Molecular Mechanisms of Flavonoids against Tumor Gamma-Herpesviruses and Their Correlated Cancers—A Focus on EBV and KSHV Life Cycles and Carcinogenesis

Epstein–Barr virus (EBV) and Kaposi sarcoma-associated herpesvirus (KSHV) are cancer-causing viruses that belong to human gamma-herpesviruses. They are DNA viruses known to establish lifelong infections in humans, with the ability to develop various types of cancer. Drug resistance remains the main barrier to achieving effective therapies for viral infections and cancer. Thus, new medications with dual antiviral and anticancer actions are highly needed. Flavonoids are secondary metabolites biosynthesized by plants with diverse therapeutic effects on human health. In this review, we feature the potential role of flavonoids (flavones, protoflavones, isoflavones, flavanones, flavonols, dihydroflavonols, catechins, chalcones, anthocyanins, and other flavonoid-type compounds) in controlling gamma-herpesvirus-associated cancers by blocking EBV and KSHV infections and inhibiting the formation and growth of the correlated tumors, such as nasopharyngeal carcinoma, Burkitt’s lymphoma, gastric cancer, extranodal NK/T-cell lymphoma, squamous cell carcinoma, Kaposi sarcoma, and primary effusion lymphoma. The underlying mechanisms via targeting EBV and KSHV life cycles and carcinogenesis are highlighted. Moreover, the effective concentrations or doses are emphasized.


Introduction
Epstein-Barr virus (EBV or human herpesvirus 4) and Kaposi sarcoma-associated herpesvirus (KSHV or human herpesvirus 8) are DNA tumor herpesviruses that belong to the Gammaherpesvirinae subfamily. Both viruses have lifelong effects on humans, forming lytic and latent infections, which undergo periodic reactivation. Inducing various types of cancer is the most significant problem reported with both viruses [1][2][3]. Moreover, both viruses can cause neurological complications by affecting the nervous system [4,5]. The human herpesvirus life cycle involves various steps, including attachment to the host cells, entry, lytic replication, latency, and reactivation [6,7]. Briefly, the virus begins the infection by adhering to the host cell via specific binding receptors and enters the cells using multiple viral entry glycoproteins. After the primary infection, the virus induces lytic replication (a multi-step process) and controls the genetic materials of the host cell and its innate immune system [8][9][10]. Subsequently, the virus moves to the latent site, forming latency, and persists in an inactive state. Recurrent infection occurs when the virus is reactivated from a latent state, and this step depends on the function of the infected host's immune system. The human herpesvirus often reactivates after immunosuppression triggered by diverse physiological and environmental factors that negatively impact the immune system [11][12][13][14][15]. Infections with human gamma-herpesviruses are mostly asymptomatic, and symptoms negatively impact the immune system [11][12][13][14][15]. Infections with human gammaherpesviruses are mostly asymptomatic, and symptoms appear once the immune system is impaired, mainly in immunocompromised patients [16]. Notably, the activation of the lytic cycle of EBV and KSHV leads to the expression of viral genes, proteins, and antigens that could promote tumorigenesis [17]. More information about EBV, KSHV, and their linked cancers is described in later sections.
So far, no drugs have been approved to treat or prevent EBV and KSHV infections effectively; however, acyclovir and its derivatives are currently prescribed to inhibit the replication of both viruses and reduce the severity and frequency of symptoms [18,19]. Moreover, the formed cancers triggered by both viruses are treated by different approaches that involve anticancer medications [20]. Nevertheless, the development of drug resistance makes the therapy for both viruses and their associated malignancies ineffective [3,21]. Therefore, the search for new sources, such as plants, that offer medicines with the ability to overcome this obstacle is highly required [22][23][24].
Consequently, this review aims to shed light on the potential of flavonoids as effective drugs against EBV and KSHV infections and their linked tumors, with a special emphasis on molecular mechanisms that affect the viral life cycle and carcinogenesis. Effective concentrations or doses are included as key factors for evaluating drug effectiveness. We performed a comprehensive literature search covering the period from 2012 to September 2022 (ten years update) using several online databases and search engines, such as Web of Science Core Collection, Scopus, PubMed, ScienceDirect, Embase, SciFinder, and Google Scholar with keywords that define anti-EBV and anti-KSHV properties of flavonoids and their protective potential against the correlated cancers. We have also included a few studies published before 2012 to provide a deeper comparison, critique, and assessment.

Flavonoids: An Overview, Anti-Herpesvirus, and Anticancer Properties
Flavonoids are a group of bioactive compounds biosynthesized by plants, including edible plants. They belong to the class of polyphenols with a basic skeleton that contains 15-carbon atoms (C6-C3-C6) ( Figure 1) [25,26]. Functionally, flavonoids participate in the development process of plants and protect them from diverse biotic and abiotic stresses, including, but not limited to, pathogens and UV radiation damage [27,28]. Chemically, they are classified into several subclasses, such as flavones, protoflavones, isoflavones, flavanones, flavonols, flavanols (including catechins), chalcones, and anthocyanins/anthocyanidins [29,30]. Some factors were found to affect the main structure of the flavonoid molecule, including the degree and pattern of alkalization, hydroxylation, prenylation, or glycosylation reactions [31,32]. The amount of flavonoids in plants varies, and might be influenced by different agricultural and environmental factors, including abiotic stresses. Moreover, the extraction methods can impact the amount yielded [33,34]. Flavonoids are known for their promising health benefits in preventing or curing different health conditions [35]. For instance, flavonoids were found to provide a valuable contribution to herpesvirus research, where these compounds showed an excellent capacity to block the infections of various types of herpesvirus by diverse mechanisms that target multiple stages of the viral life cycle. Inhibition of viral DNA replication was observed to be the main target of flavonoids [36][37][38]. In laboratory, animal, and clinical investigations, flavonoids were also observed to possess notable anticancer properties Flavonoids are known for their promising health benefits in preventing or curing different health conditions [35]. For instance, flavonoids were found to provide a valuable contribution to herpesvirus research, where these compounds showed an excellent capacity to block the infections of various types of herpesvirus by diverse mechanisms that target multiple stages of the viral life cycle. Inhibition of viral DNA replication was observed to be the main target of flavonoids [36][37][38]. In laboratory, animal, and clinical investigations, flavonoids were also observed to possess notable anticancer properties against different types of cancer cells, with the ability to stop them from multiplying by various mechanisms of action [39][40][41][42].

Chalcones
Lytic cycle. EBV-EA. [65]  Importantly, quercetin was found to inhibit an EBV infection with the ability to adversely impact this effect by promoting lytic reactivation [72]. Similarly, Huh et al. [73] also pointed out this adverse action, where quercetin was noticed to upregulate the expression of EBV lytic gene promoter BHLF1, indicating the potential negative impact on blocking EBV infection. Therefore, further investigations are required to evaluate the influencing factors that lead to such an effect.
Ito et al. [62] reported that the prenyl group on the A-ring, the prenyl group at 3 , and a hydroxyl group at 4 placed on the isoflavone skeletons of lupalbigenin, isolupalbigenin, glyurallin B, and isoangustone A are responsible for the induced inhibitory actions against EBV-EA; according to the structure-activity relationship evaluation. Moreover, the same research group showed in a previous study the importance of the presence of a prenylated isoflavone analog as a contributing factor accountable for the anti-EBV-EA activity of some examined isoflavones isolated from Millettia atropurpurea, such as 5,7,4 -trihydroxy-6,8-diprenylisoflavone (100% inhibition at 1 × 10 3 mol ratio/12-O-tetradecanoylphorbol-13-acetate (TPA)) [74]. The chemical synthesis of natural products plays a fundamental role in drug discovery [75]. In this context, an earlier study presented a synthetic analog PJ-2-genistein conjugate consisting of genistein (an isoflavone) and 3β-methoxyserrat-14en-21β-ol (PJ-2) with a promising anti-EBV effect by targeting EBV-EA. The synthesized compound displayed inhibitory action against EBV-EA activation with a 50% inhibitory concentration (IC 50 ) value of 455 mol ratio/32 pmol TPA, compared with the drug control oleanolic acid (IC 50 = 449 mol ratio/32 pmol TPA) [76].

Flavones and Prenylated Flavonols
Luteolin is a bioactive flavone distributed in various medicinal plants, fruits, and vegetables with diverse biological properties, including anticancer and antiviral activities [77][78][79]. Several experimental studies indicated the protective potential of luteolin against different types of cancer correlated with EBV infection with diverse mechanisms. For instance, Wu et al. [80] reported the results of a combined in vitro and in vivo study to reveal the effect of luteolin on NPC performed on EBV-positive NPC cells. Luteolin (0-50 µM) was discovered to inhibit EBV reactivation by repressing Rta and Zta gene expressions and lessening genomic instability caused by recurrent EBV reactivation in NPC cells. It also suppressed cell proliferation, migration, invasion, and spheroid formation (at concentrations ranging from 10 to 50 µM). Furthermore, tumor growth in a mouse model induced by EBV reactivation was effectively repressed by luteolin treatment (40 mg/kg administered intraperitoneally every 3 or 4 days for 4 weeks). compound displayed inhibitory action against EBV-EA activation with a 50% inhibitory concentration (IC50) value of 455 mol ratio/32 pmol TPA, compared with the drug control oleanolic acid (IC50 = 449 mol ratio/32 pmol TPA) [76].

Flavones and Prenylated Flavonols
Luteolin is a bioactive flavone distributed in various medicinal plants, fruits, and vegetables with diverse biological properties, including anticancer and antiviral activities [77][78][79]. Several experimental studies indicated the protective potential of luteolin against different types of cancer correlated with EBV infection with diverse mechanisms. For instance, Wu et al. [80] reported the results of a combined in vitro and in vivo study to reveal the effect of luteolin on NPC performed on EBV-positive NPC cells. Luteolin (0-50 µM) was discovered to inhibit EBV reactivation by repressing Rta and Zta gene expressions and lessening genomic instability caused by recurrent EBV reactivation in NPC cells. It also suppressed cell proliferation, migration, invasion, and spheroid formation (at concentrations ranging from 10 to 50 µM). Furthermore, tumor growth in a mouse model induced by EBV reactivation was effectively repressed by luteolin treatment (40 mg/kg administered intraperitoneally every 3 or 4 days for 4 weeks). EBV-LMP1 is known to promote cell proliferation and progression of NPC cells [81]. According to the findings obtained by Lo and colleagues [82], a new function of LMP1 (expressed in EBV-positive NPC cells) in enhancing lipogenesis was discovered. LMP1 was observed to elevate the expression, maturation, and activation of sterol regulatory elementbinding protein 1 (SREBP1), a main regulator of lipogenesis, and its downstream target fatty acid synthase (FASN). Remarkably, luteolin (20 µM) has notably hindered the lipogenesis and proliferation of NPC cells by suppressing LMP1, SREBP1, and FASN expressions. Moreover, luteolin has effectively inhibited NPC growth and induced apoptosis in mice at a dose of 20 mg/kg administered every 2-3 days for 3 weeks.
Another study demonstrated the promising anticancer effect of luteolin against NPC, by preventing the infection of EBV by targeting lytic replication. The underlying mechanism was explored by impeding Rta and Zta expressions and downregulating SP1 activity [56].
Treatment of EBV-positive NPC cells with baicalein, a bioactive constituent of Scutellaria baicalensis, at concentrations of 0, 15, 30, and 60 µM, significantly diminished the expressions of Sp1, EBNA1, and EBNA1 Q-promoter. Moreover, in a mouse xenograft model of EBV-positive NPC, baicalein has notably decreased tumor progression at a low dose of 15 mg/kg/day or a high dose of 30 mg/kg/day for 21 days [84]. By targeting EBV-positive B-cell tumors, additional anticancer mechanisms of baicalein (100 µM) were determined in vitro. Baicalein-mediated signal-regulating kinase 1/c-Jun N-terminal kinase (ASK1/JNK) activation was found to regulate the mitochondria-dependent apoptosis pathway by upregulating transcriptionally active p63 (TAp63) expression and downregulating NF-κB and proteins CD74/CD44 expressions [85].
Wu and coworkers [86] studied in vitro the therapeutic potential and underlying molecular mechanisms of icaritin, a major prenylated flavonol of the Epimedium genus, against EBV-positive extranodal NK/T-cell lymphoma (ENKL), an aggressive hematological tumor. The acquired results showed that icaritin (16-50 µM) impedes ENKL cell proliferation and generates apoptosis and cell cycle arrest at the G 2 /M phase. It was also found to upregulate Bax (pro-apoptotic protein), downregulate Bcl-2 (anti-apoptotic protein) and pBad (protein Bad), and activate caspase-3 and caspase-9. The mechanism responsible for the induced anti-proliferative and pro-apoptotic activities was ascertained to be mediated by suppressing signal transducer and activator of transcription 3 (STAT3) and AKT pathways through LMP1 downregulation. Significantly, combined treatment of icaritin (50 µM) with the antiviral drug ganciclovir (25 µg/mL) induced potent ENKL cell apoptosis. The chemical structures of the reviewed flavones and prenylated flavonols are shown in Figure 3. apoptosis in mice at a dose of 20 mg/kg administered every 2-3 days for 3 weeks.
Another study demonstrated the promising anticancer effect of luteolin against NPC, by preventing the infection of EBV by targeting lytic replication. The underlying mechanism was explored by impeding Rta and Zta expressions and downregulating SP1 activity [56].
Treatment of EBV-positive NPC cells with baicalein, a bioactive constituent of Scutellaria baicalensis, at concentrations of 0, 15, 30, and 60 µM, significantly diminished the expressions of Sp1, EBNA1, and EBNA1 Q-promoter. Moreover, in a mouse xenograft model of EBV-positive NPC, baicalein has notably decreased tumor progression at a low dose of 15 mg/kg/day or a high dose of 30 mg/kg/day for 21 days [84]. By targeting EBVpositive B-cell tumors, additional anticancer mechanisms of baicalein (100 µM) were determined in vitro. Baicalein-mediated signal-regulating kinase 1/c-Jun N-terminal kinase (ASK1/JNK) activation was found to regulate the mitochondria-dependent apoptosis pathway by upregulating transcriptionally active p63 (TAp63) expression and downregulating NF-κB and proteins CD74/CD44 expressions [85].
Wu and coworkers [86] studied in vitro the therapeutic potential and underlying molecular mechanisms of icaritin, a major prenylated flavonol of the Epimedium genus, against EBV-positive extranodal NK/T-cell lymphoma (ENKL), an aggressive hematological tumor. The acquired results showed that icaritin (16-50 µM) impedes ENKL cell proliferation and generates apoptosis and cell cycle arrest at the G2/M phase. It was also found to upregulate Bax (pro-apoptotic protein), downregulate Bcl-2 (antiapoptotic protein) and pBad (protein Bad), and activate caspase-3 and caspase-9. The mechanism responsible for the induced anti-proliferative and pro-apoptotic activities was ascertained to be mediated by suppressing signal transducer and activator of transcription 3 (STAT3) and AKT pathways through LMP1 downregulation. Significantly, combined treatment of icaritin (50 µM) with the antiviral drug ganciclovir (25 µg/mL) induced potent ENKL cell apoptosis. The chemical structures of the reviewed flavones and prenylated flavonols are shown in Figure 3.
Glyurallin B, an isoflavone isolated from Derris scandens (Roxb.) Benth., demonstrated inhibitory actions against EBV and its associated squamous cell carcinoma, also known as epidermoid carcinoma. Glyurallin B efficiently inhibited an EBV-EA activation with an IC 50 value of 278 mol ratio/32 pmol TPA. It also exposed in vitro cytotoxicity and antiproliferative activities against squamous cell carcinoma at a concentration of 5 µM. The underlying mechanism was observed to relate to disturbing the mitochondrial pathway [62]. The chemical structures of the reviewed isoflavones are displayed in Figure 4. species (ROS) production, guiding to activating caspases-3, -8, and -9 apoptotic processes [61].
Glyurallin B, an isoflavone isolated from Derris scandens (Roxb.) Benth., demonstrated inhibitory actions against EBV and its associated squamous cell carcinoma, also known as epidermoid carcinoma. Glyurallin B efficiently inhibited an EBV-EA activation with an IC50 value of 278 mol ratio/32 pmol TPA. It also exposed in vitro cytotoxicity and antiproliferative activities against squamous cell carcinoma at a concentration of 5 µM. The underlying mechanism was observed to relate to disturbing the mitochondrial pathway [62]. The chemical structures of the reviewed isoflavones are displayed in Figure 4.

Flavonols
Quercetin is a biologically active flavonol identified in various vegetables and fruits, including lovage, cilantro, dill, onions, apples, and berries. It possesses multiple bioactivities against a wide range of diseases, including cancer and viral infections [87][88][89]. The direct anticancer activity of quercetin on EBV-associated cancers has been revealed in numerous laboratory and animal experiments with various mechanisms. For example, at an effective dose of 30 mg/kg/day for 2 weeks, quercetin displayed a potent anticancer effect in mice xenograft models infected with EBV-human gastric carcinoma. The molecular mechanism responsible for the induced anticancer action was explored by interfering with the expressions of viral EBNA1 and LMP2. Moreover, quercetin has significantly generated p53-dependent apoptosis in EBV-human gastric carcinoma, which was found to be linked with overexpression of the cleaved forms of caspase-3, -9, and Parp [90]. An additional in vitro experiment showed the capacity of quercetin (62 µM) to induce

Flavonols
Quercetin is a biologically active flavonol identified in various vegetables and fruits, including lovage, cilantro, dill, onions, apples, and berries. It possesses multiple bioactivities against a wide range of diseases, including cancer and viral infections [87][88][89]. The direct anticancer activity of quercetin on EBV-associated cancers has been revealed in numerous laboratory and animal experiments with various mechanisms. For example, at an effective dose of 30 mg/kg/day for 2 weeks, quercetin displayed a potent anticancer effect in mice xenograft models infected with EBV-human gastric carcinoma. The molecular mechanism responsible for the induced anticancer action was explored by interfering with the expressions of viral EBNA1 and LMP2. Moreover, quercetin has significantly generated p53-dependent apoptosis in EBV-human gastric carcinoma, which was found to be linked with overexpression of the cleaved forms of caspase-3, -9, and Parp [90]. An additional in vitro experiment showed the capacity of quercetin (62 µM) to induce cytotoxic effect, apoptosis, and cell cycle arrest against EBV-associated gastric carcinoma cells. It also prevented an EBV infection by interacting with two steps of the viral life cycle, such as entry and latency, and inhibiting the expression of EBNA1 at a concentration of 62 µM [72].
In further in vivo study, the antitumor action of quercetin against EBV-associated gastric carcinoma performed on a mouse xenograft model was enhanced via a co-treatment with Ganoderma lucidum extract, administered at effective doses of 10 mg/kg/23 days for quercetin and 10 mg/kg/23 days for Ganoderma lucidum extract [73].
The chemopreventive activity of quercetin (100 µM) against NPC was reported in vitro via inhibiting cell proliferation and reducing the expression of FASN in EBV-positive NPC cells [92]. In any case, further protective action of quercetin (85 nmol; used as standard drug) against papilloma formation in two-stage mouse skin carcinogenesis was unveiled [93].
During EBV lytic reactivation, several oncogenic pathways are activated to promote viral carcinogenesis. Consequently, Granato et al. [94] conducted an in vitro investigation using quercetin to explore the possibility of inhibiting these pathways. The results indicated that quercetin (10 µM) could suppress the activation of STAT3 pathway generated by EBV infection and diminish the production of interleukin-6 (IL-6) and ROS. Interestingly, quercetin was observed to stimulate autophagy and neutralize the accumulation of sequestosome1/p62 (SQSTM1/p62), leading to stopping B cell immortalization. The study authors concluded that quercetin is a promising natural anticancer agent for treating EBV-associated malignancies and counteracting EBV-driven lymphomagenesis.
Fisetin, a flavonol molecule, has been isolated from numerous fruits, vegetables, and medicinal trees with proven dietary and pharmacological properties against several health conditions [95,96]. Its efficacy against cancers developed by EBV infections has been reported in some investigations. At various concentrations in micromolar ranges, fisetin in vitro prevented the metastasis of NPC generated by EBV infection by multiple mechanisms through suppressing the migration and invasion of LMP1-expressing NPC cells. The mechanisms were mediated via repressing molecular changes linked with the epithelial-mesenchymal transition (EMT) promoted by LMP1 and upregulating the epithelial marker and E-cadherin protein. Moreover, the levels of mesenchymal marker, vimentin protein, and twist protein (an EMT regulator) were successfully downregulated by fisetin [97]. Similarly, Li et al. [98] elucidated in vitro other mechanisms by which fisetin regulates cancer growth examined in EBV-infected NPC cells. According to their finding, fisetin (6.25-100 µM) was noted to interfere with targets of the NF-κB signal transduction pathway (p65, IκBα, and CyclinD1) activated by EBV-LMP1. The chemical structures of the reviewed flavonols are shown in Figure 6. health conditions [95,96]. Its efficacy against cancers developed by EBV infections has been reported in some investigations. At various concentrations in micromolar ranges, fisetin in vitro prevented the metastasis of NPC generated by EBV infection by multiple mechanisms through suppressing the migration and invasion of LMP1-expressing NPC cells. The mechanisms were mediated via repressing molecular changes linked with the epithelial-mesenchymal transition (EMT) promoted by LMP1 and upregulating the epithelial marker and E-cadherin protein. Moreover, the levels of mesenchymal marker, vimentin protein, and twist protein (an EMT regulator) were successfully downregulated by fisetin [97]. Similarly, Li et al. [98] elucidated in vitro other mechanisms by which fisetin regulates cancer growth examined in EBV-infected NPC cells. According to their finding, fisetin (6.25-100 µM) was noted to interfere with targets of the NF-κB signal transduction pathway (p65, IκBα, and CyclinD1) activated by EBV-LMP1. The chemical structures of the reviewed flavonols are shown in Figure 6.

Dihydroflavonols
Yun and colleagues [99] explored the anticancer mechanism of action of ampelopsin (also known as dihydromyricetin; Figure 7), an active constituent of Ampelopsis grossedentata, against EBV-positive human Burkitt's lymphoma cells. The results of in vitro assays indicated that ampelopsin (0-50 µM) caused the apoptosis of EBV-infected cells via upregulating tumor necrosis factor-related apoptosis-inducing ligand/death receptor 5

Dihydroflavonols
Yun and colleagues [99] explored the anticancer mechanism of action of ampelopsin (also known as dihydromyricetin; Figure 7), an active constituent of Ampelopsis grossedentata, against EBV-positive human Burkitt's lymphoma cells. The results of in vitro assays indicated that ampelopsin (0-50 µM) caused the apoptosis of EBV-infected cells via upregulating tumor necrosis factor-related apoptosis-inducing ligand/death receptor 5 (TRAIL/DR5) and activating the p38 signaling pathway.

Dihydroflavonols
Yun and colleagues [99] explored the anticancer mechanism of action of ampelopsin (also known as dihydromyricetin; Figure 7), an active constituent of Ampelopsis grossedentata, against EBV-positive human Burkitt's lymphoma cells. The results of in vitro assays indicated that ampelopsin (0-50 µM) caused the apoptosis of EBV-infected cells via upregulating tumor necrosis factor-related apoptosis-inducing ligand/death receptor 5 (TRAIL/DR5) and activating the p38 signaling pathway.
Isoliquiritigenin is a chalcone-type molecule known to cause cell cycle arrest and apoptosis of various cancer cells. This compound is commonly scattered in various species of the genus Glycyrrhiza, including Glycyrrhiza uralensis, Glycyrrhiza radix, and Glycyrrhiza glabra [100,101]. In an in vitro assay, isoliquiritigenin (45 µM) demonstrated antitumor properties against EBV-associated gastric carcinoma cells by causing cytotoxicity, apoptosis, and cell cycle arrest [72]. Another in vitro investigation showed the capacity of isoliquiritigenin (50 µM) to induce cytotoxic and antiproliferative effects on EBV-transformed lymphoblastoid cell lines through molecular mechanisms that involve microRNAs by targeting the p53 signaling pathway and cell cycle [102]. Isoliquiritigenin is a chalcone-type molecule known to cause cell cycle arrest and apoptosis of various cancer cells. This compound is commonly scattered in various species of the genus Glycyrrhiza, including Glycyrrhiza uralensis, Glycyrrhiza radix, and Glycyrrhiza glabra [100,101]. In an in vitro assay, isoliquiritigenin (45 µM) demonstrated antitumor properties against EBV-associated gastric carcinoma cells by causing cytotoxicity, apoptosis, and cell cycle arrest [72]. Another in vitro investigation showed the capacity of isoliquiritigenin (50 µM) to induce cytotoxic and antiproliferative effects on EBVtransformed lymphoblastoid cell lines through molecular mechanisms that involve microRNAs by targeting the p53 signaling pathway and cell cycle [102]. Figure 8 displays the chemical structures of the reviewed chalcones.   3.2.7. Other Flavonoid-Type Compounds Silibinin (Figure 9), a flavonolignan, has been extracted from Silybum marianum (L.) Gaertn. with potential therapeutic effect on clinically isolated EBV-positive NPC cells. Silibinin (0-200 µM) remarkably downregulated programmed death-ligand 1 (PD-L1) expression by inhibiting hypoxia-inducible factor-1α/lactate dehydrogenase-A (HIF-1α/LDH-A) mediated cell metabolism in EBV-positive NPC [103].

Flavonoids Target KSHV and Its Correlated Cancers
KSHV is a pathogenic gamma-herpesvirus that was first identified in Kaposi sarcoma (KS), a cancer that develops from the cells that line lymph or blood vessels. This virus is also associated with other cancerous tumors, such as primary effusion lymphoma (PEL) and various B-cell cancers. The virus is transmitted by sex, blood, and infected saliva [2,[104][105][106]. Numerous genes and proteins are encoded by KSHV, which play a critical role in the viral life cycle and contribute to cell transformation, induction, and progression of the associated malignancies [14,[107][108][109]. Therefore, new strategies for managing KSHV-related malignancies were recently proposed by targeting the KSHV life cycle (mainly the lytic reactivation) and the expressions of latent and lytic genes/proteins along with other correlated signaling pathways [3,110,111].

Flavonoids with Anti-KSHV Properties
Over the past ten years, the direct inhibitory effect of flavonoids on the KSHV life cycle has been reported in a decent amount of studies. In Table 2, we document these findings that feature the anti-KHSV efficacy of flavonoids, and their chemical structures are displayed in Figure 10. According to the results obtained by [112][113][114][115][116], flavonoids

Flavonoids Target KSHV and Its Correlated Cancers
KSHV is a pathogenic gamma-herpesvirus that was first identified in Kaposi sarcoma (KS), a cancer that develops from the cells that line lymph or blood vessels. This virus is also associated with other cancerous tumors, such as primary effusion lymphoma (PEL) and various B-cell cancers. The virus is transmitted by sex, blood, and infected saliva [2,[104][105][106]. Numerous genes and proteins are encoded by KSHV, which play a critical role in the viral life cycle and contribute to cell transformation, induction, and progression of the associated malignancies [14,[107][108][109]. Therefore, new strategies for managing KSHV-related malignancies were recently proposed by targeting the KSHV life cycle (mainly the lytic reactivation) and the expressions of latent and lytic genes/proteins along with other correlated signaling pathways [3,110,111].

Flavonoids with Anti-KSHV Properties
Over the past ten years, the direct inhibitory effect of flavonoids on the KSHV life cycle has been reported in a decent amount of studies. In Table 2, we document these findings that feature the anti-KHSV efficacy of flavonoids, and their chemical structures are displayed in Figure 10. According to the results obtained by [112][113][114][115][116], flavonoids were found to target the KSHV life cycle by various mechanisms via impeding the lytic DNA replication, virus progeny production, and the expressions of hypoxia-inducible factor 1α (HIF1α), immediate-early protein ORF45, late protein K8.1, and Rta. Moreover, targeting ROS was described as a mechanism that suppresses the KSHV lytic cycle. Table 2. Flavonoids with anti-KSHV properties and their molecular mechanisms evaluated in vitro.

Compound, Concentration, and Source Chemical Class Molecular Mechanisms (Inhibition) Reference
Hesperetin.

Compound, Concentration, and Source Chemical Class Molecular Mechanisms (Inhibition) Reference
Quercetin.

Flavones
Zhu et al. [117] studied, in vitro and in vivo, the potential anticancer properties of oroxylin A, an active ingredient of Oroxylum indicum, against KSHV-associated cancers, including KS and other aggressive angiomas. They found that oroxylin A (50 µM) inhibited lymphatic reprogramming of the endothelial cells generated by KSHV oncogene vIL-6, leading to cell invasion blocking, apoptosis, and neovascularization inhibition via modulating PPARγ/Prox1 axis.
By utilizing KSHV-positive PEL cells assayed in vitro, apigenin (12.5 and 25 µM) exposed anticancer activity by causing PEL cell death and autophagy, as well as by reducing intracellular ROS production. The mechanisms were determined by activating p53, which promotes catalase activity (a ROS scavenger enzyme) and impeding STAT3, a critical pro-survival pathway in PEL [118]. The chemical structures of the reviewed flavones are presented in Figure 11.

Flavones
Zhu et al. [117] studied, in vitro and in vivo, the potential anticancer properties of oroxylin A, an active ingredient of Oroxylum indicum, against KSHV-associated cancers, including KS and other aggressive angiomas. They found that oroxylin A (50 µM) inhibited lymphatic reprogramming of the endothelial cells generated by KSHV oncogene vIL-6, leading to cell invasion blocking, apoptosis, and neovascularization inhibition via modulating PPARγ/Prox1 axis.
By utilizing KSHV-positive PEL cells assayed in vitro, apigenin (12.5 and 25 µM) exposed anticancer activity by causing PEL cell death and autophagy, as well as by reducing intracellular ROS production. The mechanisms were determined by activating p53, which promotes catalase activity (a ROS scavenger enzyme) and impeding STAT3, a critical pro-survival pathway in PEL [118]. The chemical structures of the reviewed flavones are presented in Figure 11.
Zhu et al. [117] studied, in vitro and in vivo, the potential anticancer properties of oroxylin A, an active ingredient of Oroxylum indicum, against KSHV-associated cancers, including KS and other aggressive angiomas. They found that oroxylin A (50 µM) inhibited lymphatic reprogramming of the endothelial cells generated by KSHV oncogene vIL-6, leading to cell invasion blocking, apoptosis, and neovascularization inhibition via modulating PPARγ/Prox1 axis.
By utilizing KSHV-positive PEL cells assayed in vitro, apigenin (12.5 and 25 µM) exposed anticancer activity by causing PEL cell death and autophagy, as well as by reducing intracellular ROS production. The mechanisms were determined by activating p53, which promotes catalase activity (a ROS scavenger enzyme) and impeding STAT3, a critical pro-survival pathway in PEL [118]. The chemical structures of the reviewed flavones are presented in Figure 11.

Flavonols
At a concentration of 50 µM, quercetin ( Figure 12) in vitro was observed to interfere with multiple cancer-related pathways, inducing apoptosis and autophagy in KSHVpositive PEL cells. It repressed PI3K/AKT/mTOR and STAT3 signaling pathways and downregulated the expression of the pro-survival cellular proteins such as c-FLIP, cyclin D, and cMyc. Quercetin was also found to reduce the expression of lytic KSHV proteins implicated in PEL tumorigenesis, such as K-bZIP, and upregulate HLA-DR and calreticulin expressions. Importantly, quercetin caused pro-survival autophagy in KSHV-positive PEL cells and improved the cytotoxic action of bortezomib, a proteasome inhibitor, against these cells [119]. downregulated the expression of the pro-survival cellular proteins such as c-FLIP, cyclin D, and cMyc. Quercetin was also found to reduce the expression of lytic KSHV proteins implicated in PEL tumorigenesis, such as K-bZIP, and upregulate HLA-DR and calreticulin expressions. Importantly, quercetin caused pro-survival autophagy in KSHVpositive PEL cells and improved the cytotoxic action of bortezomib, a proteasome inhibitor, against these cells [119]. Another anticancer mechanism of quercetin was explored against KSHV-positive PEL cells using in vitro experiments. Quercetin (15 µM) effectively reduced ROS production by downregulating p62/Sequestosome1 (p62/SQSTM1) protein expression, leading to cell apoptosis and inhibition of KSHV lytic replication [115]. Further in vitro research disclosed the ability of quercetin (10 µM) to decrease the level of ROS production, specifically H2O2, in KSHV-positive PEL cells, leading to inhibiting the KSHV lytic cycle and boosting cell death [120].  Figure 13), a principal compound of Camellia sinensis, has proven cancer chemopreventive properties against KSHV-infected PEL cells, and its molecular mechanisms were detailed using in vitro experiments. EGCG caused cell cycle arrest and apoptosis in KSHV-infected PEL cells in a dose-dependent manner (20 µg/mL) by a mechanism that regulates ROS generation [114]. Additional mechanisms were reported by Yeh et al. [121], where EGCG (20 µg/mL) induced cell death in KSHV-infected PEL cells by upregulating glucose transporter GLUT3 expression and decreasing the expressions of pyruvate dehydrogenase E1-alpha (PDHA1), the main regulator of pyruvate dehydrogenase (PDH), and glutamate dehydrogenase 1 (GDH1). In addition, EGCG lessened the levels of oncometabolite D-2-hydroxyglutarate (D2HG).  Another anticancer mechanism of quercetin was explored against KSHV-positive PEL cells using in vitro experiments. Quercetin (15 µM) effectively reduced ROS production by downregulating p62/Sequestosome1 (p62/SQSTM1) protein expression, leading to cell apoptosis and inhibition of KSHV lytic replication [115]. Further in vitro research disclosed the ability of quercetin (10 µM) to decrease the level of ROS production, specifically H 2 O 2 , in KSHV-positive PEL cells, leading to inhibiting the KSHV lytic cycle and boosting cell death [120].

Catechins
EGCG (Figure 13), a principal compound of Camellia sinensis, has proven cancer chemopreventive properties against KSHV-infected PEL cells, and its molecular mechanisms were detailed using in vitro experiments. EGCG caused cell cycle arrest and apoptosis in KSHVinfected PEL cells in a dose-dependent manner (20 µg/mL) by a mechanism that regulates ROS generation [114]. Additional mechanisms were reported by Yeh et al. [121], where EGCG (20 µg/mL) induced cell death in KSHV-infected PEL cells by upregulating glucose transporter GLUT3 expression and decreasing the expressions of pyruvate dehydrogenase E1-alpha (PDHA1), the main regulator of pyruvate dehydrogenase (PDH), and glutamate dehydrogenase 1 (GDH1). In addition, EGCG lessened the levels of oncometabolite D-2-hydroxyglutarate (D2HG). apoptosis in KSHV-infected PEL cells in a dose-dependent manner (20 µg/mL) by a mechanism that regulates ROS generation [114]. Additional mechanisms were reported by Yeh et al. [121], where EGCG (20 µg/mL) induced cell death in KSHV-infected PEL cells by upregulating glucose transporter GLUT3 expression and decreasing the expressions of pyruvate dehydrogenase E1-alpha (PDHA1), the main regulator of pyruvate dehydrogenase (PDH), and glutamate dehydrogenase 1 (GDH1). In addition, EGCG lessened the levels of oncometabolite D-2-hydroxyglutarate (D2HG).

Conclusions and Future Directions
The incidence of infectious diseases has become one of the most significant global health threats of the 21st century; this has been observed during the latest pandemic of coronavirus disease 2019 . Moreover, studies of viral infections that lead to cancer have become one of the most important goals in modern medicine, where cancer is a leading cause of death. For years, many hurdles, including the problem of drug resistance, have restricted achieving efficient treatments for viral diseases and cancer.

Conclusions and Future Directions
The incidence of infectious diseases has become one of the most significant global health threats of the 21st century; this has been observed during the latest pandemic of coronavirus disease 2019 . Moreover, studies of viral infections that lead to cancer have become one of the most important goals in modern medicine, where cancer is a leading cause of death. For years, many hurdles, including the problem of drug resistance, have restricted achieving efficient treatments for viral diseases and cancer. Therefore, new medications are necessary. Flavonoids are a rich source of compounds with structural and chemical diversity for drug discovery. In this paper, we provided a critical assessment of the protective capability of flavonoids and how they might manage human gammaherpesvirus infections and their associated tumors by diverse molecular mechanisms. The reviewed flavonoids were examined by different experimental methodologies performed in various in vitro, in vivo, and in silico models. In addition, this review emphasized several strategies to improve the efficacy of flavonoids against EBV and its linked cancers, such as chemical synthesis, microbial transformation, and combinatory treatment with standard antiviral drugs such as ganciclovir. Moreover, quercetin in combination with bortezomib, a proteasome inhibitor, boosted the cytotoxicity against KSHV-positive PEL cells and, hence, improved treatment efficacy. The molecular mechanisms of luteolin, baicalein, wogonin, 6-Prenylnaringenin, quercetin, and 6 ,7 -dihydro-7 -hydroxyxanthoangelol F against EBV-associated tumors have been validated by animal experiments. In addition, the effectiveness of oroxylin A against KSHV-related malignancies, including KS, was confirmed in an animal study. Accordingly, these compounds merit special consideration to be engaged in more animal investigations and subsequently in clinical studies.
Concerning future directions, investigations on nano-drug delivery combined with pharmacokinetic and pharmacodynamic evaluations should be developed to improve the bioavailability and the activities of the reviewed flavonoids against EBV, KSHV, and their connected tumors. When it comes to clinical application, the involvement of the reviewed flavonoids in clinical studies faces various challenges. Aside from other obstacles related to financial support, the challenging regulatory requirements, and among others, the safety profile of the test compound, its bioavailability, and finding a sustainable and economically viable supply of sufficient quantities of the test compound are limiting factors. So far, no clinical trials have been conducted on flavonoids against human gamma-herpesviruses; however, dietary flavonoids such as quercetin, EGCG, and luteolin have proven sufficient effectiveness in preliminary clinical investigations against various types of cancer, including malignancies that might be generated by gamma-herpesvirus infections. In general, the participation of flavonoids in clinical studies is still challenging, and more efforts should be made to achieve this goal.