostate [147] cancer cells, while it enhances pancreatic cell migration [148] and salivary cancer cell angiogenesis and metastasis [149]. However, the apoptosis function of exosomal miR-421 has rarely been reported. A natural product investigation validated the fact that apoptosis induction results from the modulation of miR-23b-3p (Table 3). 10-Hydroxycamptothecin, a Nothapodytes nimmoniana-derived natural product, causes the apoptosis of fibroblasts by upregulating miR-23b-3p [127].
Exosomal miR-382-5p from cancer-associated fibroblasts enhances the migration of oral cancer cells [150]. A recent natural product study demonstrated the novel function of apoptosis induction through the modulation of miR-382-5p (Table 3). Polydatin, a metabolite of trans-resveratrol, inhibits the proliferation and causes the apoptosis of colon cancer cells by upregulating miR-382-5p and downregulating its target, programmed cell death ligand 1 (PD-L1) [129].
Some miRNAs showing resistance-promoting effects are downregulated by several natural products. miR-21-5p has been identified in exosomes from hypoxic oral cancer cells. miR-21-5p-containing hypoxic oral cancer exosomes also exhibit cisplatin resistance in oral cancer cells, as evidenced by exosome transfer experiments [153]. Natural products may inhibit drug resistance in cancer cells by downregulating miR-21-5p. Tricin, an Allium atroviolaceum-derived compound, sensitizes the docetaxel response to prostate cancer cells by downregulating miR-21-5p [93] (Table 3).
All this warrants a detailed assessment of all the miRNA-modulating effects of these natural products that are employed in cancer studies in the future.
Cancer-associated fibroblasts enhance the progression of head and neck cancer cells by downregulating exosomal miR-3188 [164]. Without considering exosomes, other cancer studies also reported the tumor suppressive function of miR-3188. miR-3188 inhibits the proliferation of nasopharyngeal [165] and lung [166] cancer cells by targeting mTOR. A natural product study showed that pinolenic acid, a Pinus-species-derived natural product, upregulates miR-3188 to target the pyruvate dehydrogenase Kinase 4 (PDK4) and the mitochondrially encoded ATP synthase membrane subunit 6 (MT-ATP6) genes, showing anti-inflammatory effects in rheumatoid arthritis patients [103] (Table 3).
Some natural product studies showed that miRNAs can modulate liver fibrosis. Thymoquinone, a black-seed-oil-derived compound, suppresses liver fibrosis by upregulating miR-30a-5p to inhibit its target, such as snail family transcriptional repressor 1 (SNAI1), suppressing EMT [111]. Astaxanthin, a xanthophyll carotenoid, inactivates liver-fibrosisassociated hepatic stellate cells by downregulating miR-382-5p [128]. This warrants a detailed evaluation of all the miRNA-modulating effects of the aforementioned natural products employed in cancer studies.
Furthermore, several exosomal miRNAs have been identified in a number of cancer cells, but no natural product studies have been reported to date. Some exosomal miRNAs show proliferation-/invasion-promoting effects. Exosomal miR-626 enhances the proliferation and migration of oral cancer cells by targeting nuclear factor I/B (NFIB) [177]. miR-10b-5p shows a higher expression in metastatic breast cancer cells than in non-metastatic breast cancer or normal cells. Exosomal miR-10b-5p transmission enhances the invasion capacity of normal breast cancer [178]. The delivery of exosomal miR-10b-5p from gastric cancer cells also improves the proliferation of fibroblasts [179].

Overview of the Natural Products and Their Modulating Exosomal miRNAs That Regulate Exosomal Processing
The connections of natural products with their exosomal miRNA-regulated cell functions are summarized in Table 3. However, the impacts of exosomal processing, their genes related to these natural products, and their modulated exosomal miRNAs remain unclear. Utilizing the miRDB database [31], the target prediction of the exosome-processing genes for these natural-product-modulated exosomal miRNAs (Table 3) was performed. From exosomal assembly to secretion, the exosomal processing genes targeted by natural-productmodulated exosomal miRNAs were plotted (Figure 3). This warrants a careful investigation of the predicted targets of these natural-product-modulated exosomal miRNAs based on experiments in the future.  Overview of natural products modulating exosomal miRNAs through exosomal processing genes and exosomal assembly and secretion. The targets of exosomal processing genes with respect to natural-product-modulated miRNAs (Table 3) were predicted using the miRDB database. Some natural products (Table 3) are not shown here because the exosomal processing gene targets of their modulated miRNAs could not be identified in miRDB.

Conclusions
Tumor-derived exosomes containing many biomolecules can regulate sophisticated cell functions. This review focused on our understanding of the roles of exosomal miRNAs in controlling cancer cell functions. The impacts of the modulating effects of natural products in regulating exosome processing and exosomal miRNAs were also summarized.
Many natural products exhibit diverse functions and affect the expression of many genes, but they the impacts of natural products on exosome biogenesis have been overlooked. By examining exosomal processing information derived from the GO database and PubMed/Google scholar searches, we noted that some of the altered genes belong to Overview of natural products modulating exosomal miRNAs through exosomal processing genes and exosomal assembly and secretion. The targets of exosomal processing genes with respect to natural-product-modulated miRNAs (Table 3) were predicted using the miRDB database. Some natural products (Table 3) are not shown here because the exosomal processing gene targets of their modulated miRNAs could not be identified in miRDB.

Conclusions
Tumor-derived exosomes containing many biomolecules can regulate sophisticated cell functions. This review focused on our understanding of the roles of exosomal miRNAs in controlling cancer cell functions. The impacts of the modulating effects of natural products in regulating exosome processing and exosomal miRNAs were also summarized.
Many natural products exhibit diverse functions and affect the expression of many genes, but they the impacts of natural products on exosome biogenesis have been overlooked. By examining exosomal processing information derived from the GO database and PubMed/Google scholar searches, we noted that some of the altered genes belong to the classification of exosomal processing. Accordingly, this work represents a novel contribution to the study of the exosomal processing of natural products.
Similarly, many exosomal miRNAs have been reported but lack detailed investigations of their regulation of exosomal processing. By utilizing the miRDB database, the potential impacts of exosomal processing genes were predicted to be targeted by exosomal miRNAs. This prediction further provides a direction for future research, which should aim to assess the detailed mechanisms of exosomal miRNAs, although further experiments are still required to confirm them.
Finally, we collected and organized several natural products and their associated modulations of exosomal miRNAs and cell functions, such as proliferation, apoptosis, migration, the tumor immune microenvironment, and other diverse effects. The potential roles of exosomal processing in these natural product investigations were further assessed using information retrieved from the miRDB database. Similarly, we demonstrated that some natural-product-modulated exosomal miRNAs overlap with tumorimmune-microenvironment-associated miRNAs. Although they are indirectly connected, this information provides a future direction for research, which should aim to validate whether these natural products can modulate exosomal miRNAs to regulate the tumor immune microenvironment.
Consequently, we offer a clear conclusion that several exosome-processing genes involved in exosomal secretion and assembly are organized in connection to natural products based on our utilization of the miRDB database to retrieve the target predictions of exosomal miRNAs. Accordingly, we filled the gaps in current knowledge between the exosomal processing of exosomal miRNAs and natural products.
Notably, the miRDB-database-predicted targets of exosomal processing genes were collected based on different cell types. Different cell types may show various miRNAs and targeting responses. This warrants careful examination based on wet experiments to validate the relationship between exosomal miRNAs and natural products in order to explore their impacts on the modulation of cancer cell functions.
This review sheds light on the connections between exosomes, exosomal miRNAs, natural products, and cancer cell functions, providing a clear direction for future research on the modulation of exosomal miRNAs by natural products.