Elevated SFXN2 limits mitochondrial autophagy and increases iron-mediated energy production to promote multiple myeloma cell proliferation.

Human sideroflexin 2 (SFXN2) belongs to the SFXN protein family, which is a mitochondrial outer membrane protein involved in mitochondrial iron metabolism. Mitochondria are indispensable for cellular energy production and iron metabolism. However, it remains elusive how SFXN2 modulates mitochondrial homeostasis and cellular iron metabolism in multiple myeloma (MM). In this study, we first found that SFXN2 was significantly elevated and correlated to poor outcomes in MM patients from clinical datasets. SFXN2 overexpression promoted MM cell proliferation and suppressed starvation-induced autophagy/mitophagy, while SFXN2 knockdown aggravated mitochondria damage and autophagic processes in ARP1 and H929 MM cell lines. Furthermore, inhibition of SFXN2 exerted effectively anti-myeloma activity in vivo by using myeloma xenograft model. Mechanism studies indicated that heme oxygenase 1 (HO1) with anti-oxidant function contributed to the process of autophagy suppression and cellular proliferation mediated by SFXN2. Our study revealed the critical role of SFXN2 in regulating mitochondrial bioenergetics, mitophagy, cellular iron metabolism, and redox homeostasis in interconnected and intricate way. Collectively, these findings not only provide insights into the metabolic reprogramming of tumor cells, but also highlight the therapeutic potential of SFXN2 in combination with iron metabolism as target for prognosis and treatment in MM patients.

A novel protein encoded by circHNRNPU promotes multiple myeloma progression by regulating the bone marrow microenvironment and alternative splicing.

BACKGROUD: Multiple myeloma (MM) is an incurable plasma cell malignancy in the bone marrow (BM), while immunoglobulin D type of MM (IgD MM) is a very rare but most severe subtype in all MM cases. Therefore, systemic study on IgD MM is purposeful to disclose the recurrent and refractory features in both IgD and other types of MM, and beneficial to the development of potent therapeutic strategy on MM. METHODS: Agilent SBC-ceRNA microarray chips were employed to examine 3 normal plasma cell samples (NPCs), 5 lgD MM samples and 5 lgG MM samples, respectively. Sanger sequencing, RNase R digestion and qPCR assays were used to detect the existence and expression of circHNRNPU. BaseScope™ RNA ISH assay was performed to test circHNRNPU levels in paraffin-embedded MM tissues. The protein encoded by circHNRNPU was identified by LC-MS/MS, which was named as circHNRNPU_603aa. The function of circHNRNPU_603aa on cellular proliferation and cell cycle was assessed by MTT test, colony formation assay, flow cytometry and MM xenograft mouse model in vivo. RIP-seq, RIP-PCR and WB analysis for ubiquitination were performed to explore the potential mechanism of circHNRNPU_603aa in MM. Exosomes were isolated from the culture supernatant of MM cells by ultracentrifugation and characterized by Transmission Electron Microscope and WB confirmation of exosomes markers Alix and CD9. RESULTS: CircHNRNPU was one of the top most abundant and differentially expressed circRNA in IgD MM relative to lgG and NPCs samples. Increased circHNRNPU was associated with poor outcomes in four independent MM patient cohorts. Intriguingly, MM cells secreted circHNRNPU, which encoded a protein named as circHNRNPU_603aa. Overexpressed circHNRNPU_603aa promoted MM cell proliferation in vitro and in vivo, in contrast knockdown of circHNRNPU_603aa by siRNA abrogated these effects. Due to circHNRNPU_603aa including RNA-binding RGG-box region, it regulated SKP2 exon skipping, thereby competitively inhibited c-Myc ubiquitin so as to stabilize c-Myc in MM. MM cells secreted circHNRNPU through exosomes to interfere with various cells in the BM microenvironment. CONCLUSION: Our findings demonstrate that circHNRNPU_603aa is a promising diagnostic and therapeutic marker in both MM cells and BM niche.

AHSA1 is a promising therapeutic target for cellular proliferation and proteasome inhibitor resistance in multiple myeloma.

BACKGROUND: Currently, multiple myeloma (MM) is still an incurable plasma cell malignancy in urgent need of novel therapeutic targets and drugs. METHODS: Bufalin was known as a highly toxic but effective anti-cancer compound. We used Bufalin as a probe to screen its potential targets by proteome microarray, in which AHSA1 was the unique target of Bufalin. The effects of AHSA1 on cellular proliferation and drug resistance were determined by MTT, western blot, flow cytometry, immunohistochemistry staining and xenograft model in vivo. The potential mechanisms of Bufalin and KU-177 in AHSA1/HSP90 were verified by co-immunoprecipitation, mass spectrometry, site mutation and microscale thermophoresis assay. RESULTS: AHSA1 expression was increased in MM samples compared to normal controls, which was significantly associated with MM relapse and poor outcomes. Furthermore, AHSA1 promoted MM cell proliferation and proteasome inhibitor (PI) resistance in vitro and in vivo. Mechanism exploration indicated that AHSA1 acted as a co-chaperone of HSP90A to activate CDK6 and PSMD2, which were key regulators of MM proliferation and PI resistance respectively. Additionally, we identified AHSA1-K137 as the specific binding site of Bufalin on AHSA1, mutation of which decreased the interaction of AHSA1 with HSP90A and suppressed the function of AHSA1 on mediating CDK6 and PSMD2. Intriguingly, we discovered KU-177, an AHSA1 selective inhibitor, and found KU-177 targeting the same site as Bufalin. Bufalin and KU-177 treatments hampered the proliferation of flow MRD-positive cells in both primary MM and recurrent MM patient samples. Moreover, KU-177 abrogated the cellular proliferation and PI resistance induced by elevated AHSA1, and decreased the expression of CDK6 and PSMD2. CONCLUSIONS: We demonstrate that AHSA1 may serve as a promising therapeutic target for cellular proliferation and proteasome inhibitor resistance in multiple myeloma.

BUB1B and circBUB1B_544aa aggravate multiple myeloma malignancy through evoking chromosomal instability.

Multiple myeloma (MM) is an incurable plasma cell malignancy in the bone marrow characterized by chromosome instability (CIN), which contributes to the acquisition of heterogeneity, along with MM progression, drug resistance, and relapse. In this study, we elucidated that the expression of BUB1B increased strikingly in MM patients and was closely correlated with poor outcomes. Overexpression of BUB1B facilitated cellular proliferation and induced drug resistance in vitro and in vivo, while genetic targeting BUB1B abrogated this effect. Mechanistic studies unveiled that enforced expression of BUB1B evoked CIN resulting in MM poor outcomes mainly through phosphorylating CEP170. Interestingly, we discovered the existence of circBUB1B_544aa containing the kinase catalytic center of BUB1B, which was translated by a circular RNA of BUB1B. The circBUB1B_544aa elevated in MM peripheral blood samples was closely associated with MM poor outcomes and played a synergistic effect with BUB1B on evoking CIN. In addition, MM cells could secrete circBUB1B_544aa and interfere the MM microenvironmental cells in the same manner as BUB1B full-length protein. Intriguingly, BUB1B siRNA, targeting the kinase catalytic center of both BUB1B and circBUB1B_544aa, significantly inhibited MM malignancy in vitro and in vivo. Collectively, BUB1B and circBUB1B_544aa are promising prognostic and therapeutic targets of MM.

Targeting RFWD2 as an Effective Strategy to Inhibit Cellular Proliferation and Overcome Drug Resistance to Proteasome Inhibitor in Multiple Myeloma.

The potential to overcome resistance to proteasome inhibitors is greatly related with ubiquitin-proteasome system during multiple myeloma (MM) treatment process. The constitutive photomorphogenic 1 (RFWD2), referred to an E3 ubiquitin ligase, has been identified as an oncogene in multiple cancers, yet important questions on the role of RFWD2 in MM biology and treatment remain unclear. Here we demonstrated that MM patients with elevated RFWD2 expression achieved adverse outcome and drug resistance by analyzing gene expression profiling. Moreover, we proved that RFWD2 participated in the process of cell cycle, cell growth and death in MM by mass spectrometry analysis. In vitro study indicated that inducible knockdown of RFWD2 hindered cellular growth and triggered apoptosis in MM cells. Mechanism study revealed that RFWD2 controlled MM cellular proliferation via regulating the degradation of P27 rather than P53. Further exploration unveiled that RFWD2 meditated P27 ubiquitination via interacting with RCHY1, which served as an E3 ubiquitin ligase of P27. Finally, in vivo study illustrated that blocking RFWD2 in BTZ-resistant MM cells overcame the drug resistance in a myeloma xenograft mouse model. Taken together, these findings provide compelling evidence for prompting that targeting RFWD2 may be an effective strategy to inhibit cellular proliferation and overcome drug resistance to proteasome inhibitor in MM.

Solutol®HS15+pluronicF127 and Solutol®HS15+pluronicL61 mixed micelle systems for oral delivery of genistein.

Purpose: We aimed to prepare two oral drug delivery systems consisting of polyoxyl 15 hydroxystearate (HS15) with pluronicF127 (F127) and HS15 with pluronicL61 (L61) to overcome the challenges of genistein's poor oral bioavailability. This provides a good strategy for enhancing the potential value of genistein. Methods: We designed two binary mixed micelle systems employing the organic solvent evaporation method using surfactants (HS15, L61, and F127). Formulations (GEN-F and GEN-L) were characterized by transmission electron microscopy. Drug content analysis, including entrapment efficiency (EE%), drug loading (DL%), and the cumulative amount of genistein released from the micelles, was performed using HPLC. The permeability of optimum formulation was measured in Caco-2 cell monolayers, and the oral bioavailability was evaluated in SD rats. Results: The solutions of GEN-F and GEN-L were observed to be transparent and colorless. GEN-F had a lower EE% of 80.79±0.55% and a DL% of 1.69±0.24% compared to GEN-L, which had an EE% 83.40±1.36% and a DL% 2.26±0.18%. TEM results showed that the morphology of GEN-F and GEN-L was homogeneous and resembled a spherical shape. The dilution and storage conditions had no significant effect on particle size and EE%. Genistein demonstrated a sustained release behavior when encapsulated in micelles. Pharmacokinetics study showed that the relative oral bioavailability of GEN-F and GEN-L increased by 2.23 and 3.46 fold while also enhancing the permeability of genistein across a Caco-2 cell monolayer compared to that of raw genistein. Conclusion: GEN-F and GEN-L as a drug delivery system provide an effective strategy for enhancing and further realizing the potential value of GEN.

Enhancing the oral bioavailability of baicalein via Solutol® HS15 and Poloxamer 188 mixed micelles system.

OBJECTIVES: To increase the solubility of baicalein (BAI) by preparing BAI-micelles (BAI-M) with Solutol HS15 (HS15) and Poloxamer 188 (F68), thereby improving its oral bioavailability. METHODS: Baicalein micelles were prepared with HS15 and F68 by thin-film dispersion method and optimized by central composite design (CCD) approach. Physicochemical, in vitro release, Caco-2 cell transport and pharmacokinetic studies of BAI-M were performed. KEY FINDINGS: The optimal formulation showed spherical shape by characterization of the transmission electron microscope with average small size (23.14 ± 1.46 nm) and high entrapment efficiency (92.78±0.98%) and drug loading (6.45±1.54%). The in vitro release study of BAI-M showed a significantly sustained release pattern compared with free BAI. Caco-2 cell transport study demonstrated that high permeability of BAI was achieved after loading it into micelles. Meanwhile, pharmacokinetics study of BAI-M showed a 3.02-fold increase in relative oral bioavailability compared with free BAI. CONCLUSIONS: Based on our findings, we concluded that HS15 can be used as a carrier in this drug delivery system that includes F68, and BAI-M has great potential in improving solubility and oral bioavailability.

Improved oral bioavailability of magnolol by using a binary mixed micelle system.

The aim of this study was to prepare two novel magnolol (MO)-loaded binary mixed micelles (MO-M) using biocompatible copolymers of Soluplus (SOL) and Solutol® HS15 (HS15), SOL and d-alpha-tocopheryl polyethylene glycol 1000 succinate (TPGS), to improve magnolol's poor solubility and its oral bioavailability. The organic solvent evaporation method was used to obtain two MO-M by optimization; one was prepared by using SOL and HS15 (MO-H), and the other was prepared by using SOL and TPGS (MO-T). The entrapment efficiency (EE%) and drug loading (DL%) of MO-T were 94.61 ± 0.91% and 4.03 ± 0.19%, respectively, and the MO-H has higher EE% and DL% (98.37 ± 1.23%, 4.12 ± 0.16%). TEM results showed that the morphology of MO-M was homogeneous and was spherical in shape. The dilution stability of MO-M did not undergo significant changes. Permeability of MO-M across a Caco-2 cell monolayer was enhanced in Caco-2 cell transport models. The pharmacokinetics study showed that the relative oral bioavailability of MO-T and MO-H increased by 2.39- and 2.98-fold, respectively, compared to that of raw MO. This indicated that MO-H and MO-T could promote absorption of MO in the gastrointestinal tract. Collectively, the mixed micelles demonstrated greater efficacy as a drug delivery system. The development of these novel mixed micelles is valuable for resolving the poor solubility and bioavailability of drugs.

Preparation, characterization, and pharmacokinetics study of a novel genistein-loaded mixed micelles system.

Genistein (GEN), is a natural dietary isoflavone, has been reported to show anticancer activities. However, its poor aqueous solubility and oral bioavailability limit its clinical application. We designed a novel genistein-loaded mixed micelles (GEN-M) system composed of Soluplus® and Vitamin E d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) were prepared by organic solvent evaporation aimed to overcome the challenges of GEN's poor solubility and then further improve its oral bioavailability. The optimized, spherical-shaped GEN-M was obtained at a ratio of 10:1 (Soluplus®:TPGS). The mean particle size of GEN-M was 184.7 ± 2.8 nm, with a narrow polydispersity index (PDI) of 0.162 ± 0.002. The zeta potential value of GEN-M was -2.92 ± 0.01 mV. The micelles solutions was transparent with blue opalescence has high the entrapment efficiency (EE) and drug loading (DL) of 97.12 ± 2.11 and 3.87 ± 1.26%, respectively. GEN-M was demonstrated a sustained release behavior when formed micelles shown in drug release in vitro. The solubility of GEN in water increased to 1.53 ± 0.04 mg/mL after encapsulation. The permeability of GEN across a Caco-2 cell monolayer was enhanced, and the pharmacokinetics study of GEN-M showed a 2.42-fold increase in relative oral bioavailability compared with free GEN. Based on these findings, we conclude that this novel nanomicelles drug delivery system could be leveraged to deliver GEN and other hydrophobic drugs.

Enhancement of oral bioavailability of magnolol by encapsulation in mixed micelles containing pluronic F127 and L61.

OBJECTIVES: We aimed to prepare novel magnolol-loaded mixed micelles (MAG-M) by pluronic F127 and L61 to overcome the challenges of magnolol's poor solubility and then further improve its oral bioavailability. METHODS: Magnolol-loaded mixed micelles containing pluronic F127 and L61 were prepared by an organic solvent evaporation method. Physicochemical, transport experiment across Caco-2 cell monolayers and pharmacokinetic studies were performed to characterize MAG-M and to determine the final improvement of the oral bioavailability. KEY FINDINGS: The MAG-M solution was transparent and colourless with average size, polydispersity index and zeta potential of 228.0 ± 2.1 nm, 0.298 ± 0.012 and -0.89 ± 0.02 mV. The micelle solution has a higher EE% and DL% of 81.57 ± 1.49% and 27.58 ± 0.53%, respectively. TEM result showed that the morphology of MAG-M was homogeneous and spherical shape. The dilution stability of MAG-M was no significant change in particle size and entrapment efficiency. MAG was demonstrated a sustained-release behaviour after encapsulated in micelles. MAG permeability across a Caco-2 cell monolayer was enhanced, and the pharmacokinetics study of MAG-M showed a 2.83-fold increase in relative oral bioavailability compared with raw MAG. CONCLUSIONS: The mixed micelles containing pluronic F127 and L61 as drug delivery system provided a well strategy for resolving the poor solubility and bioavailability problems of MAG.