Synergistically Anti-Multiple Myeloma Effects: Flavonoid, Non-Flavonoid Polyphenols, and Bortezomib

Multiple myeloma (MM) is a clonal plasma cell tumor originating from a post-mitotic lymphoid B-cell lineage. Bortezomib(BTZ), a first-generation protease inhibitor, has increased overall survival, progression-free survival, and remission rates in patients with MM since its clinical approval in 2003. However, the use of BTZ is challenged by the malignant features of MM and drug resistance. Polyphenols, classified into flavonoid and non-flavonoid polyphenols, have potential health-promoting activities, including anti-cancer. Previous preclinical studies have demonstrated the anti-MM potential of some dietary polyphenols. Therefore, these dietary polyphenols have the potential to be alternative therapies in anti-MM treatment regimens. This systematic review examines the synergistic effects of flavonoids and non-flavonoid polyphenols on the anti-MM impacts of BTZ. Preclinical studies on flavonoids and non-flavonoid polyphenols-BTZ synergism in MM were collected from PubMed, Web of Science, and Embase published between 2008 and 2020. 19 valid preclinical studies (Published from 2008 to 2020) were included in this systematic review. These studies demonstrated that eight flavonoids (icariin, icariside II, (-)-epigallocatechin-3-gallate, scutellarein, wogonin, morin, formononetin, daidzin), one plant extract rich in flavonoids (Punica granatum juice) and four non-flavonoid polyphenols (silibinin, resveratrol, curcumin, caffeic acid) synergistically enhanced the anti-MM effect of BTZ. These synergistic effects are mediated through the regulation of cellular signaling pathways associated with proliferation, apoptosis, and drug resistance. Given the above, flavonoids and non-flavonoid polyphenols can benefit MM patients by overcoming the challenges faced in BTZ treatment. Despite the positive nature of this preclinical evidence, some additional investigations are still needed before proceeding with clinical studies. For this purpose, we conclude by providing some suggestions for future research directions.


Introduction
Multiple myeloma (MM) is a clonal plasma cell tumor originating from the postgerminal lymphoid B cell line. It has an incidence of approximately 2.1 out of 100,000 people worldwide [1]. Its incidence varies notably among different races, with older individuals at higher risk [2]. Moreover, it often leads to poor outcomes in low and middle-income countries due to the lack of specialized health care, diagnosis, and advanced treatments. The 5-year survival rate for MM patients treated in Nigeria is only 7.6% [3].
At present, internal medicine intervention predominates in MM therapy. Standard treatment options include (1) induction therapy with a combination of injectable proteasome inhibitors (i.e., bortezomib (BTZ)), oral immunomodulators (i.e., lenalidomide), and dexamethasone; and (2) autologous stem cell transplantation (ASCT) with subsequent maintenance lenalidomide [4]. According to the clinical evidence, Velcade, Revlimid, and Dexamethasone (VRD)-based pre-ASCT induction therapy enhances the prognosis of MM patients [5,6]. Nevertheless, these interventions remain challenged by MM's malignant qualities. For example, ASCT is not recommended for MM patients who are over the age of 65 or who have severe comorbid conditions like renal or pulmonary impairment, cardiac disease, or hepatic disease [7]. Most significantly, intrinsic clonal heterogeneity and genomic instability of plasma cells influence both inherent and acquired drug resistance [8]. For most MM patients, after several remissions and relapses, drug resistance develops into almost all available therapies [9]. Additionally, these MM medications could also have adverse effects. BTZ, for instance, may have adverse effects on the cardiovascular system, bone marrow suppression, and peripheral neuropathy [10]. Therefore, in order to overcome these difficulties, alternative and supportive therapies are required.
In recent years, natural dietary compounds have attracted the attention of researchers. They have a wide range of biological activities, such as anti-inflammatory, antioxidant, antiaging, immune enhancement, as well as anti-tumor [11][12][13][14][15]. Among them, flavonoids, a class of polyphenolic plant secondary metabolites, are of great interest. Through inhibition of cell proliferation, stimulation of apoptosis, targeting of cancer stem cells, inhibition of angiogenesis and metastasis, and chemotherapy sensitization, flavonoids have anticancer effects [16]. In addition, the cancer prevention and treatment potential of some non-flavonoid polyphenols are equally noteworthy [17,18]. Jöhrer and Pojero reviewed the tumor activity inhibition and targeting cancer effects of some natural compounds (including polyphenols) in MM in preclinical trials, respectively [19,20]. As mentioned earlier, the main challenge MM drug interventions face is drug resistance. Therefore, exploring possible alternative treatments to overcome it is clinically valuable. This systematic review focuses on the synergistic anti-MM effects of flavonoids, other non-flavonoid polyphenols, and BTZ.

Research Methodology
The systematic review adheres to the guidance of the Preferred Reporting Items for Reference Systems Evaluation and Meta-Analysis (PRISMA) statement [21]. Preclinical studies on flavonoids or non-flavonoid polyphenols -bortezomib synergistic potentiation of anti-MM effects were collected by searching PubMed, Web of Science(WOS), and Embase for the keywords "flavonoid" or "polyphenol," "bortezomib" or "Velcade," and "Multiple myeloma" or "Myeloma, Plasma-Cell." Searching with the search formula "Multiple myeloma" or "Myeloma, plasma cell" (subject) and "Bortezomib or Velcade" (all fields) and "flavonoids or polyphenols" (all fields) were put into WOS, 33 publications were found. Performing a search at PubMed with the search formula: ((Multiple myeloma or "Myeloma, Plasma-Cell") AND (Bortezomib or Velcade)) AND (flavonoids or polyphenols), 38 publications were retrieved. Searching in Embase with the search terms: ((Multiple myeloma or "Myeloma, Plasma-Cell") AND (Bortezomib or Velcade)) AND (flavonoids or polyphenols), 144 publications were retrieved. The filters had not been applied in any of the above three databases. Endnote 20 was used to check for duplicates automatically and by hand for 215 publications, and 41 duplicates were taken out. The titles and abstracts of 174 publications were reviewed by two independent reviewers (Kaixi Ding and Wei Jiang). In case no consensus was reached, a third independent reviewer made the final decision. 121 publications that were not relevant to the topic of this review were removed.
The remaining 53 publications were then read in full by two independent reviewers. According to the inclusion and exclusion criteria: (1) treatment with flavonoid compounds or non-flavonoid polyphenolic compounds or plant extracts containing polyphenols; (2) animal studies or cell cultures; (3) interventions in animals or cell models of multiple myeloma; (4) studies included flavonoid compounds or non-flavonoid polyphenolic compounds or plant extracts containing polyphenols and BTZ synergistic anti-MM effects or mechanisms (5) Excluding review literature, meta-analyses, case reports, editorials, abstracts and conference proceedings and other types of literature. A total of 38 publications were discharged after a detailed review by the reviewers. In addition, two reviewers agreed that four publications with relevant citation searches from the research and the review literature should be added as secondary sources.

Mechanisms of MM
MM's onset, progression, metastasis, and osteolytic destruction are associated with the regulation of multiple signaling pathways ( Figure 2). Throughout the development of MM, rapid cell proliferation was mainly regulated by PI3K/Akt, Ras/Raf/MEK/Erk, JAK/STAT, Wnt/β-catenin, and RANK/RANKL/OPG signal pathways [41]. Uncontrolled cell proliferation was attributed to the inhibition of normal cell cycle regulation (e.g., FOXO1 and Cyclin D1), and the upregulation of downstream key anti-apoptotic factors (e.g., Bcl2, Bcl-xl, Mcl-1, and Caspase) [42]. Angiogenesis, mainly promoted by VEGF, provides oxygen and nutrients for tumor proliferation. Additionally, the activation of the Wnt/-catenin and RANK/RANKL/OPG signal pathways is primarily responsible for osteolytic bone disease in MM, which is brought on by an increase in osteoclast activity [41].
Finally, MM cells can further develop drug resistance, and this may be related to mechanisms such as the upregulation of permeability-glycoprotein (P-gp) (leading to reduced drug aggregation in vivo). IL-6 and insulin-like growth factor (IGF), two cytokines produced by bone marrow mesenchymal stem cells, also encourage drug resistance in MM cells(activating specific signaling pathways that lead to drug resistance) [43].

Bortezomib in MM Therapy
An important class of medications for the treatment of MM is protease inhibitors. Based on targeted inhibition of the 26S proteasome, protease inhibitors are crucial in the pathogenesis and proliferation of MM [44]. BTZ, the first protease inhibitor, approved for clinical use in 2003, has resulted in gains in overall survival, progression-free survival, and remission rates in patients with MM ( Figure 3) [45]. Its main anti-cancer mechanism is the inhibition of the chymotrypsin-like site of the 20S protein hydrolysis core within the 26S proteasome, which induces cell cycle arrest and apoptosis [46]. The main signaling mechanisms of BTZ-induced apoptosis in MM cells are NF-κB blockade and JNK activation. In addition, BTZ stabilizes various tumor suppressor proteins, such as P53, inhibiting MM cell cycle progression [47]. Currently, the drug is commonly used in MM patients in first-line, relapsed, and/or refractory settings [48]. However, drug resistance in MM therapy is the main challenge currently faced when using BTZ [43].

Exploration of Synergistic Drug Combinations in MM Therapy
When two or more medications are combined to improve their therapeutic effects, this is referred to as drug synergism [49]. For various diseases, including MM, appropriate drug combinations can reduce drug resistance or maximize efficacy [50][51][52][53]. For instance, the previously mentioned VRD is currently an effective and well-tolerated pre-ASCT induction protocol, benefiting newly diagnosed MM patients [6]. BTZ, as a clinically used protease inhibitor, is a cornerstone of VRD. However, significant obstacles to using BTZ to treat MM are drug resistance and the malignancy of MM. To cross these obstacles, researchers have investigated the BTZ resistance-reducing effects of various drug combinations and their synergistic anti-MM effects, such as daratumumab (anti-CD38 antibody), BTZ, and dexamethasone (CASTOR trial), TAK-243 (novel and specific UAE inhibitor) and BTZ, decitabine (epigenetic modulator) and BTZ [54][55][56]. Additionally, recent studies have demonstrated that some naturally occurring polyphenolic compounds have anti-MM activity. These polyphenolic compounds also have the potential to reduce BTZ resistance. Given the low toxicity of natural polyphenolic compounds, their synergistic combination with BTZ may offer MM patients extra therapeutic options [19,20].

Flavonoids and Non-Flavonoid Polyphenols in MM Therapy
Polyphenols are a class of phytochemicals divided into flavonoids and non-flavonoids. A group of compounds known as flavonoids consists of two benzene rings connected by phenolic hydroxyl groups by a central three-carbon atom. They are secondary metabolites in fruits, vegetables, and other herbal plants. The main flavonoid subgroup includes flavones, flavonols, flavan-3-ols, anthocyanins, isoflavones, and chalcones ( Figure 4) [57]. The main non-flavonoids contained phenolic, hydroxycinnamic, lignans, stilbenes, and tannins [58]. Based on the results of this systematic review, various polyphenols, including flavonols(icariin, icariside II), flavan-3-ols((-)-epigallocatechin-3-gallate), flavone(scutellarein, wogonin, morin), isoflavone(formononetin, daidzin), plant extracts rich in flavonoids(punica granatum juice) and non-flavonoid polyphenols(silibinin, resveratrol, curcumin, caffeic acid) exerted anti-MM synergistic effects in combination with BTZ in vivo and/or in vitro (Table 2) ( Figure 5).   Flavonols are a class of flavonoids with a 3-hydroxyflavonoid backbone. Two flavonols are of interest in synergistic anti-MM therapy. Icariin, an active ingredient in the stem and leaves of Epimedium, exerts significant anti-tumor effects on a variety of human tumor cells [22]. Similarly, icariside II, isolated from E. koreanum, inhibits the proliferation and induces apoptosis of many human cancer cells, such as MM, breast cancer, and prostate cancer [23].
Flavones are a class of flavonoids with a 2-phenylchromen-4-one backbone. Three flavones are of interest in synergistic anti-MM therapy. Scutellarein, obtained from S. baicalensis or S. barbata, exerts anticancer activity against various tumors, including breast, lymphoma, colorectal, lung, and liver cancers [24,25]. Similarly, wogonin, an active monoflavone from Scutellaria baicalensis with anti-angiogenic activity, is a potential anti-cancer drug with low toxicity [26]. Morin, isolated from members of the mulberry family, such as mulberry figs and old figs, has anti-proliferative activity against some tumors, such as oral squamous cell carcinoma, leukemia, and colorectal cancer [27].
In contrast to flavones, isoflavones have a chemical structure based on a 3-phenylchromium-4-one backbone. Two isoflavones are of interest in synergistic anti-MM therapy. Formononetin, mainly isolated from the roots of Astragalus membranaceus, Trifolium pratense, Glycyrrhiza glabra, and Pueraria lobate, has anti-inflammatory, antioxidant, antiviral, neuroprotective, wound healing, and antitumor biological activities [28,29]. Daidzin can be isolated from Pueraria lobate. It has demonstrated anticancer activity in preventing and treating breast and prostate cancers [30].
In addition, Punica granatum juice(PGJ), one plant extract rich in flavonoids, is also of attention. The drug activity of PGJ is related to many flavonoid phytoactive components, including the anthocyanins catechin, quercetin, kaempferol, apigenin, and lignan. The anticancer effects of PGJ are widely used to prevent and treat colon, lung, skin, and prostate cancers [31].
Finally, polyphenols other than flavonoids belong to non-flavonoid polyphenols. Four non-flavonoid polyphenols are of interest in synergistic anti-MM therapy. Silibinin, an extract of Milk Thistle, exerts very high antioxidant and antitumor properties [32,60]. Resveratrol is widely found in grapes, berries, and peanuts and has anticancer activity against most human cancers [61]. Another non-flavonoid polyphenol, curcumin, is present in turmeric root. It exerts anti-inflammatory, anti-atherosclerotic, and anti-tumor pharmacological effects [33]. In addition, caffeic acid, an active component of honeybee propolis, has cytotoxic, apoptosis, and anti-proliferation effects [34].

Icariin and Bortezomib
In KM3/BTZ-resistant cells, icariin increased the sensitivity of KM3/BTZ cells to BTZ and partially reversed the drug resistance. Its effect on changing drug resistance may be mediated by upregulating the expression of pro-apoptotic cytokine Par-4, decreasing the expression of drug resistance proteins HSP27 and P-gp [22]. Another study found that the combination of icariin and BTZ promoted apoptosis in U266 cells by blocking the JAK/STAT pathway and lowering the expression of anti-apoptotic proteins like Bcl-2, Bcl-xl, and Survivin. This combination also delayed the cell cycle progression, as shown by the result that more U266 cells were in the G0/G1 phase [23].

Icariside II and Bortezomib
While icariside II enhanced the apoptotic effect of BTZ in U266 cells, it also inhibited the JAK/STAT pathway and down-regulated the expression of STAT3 target genes Bcl-2, Bcl-xl, Survivin, cyclin D1, COX-2, and VEGF, beneficially inhibiting proliferation and promoting apoptosis [35].

Scutellarein and Bortezomib
In vitro, scutellarein alone time-dependently reduced cell viability and significantly induced apoptosis in MM.1R and IM-9 cells. In vivo, dual intervention with scutellarein and BTZ significantly reduced xenograft tumor burden in nude mice with no significant effect on mouse body weight. In parallel, protein expression levels of some apoptotic markers were altered, such as active caspase-3 (upregulated), Bax (upregulated), and Bcl-2 (downregulated) [25]. In another in vivo study, scutellarein eliminated MM cell resistance to BTZ through multiple mechanistic pathways. This result was caused by the HDAC/miR-34a-mediated epigenetic regulation of the c-Met/Akt/mTOR pathway and the NF-κB-mediated activation of the apoptosis cascade [24].

Wogonin and Bortezomib
Wogonin and BTZ synergistically inhibited the secretion levels of pro-angiogenic factors VEGF, PDGF, and bFGF in RPMI 8226 cells. The angiogenesis-inhibitory effect of wogonin may be related to its inhibition of the c-Myc/HIF-1α signaling axis [26].

Formononetin and Bortezomib
In vitro, formononetin and BTZ enhanced STAT3 inhibition and promoted the death of U266 cells [28]. Subsequently, a follow-up study by the same research group found that formononetin and BTZ exert synergistic enhancement of anti-proliferation and proapoptosis by blocking the activation of NF-κB, PI3K/AKT, and AP-1 [29].

Daidzin and Bortezomib
Daidzin synergistically increased the apoptotic and cytotoxic effects of BTZ by inhibiting the activation of STAT3 and its upstream kinases (JAK1, JAK2, and c-Src). In addition, this drug combination increased caspase-3 activation and PARP cleavage, leading to the downregulation of the expression of various oncogenic apoptotic proteins [30].

Plant Extracts and Bortezomib
In vitro, PGJ inhibited angiogenesis, microvascular growth outside of aortic rings, cell migration, and invasion in MM cells. In addition, After BTZ exposure, PGJ intervention increased the cytotoxic effects on U266 cells [31].

Synergistic Effects of Non-Flavonoid Polyphenols and Bortezomib in Anti-MM
Four flavonoids (silibinin, resveratrol, curcumin, caffeic acid) synergize with BTZ in anti-MM effect by regulating proliferation, apoptosis, and drug resistance-related signaling pathways (Table 4).

Silibinin and Bortezomib
In combination with low concentrations of BTZ, silibinin increased the cytotoxic effect of BTZ by increasing the expression of activated caspases, which promoted apoptosis [32].

Resveratrol and Bortezomib
In vitro, resveratrol induced apoptosis in MM144 cells by upregulating the Fas/CD95 signaling pathway and caspase-8 and caspase-10. Moreover, when used in combination, resveratrol, and BTZ highly enhanced U266 cell apoptosis [37].

Curcumin and Bortezomib
Curcumin enhances the apoptotic effect of BTZ on MM cells by regulating multiple signaling pathways (Figure 7). The bone marrow microenvironment, in which bone marrow stromal cells (BMSCs) interact with MM, influences the survival and growth of MM cells. In vitro, curcumin inhibited the activation of JAK/STAT and MAPK pathways in U266 cells after treatment with BMSCs cell supernatant. Additionally, curcumin and BTZ cotreatment efficiently prevented IL-6-induced STAT3 and Erk phosphorylation, increased PARP cleavage, and decreased pro-caspase-3 levels. Through these mechanisms mentioned above, this combination inhibited the growth of U266 cells and promoted apoptosis [38]. After that, in another trial, curcumin enhanced the inhibitory effect of BTZ on NF-κB activation in U266 cells, leading to increased apoptosis. In vivo, the combination group was more potent than the BTZ group in reducing tumor volume [39]. Moreover, curcumin increased the expression of cleaved caspase-3 protein by inhibiting the Notch1 signaling pathway, which increased the drug sensitivity of RPMI-8226 cells and U266 cells to BTZ [33]. A class of novel curcumin analogs known as amino acid adducts of curcumin improved the proteasomal inhibitory effect of BTZ on MM cells and they enhanced the proliferation inhibition and apoptosis induction of BTZ. Notably, similar to curcumin, the twelfth curcumin analog increased PARP and caspase-3 cleavage, enhancing BTZ-induced apoptosis. The water-soluble, highly bioavailable twelfth curcumin analog has the potential to replace curcumin in anti-MM therapy in the future [40].

Caffeic acid and Bortezomib
In vitro, caffeic acid alone inhibits NF-κB-binding activity and IL-6 levels, which are closely linked to apoptosis and growth of tumor cells. Moreover, the combination of caffeic acid and BTZ synergistically increased cytotoxic and anti-proliferative effects on ARH-77 cells [34].

Conclusions and Future Directions
Although treatment options for MM continue to be optimized, the prognosis remains unsatisfactory. The aggressiveness and drug resistance of malignant tumors hinder the current treatment with protease inhibitors-especially BTZ. In this regard, flavonoids and non-flavonoid polyphenols are potential supportive therapies to address some of the challenges faced in BTZ treatment. Flavonoids(icariin, icariside II, EGCG, scutellarein, wogonin, morin, formononetin, daidzin), plant extract rich in flavonoids(PGJ), and non-flavonoid polyphenols(silibinin, resveratrol, curcumin, caffeic acid) combined with BTZ demonstrate the synergistic anti-MM effect. These synergistic anti-MM effects were achieved by antiproliferative, pro-apoptotic, and anti-drug resistance. Based on those pieces of evidence, flavonoids, plant extract rich in flavonoids, and non-flavonoid polyphenols may benefit patients with MM, especially by overcoming the challenges faced in BTZ therapy.
According to some other relevant preclinical evidence, some polyphenols alone have anti-MM activity, such as isoginkgetin (a biflavone) and virola oleifera (a polyphenol-rich plant extract) [63,64]. Their synergistic effects, in combination with BTZ for the treatment of MM, need to be further explored. In addition, most of the synergistic anti-MM impacts of polyphenols + BTZ are only verified at the cellular level, such as icariin, icariside, EGCG, wogonin, morin, daidzin, PGJ, silibinin, and resveratrol. Their anti-MM synergistic effects in combination with BTZ need further clinically relevant animal models for validation. These polyphenols + BTZ exert synergistic effects through modulation of MMrelated signaling pathways such as NF-κB, PI3K/Akt, Ras/Raf/MEK/Erk, JAK/STAT, and Wnt/β-catenin. However, the signaling pathways of each combination of anti-MM are still rudimentary, and more research is needed to refine the signaling pathway network. Although these results show promise for a synergistic MM treatment using these polyphenolic compounds and BTZ, the evidence at this time is only preclinical. Therefore, further clinical studies must demonstrate these synergistic effects. Moreover, preclinical studies on the anti-MM synergistic effects of these polyphenols with two other clinically available protease inhibitors, carfilzomib, and estazomib, need to be conducted to provide more options for clinical combination use. Adverse effects of protease inhibitor use are another challenge in the treatment of MM, although not within the scope of the review. Some polyphenols have the ameliorative potential for the non-tumor toxicity of protease inhibitors. For instance, rutin mitigates carfilzomib-induced cardiotoxicity by inhibiting NF-κB, mast gene expression, and reducing oxidative stress [65]. Resveratrol, by activating SIRT1, improves BTZ mechanical nociceptive sensitization [66]. For the comprehensive management of MM, additional studies on the antagonism of polyphenols against protease inhibitor-associated toxicity are also of clinical value. Finally, BTZ could alleviate MMassociated bone disease by regulating the RANK/RANKL/OPG signaling pathway [67]. By concentrating on the modulation of the RANK/RANKL/OPG signaling pathway, it is possible to investigate whether combining polyphenols and BTZ has beneficial effects on MM-associated bone disease.
Some polyphenols contain catechol moieties, such as EGCG, quercetin, and myricetin. Notably, they reduce the protease inhibitory activity of BTZ by forming stable cyclic boronic esters, thereby decreasing the anti-MM effect of BTZ [68,69]. A representative potential BTZ antagonistic polyphenol, EGCG, demonstrated dose-dependent inhibition of BTZ antitumor activity [62]. An in vitro study found that, by activating Wnt/β-catenin, EGCG antagonized the antitumor effect of BTZ [70]. Therefore, clinical staff members should be cautious about BTZ drug interactions with these polyphenols in clinical use. Last but not least, the problem of bioavailability is an important reason why polyphenols do not work as well as they could in the body. In the future, Low bioavailability polyphenols will eventually need to be combined with new drug delivery systems or other derivatives to utilize their anticancer capabilities fully.