Bioactive Compounds from Herbal Medicine Targeting Multiple Myeloma from

: Multiple myeloma (MM) is one of the most widespread hematological cancers. It is characterized by a clonal proliferation of malignant plasma cells in the bone marrow and by the over-production of monoclonal proteins. In recent years, the survival rate of patients with multiple myeloma has increased significantly due to the use of transplanted stem cells and of the new therapeutic agents that have significantly increased the survival rate, but it still cannot be completely cured and therefore the development of new therapeutic products is needed. Moreover, many patients have various side effects and face the development of drug resistance to current therapies. The pur-pose of this review is to highlight the bioactive active compounds (flavonoids) and herbal extracts which target dysregulated signaling pathway in MM, assessed by in vitro and in vivo experiments or clinical studies, in order to explore their healing potential targeting multiple myeloma. Mecha-nistically, they demonstrated the ability to promote cell cycle blockage and apoptosis or autophagy in cancer cells, as well as inhibition of proliferation/migration/tumor progression, inhibition of angiogenesis in the tumor vascular network. Current research provides valuable new information about the ability of flavonoids to enhance the apoptotic effects of antineoplastic drugs, thus providing viable therapeutic options based on combining conventional and non-conventional therapies in MM therapeutic protocols. bortezomib. Combination bortezomib inhibited the activation of


Introduction
Multiple myeloma (MM) is one of the most widespread hematological cancers. It is characterized by a clonal proliferation of malignant plasma cells in the bone marrow and by the increased production of M monoclonal protein [1]. MM represents approximately 1% of all cancers [2] and 10% of all hematological tumors [2,3]. Its incidence depends on different factors, such as age, sex, and country [2] and is estimated to 6.2 in 1 × 105 individuals [4]. Moreover, MM affects people over 65 years [5], especially males, from the industrialized regions of Europe, North America, and Australia [6].
Multiple myeloma is heterogeneous cytogenetically and shows numerous genetic and epigenetic changes, in which numerous signaling pathways are involved [7]. It is characterized by chromosomal instability and frequent genetic mutations, including RB1, DIS3, KRAS, MYC, TP53, NRAS, TP53, FAM46C, BRAF, and p18 (CDKN2c) [8,9]. Due to its increased heterogeneity, MM has numerous refractory/recurrent cases. Because the bone marrow is the ideal site for homing and malignant cells progression [10], 70% of patients have bone metastases at the diagnosis, and their percentage increases to 90% during the progression of the malignancy. These patients experience bone lysis caused by inhibition of osteoblast differentiation [11]. Additionally, this condition is associated with hypercalcemia, renal failure, and anemia, which affect the quality of life as well [7,12].
In the recent years, the survival rate of MM patients has increased significantly due to the use of transplanted stem cells or new therapeutic agents, and significantly increased survival rate [13].
The main therapies used in MM are: Glucocorticoids as dexamethasone and prednisone are steroids used in the treatment of MM [14]. Glucocorticoids indirectly inhibit target genes through inhibitory interactions of glucocorticoid (GC)-glucocorticoid receptor (GR) monomers with NF-κB and activator protein-1 (AP-1) [15], the latter being important factors in the pathogenesis of multiple myeloma [16][17][18].
• Proteasome inhibitors [19][20][21]. The proteasome is a multicatalytic target responsible for the degradation of the proteins. Immunomodulatory drugs, such as pomalidomide and lenalidomide [28][29][30]. MM was one of the first tumors in which the therapeutic efficacy based on immune receptors inhibition, especially of the PD-1 axis, was evaluated in preclinical models [6]. • Monoclonal antibodies [31][32][33]. There are two monoclonal-based therapy approved for the treatment of MM: daratumumab (CD38 pathway) [34], and elotuzumab (tar-getSLAMF7 pathway) [35]. The efficacy of monoclonal antibodies is the highest, but this therapy has a high cost [6]. In 2020, the FDA approved a third monoclonal antibody for MM therapy, Sarclisa (isatuximab-irfc) in combination with pomalidomide and dexamethasone [36]. Isatuximab is a monoclonal antibody that targets the transmembrane receptor and the ectoenzyme CD38, a protein overexpressed by malignant hematological cells [37]. Isatuximab is a new MM treatment for patient's refractory to lenalidomide and proteasome inhibitor [38].
Although various pharmacological strategies for the MM clinical treatments have been developed, in most patients it eventually recurred [39]. Patients have various side effects and face the development of drug resistance [39][40][41]. One of the harmful side effects of MM therapy is neurotoxicity. It is often associated with chemotherapy-induced peripheral neuropathy and has symptoms such as hypersensitivity, dysaesthesia, and paresthesia [42,43]. More than 50% of bortezomib-treated patients experienced this side effect, leading to dose reduction or treatment discontinuation [6]. All these problems highlight the need to research new therapeutic targets to improve the treatment [44,45].
In recent years, there has been a growing interest in the development of drugs to alleviate the side effects of antineoplastic therapy and the emergence of drug resistance, through the use of biologically active compounds with low side effects and that are effective in the treatment of several cancers [46]. Bioactive compounds such as alkaloids, flavonoids, terpenoids or saponins have the ability to prevent the side effects of chemotherapeutic drugs, prolong survival and improve the quality of life of cancer patients [47][48][49][50]. It is estimated that about 70% of cancer drugs are natural products or derived from natural products [51]. However, there are limitations when using non-conventional therapies in oncohematological diseases, because in vitro or in vivo studies predominate, clinical trials are missing, or the results provide non-uniformity in their mechanism of action.
Interest in the study of phenolic compounds has increased over the last decade due to promising biological activities and potential medical applications [52][53][54]. These effects are mainly related to their antioxidant activity. Polyphenols have the ability to eliminate free radicals and chelated metal ions [55]. They can indirectly decrease the production of reactive oxygen species (ROS), either by improving the activity of antioxidant enzymes or by inhibition of pro-oxidant enzymes [56]. Phenolic compounds can induce reversal of carcinogenesis by modulating molecules involved in intracellular signaling and by blocking cancer progression [57]. The property of it to neutralize free radicals (free radical-scavenging properties) helps to prevent chronic pathologies, such as cancer, cardiovascular or neurodegenerative diseases [53]. The anticancer and pro-apoptotic properties of polyphenols have been highlighted in numerous studies [58,59], properties that qualify them to be analyzed as therapeutic options in MM. Currently, there are just a few clinical trials provided by Database ClinicalTrials.Gov. regarding synergic anti-tumor activity of herbal compounds and MM drugs (curcumin and Bioperine or Lenalidomide, EGCG and monoclonal gamopathy) and therefore it is necessary to focus on other flavonoids or plant extracts as posible options in the therapeutic management of MM.
The aim of this review is to highlight bioactive active compounds (flavonoids) and herbal extracts that target dysregulated signaling pathway in MM, assessed by in vitro and in vivo experiments or clinical studies, in order to explore their therapeutic potential in the treatment of multiple myeloma.
Activation of Bcl-2 pathways may regulate mitochondrial-mediated apoptosis. Interactions between Bcl-2 proteins can regulate permeabilization of the outer mitochondrial membrane by cytoplasmic releasing of cytochrome C, and plays a critical role in activation of the apoptosis-inherited pathway [64,65]. Moreover, Bax, Bad and Bcl-xs, may facilitate the releasing of cytochrome C or may inhibit anti-apoptotic proteins [66].
Inhibition of the PI3K/Akt/mTOR signaling pathway is crucial for the antiproliferative effect on MM cells [67,68]. Akt is the main downstream target of PI3K [69]. Inhibition of PI3K/Akt/mTOR is beneficial for improving the life quality of MM patients [70]. Phosphorylated Akt binds to PI3K-activated products, and inhibit the pro-apoptotic factors of caspase-3, -9, and Bad [71]. Moreover, MTOR is a significant downstream target of Akt [72].
STAT3 is a transcription factor that may be overexpressed in multiple myeloma. It correlates with enhanced proliferation and apoptosis resistance to therapy. STAT3 activation negatively regulated the T cell-mediated immune response [60]. Aberrant signal transducer activation and transcriptional activator 3 (STAT3) is a key event of the cancerogenesis by inducing anti-apoptosis, angiogenesis, invasion, and metastasis [73-76]. STAT3 has been shown to be constitutively active in approximately 40 to 60% of MM tumors [77][78][79][80][81].
Nuclear factor kappa B (NF-κB) is an important transcription factor involved in the proliferation and MM cells survival. NF-κB regulates the activation of various molecules involved in apoptosis, as anti-apoptotic proteins or X-linked inhibitor of apoptosis protein). XIAP is the most studied protein that inhibits apoptosis and negatively regulates caspases in MM [92].
Mitogen-activated protein kinase (MAPK) is a cell survival regulator, and Jun N-terminal kinase (JNK) signaling is a component of this pathway. PARP14 is overexpressed in over 80% of MM cells and is regulated by JNK2 [93].
The best strategy for the treatment of MM is a single agent or combination of molecules targeting several dysregulated signaling pathways.
They represent a large group of water-soluble antioxidant compounds, which frequently occur in plants as glycosides and consist of two aromatic rings linked by a carbon bond and form a heterocyclic ring [96].
Flavonoids have been extensively studied due to their antitumor activity [97-99]. Due to various bioactive activities, flavonoids are considered to be multi-targeting and have multifunctional molecules. They have the ability to kill resistant cancer cells and resensitize conventional anticancer drugs to induce multidrug resistance reversal, which highlights their role in resistant cancer treatment [100].
Flavonoids target membrane lipids and modify their physicochemical properties to exert their bioactive activities [101-104], most likely due to a membrane bilayer-mediated mechanism [105]. Therefore, it is considered that they act neither as specific regulators of target proteins, but rather as multifunctional agents that negatively regulate key factors involved in multidrug resistance [100].
Moreover, many flavonoids may function as ROS modulators (scavengers or inducers), because they affect the ROS level in cancer cells [106,107]. In order to overcome multidrug resistance, they act as inducers for ROS production, enreaching the toxic threshold for cancer cell apoptosis [106].
Flavonoids involved in management of MM are part of a different class (Table 1).

Apigenin
Apigenin is a non-mutagenic yellow crystalline solid flavonoid [108]. This is a flavonoid found in oranges, grapefruit, grapefruit, celery, parsley, onions, wheat germ, and chamomile ( Figure 1  The anti-cancer activity of apigenin was first reported in 1986 by Birt et al. [112]. Apigenin can be used in cancer prevention due to its antioxidant, anti-inflammatory, antigenotoxic effects and its ability to neutralize free radicals [113,114] Zhao et al. [122] reported cytotoxic effects of apigenin on both MM cell lines and primary MM cells, but not on normal blood mononuclear cells (PBMCs). Apigenin inhibited casein kinase 2 (CK2) activity and blocked the cell cycle in G2/M phase in U266 and RPMI 8226 cells. Additionally, it induced apoptosis and downregulated the expression of anti-apoptotic proteins in MM cells, respectively, such as Mcl-1, XIAP, Bcl-xL, Bcl-2, and survivin. Apigenin inhibited the activation of STAT3, ERK, AKT and NF-κB. Apigenin decreased Cdc37 phosphorylation, disassociated Hsp90/Cdc37/kinase complexes and degraded Hsp90/Cdc37 client proteins. Apigenin induced degradation of multiple kinases, including Src, RIP1, Raf-1, AKT and cyclin dependent kinase 4 (CDK4). In CD138+ primary MM cells, apigenin inhibited CK2 activity, and depleted Cdc37 client kinases.

Baicalein
Baicalein is one of the main biologically active compounds present in the root of Scutellariae radix [123]. The molecular formula of baicaleine is C15H10O5, as can be seen in Figure 2. The particular structure is the di-orthohydroxyl functional group, located on ring A of its molecular structure [124].  Baicalein treatment inhibited the proliferation and migration of MM cells by downregulating the expression of β-catenin, cyclin D1, c-myc, and β7 integrin [136]. Liu et al. [137] demonstrated that baicalein inhibited STAT1 phosphorylation, as well as IL-6-mediated phosphorylation of Jak, MAPK, STAT3, and Akt. Baicalein increased the U266 cell sensitivity to dexamethasone and inhibited IL-6-induced bcl-xl gene expression. Baicalein also showed a strong inhibitory effect on Erk1/2 phosphorylation. Thus, baicalein could be considered an active inhibitor of IL-6-induced protein phosphorylation, and a potential agent in the treatment of MM.
Luteolin has shown anticancer activity in many cancers, such as breast [ Luteolin inhibited the carcinogenesis progression through various pathways, such as kinase inhibition, induction of apoptotic cell death, cell cycle regulation, and decreased the transcription factors [153]. Apoptosis induction involves DNA damage, regulation of redox and protein kinases in inhibiting cancer cell proliferation [172].
Regarding the anti-myeloma activity of luteolin, it has been shown to inhibit RPMI-8226 cell proliferation by apoptosis or autophagy, being interactive or promoting each other [173].

Wogonin
Wogonin is one of the active mono-flavones extracted from the root of the Scutellaria baicalensis ( Figure 6  It has neuroprotective [184,185], anti-inflammatory, and anti-cancer activity [186][187][188][189][190][191][192]. Wogonin has been shown to induce cytotoxicity to cancer cells in vitro and inhibited tumoral growth in vivo [124,183]. Wogonin targets several signaling pathways to prevent and inhibit the development of cancer both in vitro and in vivo [193,194]. Wogonin is involved in several anti-proliferative processes mediated by ER stress, MAPK, ROS, inhibition of transcription factors, such as NF-κB or activator protein-1 (AP-1), and intracellular suppression Ca 2+ signaling [124,194]. Due to its therapeutic potential and a lower toxicity to normal cells [183], this bioactive compound is an adjuvant in chemotherapy by increasing the therapeutic effects and reducing the side effects of conventional drugs [195].
Because MM-induced angiogenesis is an essential process for cancer progession and metastasis, methods of inhibiting angiogenesis have been studied as a new anti-myeloma treatment [196,197]. Fu et al. [198] showed that wogonin inhibited MM-induced angiogenesis in both normoxic and hypoxic conditions by reducing the secretion of proangiogenic factors (VEGF, PDGF and bFGF) in U266 and RPMI 8226 cells and in co-cultures of MM and stromal cells. The antiangiogenic effect of wogonin was previously reported by Song et al. [199] in MCF-7 breast cancer cells.
The c-Myc/HIF-1α plays an essential role in activation of different pro-angiogenic factors (e.g., PDGF, VEGF, and bFGF) [200]. Wogonin induced c-Myc inhibition and stimulated HIF-1α degradation in vitro. This biologically active compound has the ability to inhibit in vivo tumor angiogenesis and growth in an experimental model of BALB/c-nude mice. Wogonin showed synergistic action against MM-dependent angiogenesis in combination with lenalidomide and bortezomib and inhibited expression of c-Myc and HIF-1α in primary MM cells [198].
Zhang et al. [201] reported a cytotoxic effect of wogonin in vitro, as demonstrated by the induction of apoptosis on RPMI8226 cells. Wogonin blocked the cells in the sub-G1 phase. Poly ADP ribose polymerase (PARP) has an important role in mediating the normal cellular response to DNA damage, and enzymatic cleavage is considered to be a marker of apoptosis. Wogonin inhibited PARP in a dose-dependent manner and induced PARP cleavage. Moreover, wogonin increased Bax expression in MM cells, suggesting its ability to stimulate apoptosis via Bax-regulating pathway. Besides, Bcl-2 expression was reduced following wogonin treatment. Wogonin inhibited Akt phosphorylation (Ser 473) in MM cells, the latter being a key event in tumorigenesis and apoptosis. Wogonin treatment did not alter the total Akt protein level. This study suggests that wogonin-induced MM cell apoptosis could be modulated by the Akt pathway, because residual phosphorylation at Ser 473 is required for Akt activation [202].

Fisetin
Fisetin is a bioactive flavonol that has a diphenylpropane structure [203] containing two aromatic rings bounded by a three carbons and supplemented with four hydroxyl group substitutions and one oxo group [204,205] (Figure 7). Fisetin is synthesized in strawberries, apples, cucumbers, and onions [206] or plants such as Acacia berlandieri, Acacia greggii, Gleditschia triacanthow, and Rhus cotinus [207]. The average daily intake of fisetin is estimated to be about 0.4 mg in humans [208]. This potent bioactive phytoconstituent acts as a coloring agent in plants [206].
Fisetin has low water solubility and poor intestinal absorption and therefore has low bioavailability [209,210]. Its solubility and bioavailability can be improved by complexing it with cyclodextrins, encapsulating with nanoparticles [211][212][213] and cocrystallization with caffeine, isonicotinamide and nicotinamide [214,215]. These changes aim to improve stability, solubility and biological effects of fisetin. The biological activity of fisetin depends on the presence of hydroxyl and oxo groups. Hydroxyl groups at C-7 and the double bond C2-C3 are essential for its antioxidant activity [216,217].
Fisetin induced apoptosis, cell cycle blocking and inhibition of CDK activity in various human cancer cell lines. It has also modulated lipid kinase and protein kinase pathways [228][229][230]. The anticancer activity of fisetin has been highlighted by its involvement in various signaling pathways, as blocking the mammalian target of rapamycin (PI3K/Akt/mTOR)/phosphatidylinositol-3-kinase/protein kinase B, mitogen-activated protein kinases)-dependent nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and p38 [231,232].
Jang et al. [207] reported that fisetin showed cytotoxic effect and induced apoptosis in U266 cells and blocked the cell cycle in sub-G1 phase. Additionally, it improved the PARP cleavage. Fisetin inhibited the expression of anti-apoptotic Bcl-2 and Mcl-1L, but not Bcl-xL, and increased the expression of pro-apoptotic Bax, Bim and Bad.
AMP-activated protein kinase (AMPK) plays an important role in inducing apoptosis [233][234][235]. Fisetin induced an increase in phosphorylated AMPK and phosphorylated acetyl-CoA carboxylase, as a major substrate of AMPK. Fisetin treatment inhibited mTOR phosphorylation and reduced phosphorylated AKT, the upstream protein of mTOR. ROS have the role of mediating apoptosis in cancers [236,237]. Thus, fisetin induced ROS growth in U266 cells [207].

Myricetin
Myricetin is a phenolic compound found in berries, grapes, wine, nuts, etc. Myricetin is also known as hydroxy quercetin due to the presence of additional -OH group and exists in two forms: a free form and a glycosidically bound form ( Figure 8) [238]. Myricetin has various pharmacological activities, such as antioxidant, anti-inflammatory, analgesic, antitumor, hepatoprotective, and antidiabetic activity [238].
The anti-cancer activity of myricetin was confirmed by a number of in vitro and in vivo cytotoxicity studies. Miricetin acted as an antioxidant, antimetastatic, immunomodulatory agent, apoptosis inducer, inhibitor of angiogenesis, and proliferation and growth promoter [238].
Akhtar et al. [243] showed that bulk and nano forms of myrcetin showed an insignificant genotoxicity level in the lymphocytes of MM patients compared to those of healthy ones. This effect was induced by altering the Bcl-2 expressions. The Bcl-2/Bax ratio decreased and the level of P53 protein increased in the lymphocytes of MM patients. Myricetin bulk and nano-treatment induced an increase in intracellular ROS levels, suggesting that modulation of apoptotic proteins triggered by myricetin occurs through P53 and oxidative stress-dependent pathways.

Quercetin
Quercetin is one of the most important bioflavonoids found in over 20 plants [244], such as berries (blueberries and cranberries), apples, green leafy vegetables, onions, broccoli, cauliflower, cabbage, as well as nuts and seeds [244,245]. It contains two benzene rings joined through a 3-carbon heterocyclic pyrone one ( Figure 9). Due to the presence in the structure of quercetin of two antioxidant pharmacophores, it has the ability to easily neutralize free radicals and can bind to metal ions. Catechol with OH group present in the C3 position is an ideal structure that favors the free radical neutralization [246].
The quercetin name derives from the Latin word "Quercetum", which means oak forest [247]. The name of the International Union of Pure and Applied Chemistry (IUPAC) of quercitin is 2-(3,4-dihydroxyphenyl) -3,5,7-trihydroxychromen-4-one [245]. Quercetin belongs to the flavonols and is not being produced by the body [247]. It is soluble in lipids and alcohol, insoluble in cold water and has a lower solubility in hot water [244,245].
Quercetin is available as a supplement and can be safely orally administrated at a dose of 1 g/ day and absorbed up to 60% [248].
Xu et al. [265] studied the effect of quercetin on multiple myeloma cell line NCI-H929. Quercetin inhibited cell proliferation, stimulated apoptosis and induced cell cycle blockade in G2/M phase. Quercetin activated caspase-3, -8, -9, and PARP apoptosis-associated proteins and inhibited BCL-2 expression. It also stimulated P21, P53, and P27 expression and inhibited CDK4 expression in NCI-H929 cells. Quercetin decreased the p-ERK and p-AKT phosphorylation in NCI-H929 cells.
He et al. [266] demonstrated that quercetin inhibited proliferation in ARP-1, RPMI8226, and MM.1R cell lines by inducing apoptosis and blocking the cell cycle in the G2/M phase. Quercetin upregulated caspase-3, -9, PARP, p21 and downregulated c-myc. The combination of quercetin with dexamethasone showed in vitro and in vivo synergistic inhibitory effect, stimulated apoptosis of MM cells and induced caspase-3 activation. Quercetin also inhibited in vivo tumor growth in vivo (ARP-1 cells in NOD-SCID mouse model).
Quercetin inhibited MM cell proliferation by downregulating the expression of IQ motif-containing GTPase activating protein 1 (IQGAP1) and the activation of extracellular signal-regulated kinase 1/2 (ERK1/2). Quercetin also inhibited activation of MAPK and the interaction between IQGAP1 and ERK1/2 in MM cells [267].

Epigallocatechin-Gallate
Epigallocatechin-gallate (EGCG) is the common catechin in green tea ( Figure 10) [268,269], representing at least 50% of the total catechins in the leaves [269] and is responsible for most biological activities, including angiogenesis [270]. The anti-proliferative, pro-apoptotic, anti-invasive and anti-angiogenic properties of EGCG have been highlighted in numerous studies [271].
EGCG has acted synergistically with other natural compounds both in vitro and in vivo in various cancers (e.g., quercetin, curcumin, genistein, and caffeine) [271,283].
EGCG inhibited cell growth by inducing apoptosis in MM cell lines or primary MM cells from patients [297,298], by caspase 3 activation and Bcl-2 and Mcl-1 reduction. Additionally, it induced loss of mitochondrial transmembrane potential and intracellular H2O2 and superoxide increasing [298].
EGCG-mediated apoptosis was the result of a direct interaction with 67LR laminin receptor and lipid-shelf clustering, which correlated with increased activation of sphingomyelinase acid (ASM) via protein kinase C delta (PKCδ). This has been demonstrated both in vitro in U266 cells and in cells harvested from patients with MM [299,300] and in vivo [297,300].
EGCG has been used in MM clinical practice in combination with phosphodiesterase 5 inhibitor vardenafil [299] or in vitro, together with L-Threo-dihydrosphingosine in vitro [303], and hydrogen sulphide donors [304]. Thus, an attempt was made to reduce the dose of EGCG and hepatic adverse effects [305][306][307][308] and to improve the anti-hepatic effects. MM ale EGCG [299,303,304].
Bae et al. [309] reported that cyclic guanosine monophosphate (cGMP) induced by EGCG activated the PKCδ/ASM singling axis in MM cells. Induction of cGMP was sufficient to induce phosphorylation of PKCδ at Ser664, the major kinase for the induction of ASM activation by EGCG. The inhibitors of the negative regulators of diacylglycerol (DAG) increased the effect of EGCG. EGCG treatment increased phospholipase C (PLC) activity. EGCG-induced ASM activation was completely suppressed by PLC inhibition. Thus, EGCG-induced cGMP activated the cGMP/PLC/PKCδ/ASM signaling axis in MM cells.
The limitations related to the use of this flavonoid in the treatment of patients with MM are represented by the fact that EGCG is active in killing the proliferation of clonal/aberrant plasma cells (aPCs) at concentrations higher than those that can be achieved by drinking tea, and MM cells present a different sensitivity [298][299][300].
Daidzin has shown preventive activities against breast and prostate cancer. This bioactive compound showed anti-proliferative activity against MCF cells by affecting the invasive potential [318,319]. Daidzin has been shown to have an anti-cancer effect in the eary stages of prostate carcinogenesis [313].
Daidzin inhibited the viability of U266 and MM1.S cells, downregulated STAT3 in U266 cells, modulated activation of upstream kinases, inhibited MM cell proliferation, stimulated apoptotic cell death, potentiated anti-inflammatory and anti-cancer properties of bortezomib [320].
Formononetin inhibited NF-κB and activated AP-1 in MM cells, attenuated activation of PI3K/AKT and MAPK pathways, decreased tumorigenic protein levels, induced apoptosis, potentiated the antitumor properties of bortezomib, and enhanced its apoptotic effects [321].
Formononetin affected MM growth through the negative regulation of (STAT) 3/5 cascades mediated via oxidative stress. Formononetin inhibited STAT activation in MM cells, reduced the ability to bind STAT3 and STAT5 to DNA, as well as the nuclear pool of p-STAT3 and p-STAT5 in U266 cells. Formononetin inhibited the activation of upstream kinases involved in the STAT3 signaling cascade in MM cells, inhibited IL-6-induced STAT3-dependent reporter gene expression, decreased the expression of proteins involved in anti-apoptosis, angiogenesis, and proliferation (CDK2, CDK4 and Cyclin E), activated caspase-3 and induced cleavage PARP in U266 cells, negatively affected the cell cycle in MM cells, reduced the expression of cyclin D1 and cyclin B1 associated with cell cycle regulation, inhibited cell viability in MM cells, inhibited glutathione reductase expression in U266 cells, induced antitumor effects in the MM model xenograft, inhibited the growth of MM in vivo and inhibited the activation of STAT3 / 5 in tumor tissues, activated caspase-3 and induced cleavage of MM tissular PARP and reduced the expression of apoptotic proteins in tumor tissues [331].
Genistein decreased cell mitosis, induced apoptosis and increased caspase-3 activity and inhibited NF-κB expression in U266 cells. Genistein treatment inhibited human MM cell proliferation by miR-29b upregulation [344].
Genistein inhibited NF-κB expressed by MM cells, inhibited Akt phosphorylation, decreased the expression of NF-κB-regulated genes, inhibited MM cell proliferation, and induced apoptosis in MM cells [345].

Chalcones
Chalcones are polyphenolic compounds that belong to flavonoids [346]. They are considered to be precursors of flavonoids and isoflavonoids [347,348].
Chalcone is an α, β-unsaturated ketone [349]. Chemically, chalcones are formed by openchain flavonoids in which the two aromatic rings bind three carbons in an α, -unsaturated system [350]. Chalcones have a diverse structure due to the number and position of different substituents, such as hydroxy or methoxy groups on rings A or B. Chalcones are also C-prenylated and, less frequently, O-prenylated. The most common type of prenylation is the substitution of 3,3-dimethylallyl (prenyl group). In addition, chalcones also have isopentenyl, dimethylchromano, furano, geranyl, and farnesyl groups [351].
Chalcones act as chemopreventive agents of cancer. They have been shown to have antioxidant properties and an anticancer effect [360]. Compounds containing chalcone moiety have the ability to induce apoptosis and are blocked in the G2/M phase by triggering mitochondrial apoptotic pathway through activation of caspases and blcl-2 family [361].
Zhu et al.

Butein
Butein is a chalcone isolated from Toxicodendron vernicifluum, Semecarpus anacardium Butea monosperma, or Dalbergia odorifera [362], with a central core structure that has been shown to give it anti-cancer properties [363]. The IUPAC name of this compound is (E) - Figure 14) [362]. Butein prevented cancer metastasis via MMP-9 and urokinase plasminogen activator (uPA) repression [364]. Butein has been shown to inhibit survival, angiogenesis, proliferation, invasion and metastasis, chemoresistance in different types of cancer, such as breast cancer, leukemia, colorectal cancer, prostate cancer, pancreatic cancer, etc., by modulating several signaling pathways. These studies highlighted that butein acts as a multitargeted agent for both cancer prevention and treatment [362].
Butein inhibited constitutive and interleukin-6-inducible STAT3 activation in MM cells. This was mediated by inhibition of the activation of the upstream kinases c-Src, Janus-like kinase (JAK) 1, and JAK2. Butein induced SHP-1 protein expression and reversed inhibition of STAT3 activation in U266 cells [365].
STAT3 activation regulates the gene expression involved in cell survival and proliferation, as antiapoptotic proteins Bcl-2, Bcl-xL, and Mcl-1 and cyclin D1 [366]. Butein down-regulated the expression of these proteins and led to the suppression of proliferation and induction of apoptosis. Overexpression of STAT3 significantly reduced buteineinduced apoptosis. Butein potentiated the apoptotic effects of thalidomide and Velcade in MM cells [366], which are two drugs used in the clinical therapy of MM [367].
Cardamonin inhibited the growth and proliferation of U266, RPMI 8226 and ARH-77 cells. This calcone induced apoptosis in these cell lines and induced the activation of caspase-3 and PARP. Cardamonin down-regulated the expression of anti-apoptotic gene products in MM cells, inhibited constitutively active NF-κB in MM cells, repressed the expression of IKKs and inhibited phosphorylation of IκBα in MM cells, and down-regulated NF-κB -regulated gene product expression in MM cells [375].
Isobavachalcone has been shown to have anticancer activity [394,395]. It inhibited skin tumors in vivo [396], induced apoptosis in multiple myeloma, neuroblastoma, prostate cancer, breast cancer, ovarian cancer, and lung cancer [394,395,397,398].
Isobavachalcone showed reduced toxicity to normal cells. Isobavachalcone inhibited the proliferation of MM H929 cells by inducing apoptosis and autophagy. Mitochondrial cell death pathway was involved in its anti-proliferative activity. Protelytic activation of PKCδ was involved in chloroquine plus isobavachalcone-induced cell death. The combination of chloroquine with isobavachalcone showed low toxicity for normal PBMCs [397].
Chen et al. [405] studied the effect of isoliquiritigenin on MM cells, both in vitro and in vivo. Isoliquiritigenin inhibited the growth of MM cells and induced their apoptosis. In MM xenograft models, isoliquiritigenin showed significant antitumor activity and potentiated the anti-MM activity of adriamycin. Isoliquiritigenin downregulated IL-6 expression and reduced levels of phosphorylated ERK and STAT3. Isoliquiritigenin inhibited phosphorylation levels of ERK and STAT3 induced by recombinant human IL-6, which are critical signaling proteins in IL-6 signaling regulation networks.
Xanthohumol showed cytostatic and cytotoxic effects in MM cells and slightly affected the viability of PBMCs. Xanthohumol treatment induced cell cycle blockade in the G1 phase, induced cell death in RPMI8226 cells by the apoptotic mechanism, affected the expression of apoptosis-related and cell cycle-associated, induced ROS generation in RPMI8226 cells, and modulated MAP phosphorylation kinases. Xanthohumol-induced apoptosis in RPMI8226 cells involved in the production of ROS and the activation of ERK1/2 and JNK1/2. Xanthohumol decreased sIL6R and VEGF production in RPMI8226 cells [414].
Jung et al. [428] demonstrated that icariin had the ability to potentiate the anti-proliferative effects of bortezomib by inhibiting STAT3 activation in MM cells. Icariin inhibited STAT3 phosphorylation in U266 and MM.1S cells. Icariin inhibited STAT3 DNA binding activity and nuclear translocation in MM cells, inhibited JAK1, JAK2 and Src, inhibited inducible activation of STAT3 and upstream kinases in MM.1S cells, inhibited the IL-6induced STAT3-dependent reporter gene, inhibited anti-apoptosis, proliferation, angiogenesis, and metastasis related proteins. Icariin activated caspase-3 and induced PARP cleavage, blocked the cell cycle and stimulated apoptosis in U266 cells, and inhibited the viability of MM cells. This bioactive compound enhanced the cytotoxic effect of bortezomib. Combination treatment of icariin with bortezomib inhibited the activation of STAT3 and its upstream kinases, facilitated the blockade of cell cycle growth in the G0/G1 phase and cellular apoptosis. Icariin showed a synergistic effect with bortezomib in inhibiting the expression of various oncogenic proteins. Inhibition of STAT3 by siRNA reversed the pro-apoptotic effects of icariin.
Li et al. [456] showed that icaritin inhibited cell proliferation, induced apoptosis of MM cell line KM3/BTZ and induced the reverse multidrug resistance. The mechanisms involved are thought to be related to down-regulation of heat shock protein 27 (HSP27), P-glycoprotein (P-gp) expression, and up-regulation of Par-4 expression.
Previously, Zhu et al. [452] showed the effect of icaritin on MM cell line (U266) and primary MM cells inhibition, due to S-phase cell cycle blockade by targeting cyclin-related proteins and CDK2 and apoptosis by IL-6/JAK2/STAT3 signaling inhibition. The effects of icaritin on the inhibition of proliferation and induction of apoptosis in MM cells were independent of estrogen receptor blockade. Icaritin showed anti-myeloma activity in vivo, underlined by inhibition of tumor growth, downregulation of p-JAK2, p-STAT3 and VEGF expression and decreased serum levels of IL-6 and IgE. The antimyeloma effect of icaritin in vivo is thought to be mediated by inhibition of the p-JAK2/p-STAT3/VEGFmediated signaling pathway.
Icariside II inhibited the STAT3 signaling pathway in U266 cells, inhibited proliferation, induced apoptosis, and suppressed STAT3-related gene products in U266 cells. Icariside II amplified the effect of bortezomib and thalidomide on the induction of apoptosis in U266 cells [460].

Plant Extracts in Multiple Myeloma
In recent years, research has focused on the discovery of new plant extract metabolites that act as antitumor agents in various cancers, including hematological cancers [461]. Plant extracts target different signaling pathways in cancer cells, such as proliferation, differentiation, and apoptosis [462], acting through synergistic and/or additive effects [463][464][465].
Concomitant administration of plant extracts and anti-cancer drugs has increased the therapeutic efficacy of treatment by increasing the sensitivity of cancer cells to drugs and overcoming drug-induced resistance to cancer [466]. Currently in clinical practice a significant number of plant-isolated compounds are used in the treatment of cancer in combination with other drugs [467], against hematological malignancies [468][469][470][471][472].
The main pharmacological effects exerted by herbal extracts are summarized in Table 2.

Conclusions and Therapeutic Perspectives
In this review, we presented 21 flavonoids and 16 plant extracts that have shown an antitumor potential in the experimental studies on MM cell lines, preclinical and clinical studies on MM patients, results that could propose them in subsequent therapeutic protocols of MM. Mechanistically, they demonstrated the ability to induce cell cycle blockage and apoptosis or autophagy in cancer cells, as well as the inhibition of proliferation/migration and tumor progression, and inhibition of angiogenesis in the tumor vascular network. However, there are currently ongoing clinical trials only for curcumin and EGCG in combination with conventional drugs. Meanwhile, current research provides valuable new information about the ability of flavonoids to enhance apoptotic effects of antineoplastic drugs (baicalein/quercetin/dexamethasone; EGCG/doxorubicin, cisplatin, sunitinib, phosphodiesterase 5 inhibitor vardenafil, safingol; Butein/thalidomide and Velcade; Icariin/bortezomib), thus providing viable therapeutic options based on combining conventional and non-conventional therapies in MM therapeutic protocols.