Formononetin Regulates Multiple Oncogenic Signaling Cascades and Enhances Sensitivity to Bortezomib in a Multiple Myeloma Mouse Model

Here, we determined the anti-neoplastic actions of formononetin (FT) against multiple myeloma (MM) and elucidated its possible mode of action. It was observed that FT enhanced the apoptosis caused by bortezomib (Bor) and mitigated proliferation in MM cells, and these events are regulated by nuclear factor-κB (NF-κB), phosphatidylinositol 3-kinase (PI3K)/AKT, and activator protein-1 (AP-1) activation. We further noted that FT treatment reduced the levels of diverse tumorigenic proteins involved in myeloma progression and survival. Interestingly, we observed that FT also blocked persistent NF-κB, PI3K/AKT, and AP-1 activation in myeloma cells. FT suppressed the activation of these oncogenic cascades by affecting a number of signaling molecules involved in their cellular regulation. In addition, FT augmented tumor growth-inhibitory potential of Bor in MM preclinical mouse model. Thus, FT can be employed with proteasomal inhibitors for myeloma therapy by regulating the activation of diverse oncogenic transcription factors involved in myeloma growth.


Introduction
Multiple myeloma (MM) is a bone marrow based severe malignancy, which is extremely difficult to cure [1][2][3][4][5]. Multiple myeloma is the second most common blood cancer comprising around 10% of all hematological malignancies [6][7][8]. Although various pharmacological strategies have been developed to cure myeloma patients, including the application of proteasome inhibitors, immunomodulatory agents, and alkylating agents, there still remains an unmet need to improve the therapeutic outcome for myeloma patients [9][10][11].
Nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) are major transcription factors implicated in the regulation of inflammation, immunomodulation, angiogenesis, and tumorigenesis [12][13][14][15]. It is made up of p50 and p65 subunits, and these are kept in a dormant state by a family of inhibitory proteins consisting of Inhibitory Subunit of NF Kappa B Alpha (IκBα), IκBβ, and other members [13,16]. Generally, NF-κB in the inactive form is made up of p50, p65, and IκBα and resides in the cytoplasm [17]. However, processing of IκBα can cause p50-p65 dimer to move into the nucleus, bind to the DNA, and modulate transcription [14,18,19]. AP-1 is also mainly composed of Jun, Fos, and Activating transcription factor (ATF) protein dimers [20]. Two transcription factors can regulate proinflammatory gene products in response to cytokines, growth factors, stress signals, bacterial, and viral infections, as
Human multiple myeloma cell lines U266 and RPMI 8226 were obtained from the American Type Culture Collection (Manassas, VA, USA). U266 and RPMI 8226 cells were cultured in RPMI 1640 medium containing 10% FBS supplemented with 100 U/mL of penicillin and 100 µg/mL of streptomycin.

Western Blotting
After the cells were treated with the indicated concentrations of FT, Western blot analysis was done as elaborated upon previously [33]. Briefly, cell lysates were separated by SDS-PAGE and transferred onto a nitrocellulose membrane. The membrane was blocked and probed with various antibodies. The proteins were detected with enhanced chemiluminescence (Millipore, Bedford, MA, Biomolecules 2019, 9, 262 3 of 17 USA). The bands were quantified using an Image J software (v1.8.0, National Institutes of Health, Bethesda, MD, USA).

Reverse Transcription Polymerase Chain Reaction (RT-PCR)
Total RNA was extracted according to the manufacturer's instructions (Invitrogen, Life Technologies, Carlsbad, CA, USA), and RT-PCR was carried out as indicated before [33].

Immunocytochemistry for p65, c-Fos, and c-Jun Localization
After the U266 cells were treated with 100 µM of FT, immunocytochemistry for various proteins was performed as described before [48].

TUNEL Assay
U266 and RPMI 8226 cells were subjected to Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, as described earlier [48]. To determine the effect of FT on the late apoptotic cell death, cells were quantified using TUNEL assay kit (Roche Diagnostics GmbH, Penzberg, Germany) and then analyzed by flow cytometry using FACScan Calibur flow cytometer and CellQuest software (Version 4.0, BD Biosciences, Becton-Dickinson, Franklin Lakes, NJ, USA).

MTT Assay
Cell viability was measured by an MTT assay to detect NADH-dependent dehydrogenase activity as done before [33]. Drug combinations were evaluated using CalcuSyn (Version 2.0, BIOSOFT, Ferguson, MO, USA) software based on the multiple drug-effect equation of Chou-Talalay.

Flow Cytometry
The effect of FT on cell cycle distribution was determined using flow cytometry following staining with Propidium iodide (PI). Cells were treated with 50 µM of FT and 10 nM of Bor for 24 h. The cells were then collected and washed with PBS, fixed in 70% cold ethanol at 4 • C overnight. After addition of 25 µg/mL of PI to the cells for 30 min in the dark, the apoptotic Sub-G1 cell population was analyzed on a FACScan Calibur flow cytometer and CellQuest software (BD Biosciences, Becton-Dickinson, Franklin Lakes, NJ, USA).

Animals
All procedures involving animals were reviewed and approved by KHU Institutional Animal Care and Use Committee [KHUASP(SE)-17-110]. All the in vivo experiments were conducted strictly in accordance with the institutional guidelines and monitored regularly. Five-week-old athymic nu/nu female mice (NARA Biotech, Korea) were implanted subcutaneously in the right flank with U266 cells. Tumors were allowed to grow to a maximum diameter of 1 to 1.5 cm and were then sacrificed.

Experimental Protocol
U266 cells [1 × 10 7 /100 µL PBS:Matrigel (1:1)] were injected subcutaneously into the right flank of the mice, as described previously [51]. When tumors have reached 0.5 cm in diameter, the mice were randomized into four treatment groups (n = 5/group). Group I was given PBS (200 µL, i.p. thrice/week), group II was given FT (20 mg/kg body weight, i.p. thrice/week), group III was given Bor (0.25 mg/kg body weight, i.p. thrice/week), group IV was given FT (20 mg/kg body weight, i.p. thrice/week) and Bor (0.25 mg/kg body weight, i.p. once/week). Therapy was continued for 21 days, and the animals were euthanized one week later. Primary tumors were excised, and the final tumor volume was measured as V = 4/3 πr 3 , where r is the mean radius of the three dimensions (length, width, and depth), and thereafter tissues were processed as described before [48].

Immunohistochemical and Western Blot Analysis of Multiple Myeloma Tumor Samples
Immunohistochemical and Western blot analysis for tumor samples was done as described previously [48].

Statistical Analysis
All numeric values are represented as the mean ± standard error (SE). Statistical significance of the data was determined by GraphPad Prism version 5 (GraphPad Software, La Jolla, CA, USA) using one-way ANOVA followed by Tukey's posthoc test. Significance was set at p < 0.05.

Formononetin Inhibits NF-κB and AP-1 Activation in Multiple Myeloma Cells
NF-κB activation can correlate with the resistance to apoptotic cell death and also regulates the tumorigenic process in various hematological malignancies, including multiple myeloma [52][53][54]. Hence, we investigated whether FT has the potential to inhibit NF-κB activation, and consequently causing programmed cell death. The structure of FT is shown in Figure 1A. Cytoplasmic (CE) and nuclear extract (NE) were generated from myeloma cells exposed to FT and examined for various assays. First, in NE, we assessed NF-κB activity by performing EMSA assay and noted that FT substantially reduced NF-κB-DNA binding ability ( Figure 1B,C). We deciphered the action of FT on the activation of IKK that is required for IκBα phosphorylation. CEs were prepared and assayed by Western blot. As shown in Figure 1D and Supplementary Figure S1, FT abrogated the activation of IKKα/β in U266 cells with no effect on the IKKα/β levels (first and second panel). Moreover, FT effectively reduced the IκBα phosphorylation with minimal effect on IκBα protein levels ( Figure 1D, third and fourth panel). Additionally, the levels of p-p65 and p65 were inspected in NEs using Western blot. FT mitigated the levels of p-p65 and p65 in MM cells ( Figure 1E).
To determine the effects of FT on the activation of another oncogenic transcription factor, AP-1 [55], EMSA was again performed. As shown in Figure 1F,G, FT substantially attenuated AP-1-DNA binding activities that also led to the reduction in the protein ( Figure 1H) and mRNA ( Figure 1I) levels of c-Jun and c-Fos. Moreover, as shown in Figure 1J, immunocytochemistry data clearly demonstrated that FT also reduced the translocation of p65, c-Fos, and c-Jun into the nuclear compartment.   Figure S1). (F,G) FT suppresses AP-1 binding activity. Cells were treated as described above in B, and EMSA was performed. U266 cells were treated as described above, and (H) Western blotting and (I) RT-PCR were done. (J) U266 cells were treated as described above, and immunostaining was carried out. The third panel shows the merged images of the first and second panels. The results shown are representative of three independent experiments. For band density, densitometric analysis was performed using an Image J software, and numbers on the bottom of the bands represent fold change in expression level relative to controls. NT: non-treated; CE: cytoplasmic extract; NE: nuclear extract.

Formononetin Mitigates the Activation of PI3K/AKT and MAPK Pathways
We next deciphered if FT could also alter the phosphorylation of PI3K/AKT, which can also regulate aberrant tumor growth. As shown in Figure 2A, PI3K, as well as AKT phosphorylation, was affected by FT. Interestingly, it was discovered that FT also altered the levels of another important set of tumor-promoting proteins, namely, p-p38(Thr180/Tyr182), p-ERK1/2(Thr202/Tyr204), and p-JNK(Thr183/Tyr185), in tumor cells ( Figure 2B).

Formononetin Downregulates the Levels of Tumorigenic Proteins and Causes Apoptosis
The survival proteins (Bcl-xL and IAP-1) can mediate resistance to apoptosis and drive the process of carcinogenesis [56]; hence, the effect of FT on these proteins was examined. The levels of Bcl-xL and IAP-1 in U266 cells were attenuated by FT exposure (Figure 2C,E). We deciphered the actions of FT also on the constitutive levels of COX-2 and MMP-9 proteins that can regulate the invasive ability of tumor cells. As depicted in Figure 2C,E, FT exposure induced a notable reduction in the levels of COX-2 and MMP-9. Figure 2D shows that FT can also augment the protein levels of p53 and p21, and the results of TUNEL assay further confirmed that FT could induce notable apoptosis in myeloma cells ( Figure 2F).

Formononetin Causes Potentiation of the Anti-Tumorigenic Actions of Bortezomib
We noted that FT could augment the cytotoxic effects of Bor against MM cells (U266 and RPMI 8226), and the combination index (CI) values suggested that FT (50 µM)-Bor (10 nM), as well as FT (75 µM)-Bor (10 nM) concentrations synergistically attenuated cellular growth ( Figure 3A). This action was mediated by its ability to enhance the effects of Bor in reducing p-p38 and p-ERK1/2 levels, and also causing an upregulation of p-JNK expression ( Figure 3B). We also observed that combination treatment mitigated the p-IKKα/β, p-IκBα, p-p65 ( Figure 3C), and c-Fos and c-Jun proteins ( Figure 3D) levels which might also explain the action of FT in elevating the cytotoxic effects of Bor.

Formononetin Enhances the Apoptotic Effects of Bortezomib by Diverse Mechanisms
As illustrated in Figure 4A, we noted that FT and Bor combination indeed caused the assembly of cells in sub-G1 stage up to 38%, and these findings were further confirmed by TUNEL assay ( Figure 4B). Interestingly, the combination treatment not only escalated the levels of pro-apoptotic protein, caspase-3, as well as caused Poly (ADP-ribose) polymerase (PARP) cleavage ( Figure 4C) but also mitigated the levels of various cancer-promoting proteins in myeloma cells ( Figure 4D).

Formononetin Affects the Antitumor Actions of Bortezomib In Vivo
We examined the efficacy of both FT and Bor to affect tumor growth in the MM model based on protocol exhibited in Figure 5A. We noted that FT treatment alone, as well as in combination with Bor, significantly attenuated tumor growth and burden ( Figure 5B-D) without affecting the body weight of the treated mice ( Figure 5E).

Formononetin Affects the Levels of Oncogenic Biomarkers in Tumor Tissues
As observed in myeloma cell lines, we also noticed that combination treatment down-modulated the levels of p65, c-Fos, and c-Jun in tumor tissues analyzed ( Figure 6A). In addition, a significant reduction in both Ki-67 and Vascular endothelial growth factor (VEGF) expression was noticed upon exposure to FT and Bor ( Figure 6B), which indicated the efficacy of combination to mitigate the tumor growth and angiogenesis. Furthermore, combination treatment not only reduced DNA binding activities ( Figure 6C) but also caused substantial apoptosis as observed in tumor tissues ( Figure 6D, first and second panels). Also, it caused an attenuation in the levels of different biomarkers in tissues ( Figure 6E and Supplementary Figure S2). Both FT and Bor were only marginally active when applied as single agents under in vivo settings.   As illustrated in Figure 4A, we noted that FT and Bor combination indeed caused the assembly of cells in sub-G1 stage up to 38%, and these findings were further confirmed by TUNEL assay ( Figure  4B). Interestingly, the combination treatment not only escalated the levels of pro-apoptotic protein, caspase-3, as well as caused Poly (ADP-ribose) polymerase (PARP) cleavage ( Figure 4C) but also mitigated the levels of various cancer-promoting proteins in myeloma cells ( Figure 4D).

Discussion
Targeting multiple signal transduction cascades (NF-κB, PI3K/AKT, and AP-1) can constitute an important pharmacological strategy as deregulation of these oncogenic pathways has been reported to mediate both initiation and progression of MM [33,53]. We noted that FT exerted substantial inhibitory effects on NF-κB, AP-1 (c-Fos and c-Jun), PI3K (tyrosine residue 458), and AKT (serine residue 473) activation. It was discovered that FT also augmented the apoptosis induced by a proteasomal blocker (Bor) and abrogated the growth of myeloma cells. FT also reduced the level of diverse tumorigenic proteins and enhanced the anti-tumor activity of Bor significantly in the preclinical model.
It was noticed that FT could suppress NF-κB and AP-1 in myeloma cells. It was noted that FT induced its suppressive action against NF-κB pathway by attenuating the phosphorylation of upstream IKK and thereby negatively regulating IκBα phosphorylation and nuclear translocation of p65 and its DNA binding affinity. Interestingly, Bor can regulate apoptosis of tumor cells by causing both inactivation as well as activation of NF-κB signaling machinery [57][58][59]. Moreover, in myeloma, an enhancement in the expression of receptor activator of nuclear factor kappa B ligand (RANKL) may lead to NF-κB and AP-1-mediated increased bone resorption [60,61]. In an interesting study, Bor was reported to abrogate both osteoclast differentiation as well as its bone resorption activity through the modulation of transcription factor-AP-1 [62]. Moreover, the activation of c-Fos and c-Jun can lead to tumorigenesis as well as resistance to chemotherapy. We noted that FT mitigated c-Fos and c-Jun activation at both protein and mRNA levels. Additionally, AP-1 DNA-binding affinity and nuclear translocation of c-Fos and c-Jun was also affected in myeloma cells.
The phosphorylation status of p38, ERK1/2, and JNK MAPKs was next examined in FT treated myeloma cells. It was noted that FT affected the activation of MAPKs by reducing the phosphorylation of p38 and ERK1/2, but enhancing the activation of JNK. Interestingly, the p38 pathway can be activated in monocytic precursors upon stimulating with different cytokines, and treatment with its pharmacological blocker SB203580 can lead to a reduced osteoclast differentiation [63,64]. On the contrary, JNK activation induced by a variety of mechanism(s), including the targeting of sphingolipid signaling, may regulate the survival of myeloma cells [65]. Furthermore, FT has been found to negatively affect the activation of PI3K/AKT cascade that can regulate ABC subfamily G member 2 (ABCG2) expression and mediate chemoresistance in myeloma cells [66], and we also observed a similar effect on PI3K/AKT axis in our experiments [66].
The negative regulation of AP-1 and NF-κB has been linked to the down-modulation of various oncogenic as well as metastatic proteins [67][68][69][70][71][72]. We noted that FT treatment caused a downregulation in the levels of diverse tumorigenic genes controlled by these transcription factors, thereby modulating the growth as well as inducing apoptosis in myeloma cells. It also caused an elevation in the levels of p53 and p21 gene products that can regulate both cell apoptosis and cell cycle progression. However, the direct mechanism of action of FT in tumor cells is still not clear from our present study, and the in silico docking analysis to decipher the possible interactions of FT with various proteins in different biochemical pathways will be carried out in future studies.
The application of Bor for myeloma therapy [73] can result in severe side effects and also drug resistance. Thus, overall, its effectiveness is greatly limited by its toxicity and development of chemoresistance [74]. The combination treatment of MM with Bor and other chemotherapeutic agents (e.g., doxorubicin and dexamethasone) has been used in clinical settings [75]. The strategy of employing natural products in combination with chemotherapy can form the basis of an important blueprint for treatment. We further demonstrated that the combination of FT and Bor treatment elicited a significantly greater antitumor effect in the preclinical model as compared with each treatment only. The combinatorial therapy used enhanced Bor-induced apoptosis through the downregulation of various pro-survival transcription factors and oncogenic proteins. In addition, our findings conclusively demonstrate that FT and Bor, when applied in conjunction, may exhibit significant anti-neoplastic effects in the preclinical model.
Overall, our findings in cell lines and in vivo model suggest that anti-tumoral actions of FT in myeloma model can be arbitrated via the regulation of multiple oncogenic cascades and gene products. Overall, the novel pharmacological combination of FT and Bor can be effectively used to supplement available treatment modalities for myeloma patients after completion of clinical trials.

Conflicts of Interest:
The authors declare no conflict of interest.