Application of combination of nano-silver and NRF2 inducer in treatment of multiple myeloma

Abstract

The invention provides application of combination of nano-silver and an NRF2 inducer in treatment of multiple myeloma. Specifically, nano-silver and an NRF2 inducer are combined for application, the effect of combination of the nano-silver and the NRF2 inducer on multiple myeloma is discussed in combination with in-vivo and in-vitro experiments, and it is found that the application of the NRF2 inducer promotes the inhibiting effect of the nano-silver on the multiple myeloma. The combined application of the nano-silver and the dimethyl fumarate can significantly increase the expression level of ROS in cells, so that the multiple myeloma cells are in an over-oxidative stress state to promote cell apoptosis, which may provide a new choice for the treatment of multiple myeloma in the future.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the application of nanosilver combined with NRF2 inducer in treating multiple myeloma. Background Art

[0002] Multiple Myeloma (MM) is a malignant tumor caused by the unlimited clonal proliferation of plasma cells that produce M protein in the bone marrow. It is the second most common hematological malignancy in the world and can cause hematopoietic dysfunction. Multiple myeloma patients show symptoms such as anemia, osteolytic lesions, hypercalcemia, abnormal renal function, and recurrent infections.

[0003] With the continuous research on the treatment of multiple myeloma, the emergence and application of new drugs and emerging treatment methods have significantly improved the cure rate and prognosis of multiple myeloma. Today, the first-line drugs for the treatment of multiple myeloma include bortezomib, lenalidomide, etc. Treatment methods include autologous stem cell transplantation, targeted therapy, surgery, etc. However, multiple myeloma still has the risk of recurrence, cannot be completely cured, and may even develop drug resistance, resulting in reduced efficacy and causing a series of toxic side effects. Therefore, there is an urgent need to find new drugs and treatments to improve the treatment effect and improve the quality of life of patients. Summary of the invention

[0004] The technical problem to be solved by the present invention is to propose a new method for treating multiple myeloma, which combines nanosilver and dimethyl fumarate, and combines in vivo and in vitro experiments to explore the effect of the combination of nanosilver and NRF2 inducers (especially dimethyl fumarate) on multiple myeloma, aiming to provide new ideas for the treatment of multiple myeloma.

[0005] Nanosilver (AgNPs) refers to single silver particles with a diameter of 1-100nm prepared by certain chemical routes or physical means. Due to its unique physical and chemical properties, it is widely used in many fields. Among them, it is used for antibacterial and treatment of breast cancer, cervical cancer, ovarian cancer, and lung cancer in the medical field. Relatively smaller AgNPs usually have stronger cell penetration and higher cytotoxicity. However, in general, nanoparticles with a size of 10-100 nanometers are considered to be the best choice for cancer treatment, because particles of this size can penetrate the tumor vasculature (EPR effect) but not the normal vasculature, thereby avoiding rapid renal clearance.

[0006] Dimethyl fumarate (DMF) is a member of the fumarate family and is an α, β unsaturated carboxylic acid ester. It is an inducer of the antioxidant group Nrf2, which can activate the Nrf2 signaling pathway and enhance the antioxidant capacity of cells. Dimethyl fumarate has been clinically approved for the treatment of multiple sclerosis (MS) and can also be used to treat psoriasis vulgaris (PSO).

[0007] In the present invention, through in vitro experiments, we tested several human myeloma cell lines with 1.9nm nanosilver, and then detected and evaluated its toxicity by CCK-8 kit and flow cytometry, and used DCFH-DA dye to detect its reactive oxygen species (ROS) level. And analyzed the nuclear factor E2-related factor 2 (Nuclear factor (erythroid-derived 2)-like 2, NRF2) protein level and its downstream gene expression. Subsequently, RNA interference silencing technology and pharmacological inducers were used to study the functional role of NRF2 in the process of nanosilver acting on MM cells. Finally, we tested the effect of nanosilver and NRF2 inducer dimethylfumarate (DMF) alone or in combination on the in vivo growth of MM cells in xenograft tumor model mice.

[0008] Results: We found that low concentrations of nanosilver can cause significant toxicity to MM cells, and their basal ROS levels are significantly higher than those of various other cancer cells. Nanosilver treatment further aggravated the oxidative damage caused by ROS and led to the accumulation of NRF2 protein in MM cells. In addition, we found that DMF aggravated the toxicity induced by nanosilver on MM cells both in vitro and in vivo.

[0009] Subsequently, verification experiments were carried out using 5nm nanosilver and 30nm nanosilver, and similar results were obtained.

[0010] Conclusion: Activation of NRF2 activity may aggravate the cytotoxicity of nanosilver in MM. The combined use of nanosilver and DMF provides a new strategy for the treatment of MM. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is the particle size distribution curve of 1.9nm nanosilver.

[0012] Figure 2 This is a microscope photo of 5nm nanosilver.

[0013] Figure 3 This is the particle size distribution curve of 5nm nanosilver.

[0014] Figure 4 This is a microscope photo of 30nm nanosilver.

[0015] Figure 5 This is the particle size distribution curve of 30nm nanosilver.

[0016] Figure 6 Figure 1 shows the cytotoxicity of MM cells induced by low concentration of 1.9 nanosilver; (A): Cell survival rate of MM cell lines U266, MM1S, RPMI8226, ARP-1 and H929 after treatment with nanosilver (1-4ug / ml) for 24h; (B): Cell survival rate of A549, HepG2, MG63 and GC7901 after treatment with nanosilver (1-4ug / ml) for 24h; n=4 in each group. (C) The percentage of apoptotic MM1S and U266 cells after treatment with nanosilver (1μg / mL and 2μg / mL, respectively) for 24h was determined by flow cytometry. (D) Representative morphology of U266 and MM1S cells treated with nanosilver for 12h. Arrows indicate morphological damage. Scale bar: 50μm. Data represent mean ± standard deviation. **P<0.01, ***P<0.001.

[0017] Figure 7 Figure 1 shows the 1.9 nm silver nanoparticles-induced MM cell death mediated by ROS activation; (A) The production of intracellular ROS was measured using the DCFH-DA probe. The ROS levels in MM cells (ARP-1, MM1S, and U266) and other cancer cells (MG63, A549, and HepG2) were observed under a fluorescence microscope. Scale bar: 50 μm; (B) Relative ROS levels in MM1S cells and U266 cells after 6 hours of silver nanoparticle treatment; Cell viability (C) and apoptosis (D) were evaluated after MM1S and U266 cells were treated with silver nanoparticles in the presence or absence of NAC (2 mM); (E) Representative morphologies of U266 and MM1S cells treated with silver nanoparticles in the presence or absence of NAC (2 mM), and arrows indicate morphological changes. Scale bar: 50 μm**P<0.01, ***P<0.001.

[0018] Figure 8 Figure 1 shows the cytotoxicity of MM cells induced by 1.9 nm silver nanoparticles mediated by NRF2 accumulation; (A) Western blot analysis of NRF2 accumulation in U266 cells treated with silver nanoparticles for 6 hours. (B) qPCR analysis of mRNA expression of NRF2 target genes HO-1 and NQO-1 in U266 cells exposed to silver nanoparticles for 6 hours.

[0019] Fig. 9NRF2 activation promotes 1.9 nm nanosilver-induced cytotoxicity in MM cells; (AC) Cell viability and apoptosis rate of MM cells (MM1S, U266, and MCC1) or other cancer cells (A549 and HepG2) were measured in the indicated cells treated with nanosilver with or without DMF (20 μM) (C). (D) Cell survival was assessed in MM1S and U266 cells treated with nanosilver with or without NK-252 (2 μM). (E) Relative intracellular ROS levels after 6 h of nanosilver treatment of U266 and MM1S cells with or without DMF (20 μM). Data represent mean ± SD. n = 4 per group. ns, not significant, **P < 0.01, ***P < 0.001.

[0020] Fig.10 Figure 1 shows the synergistic inhibition of MM growth by 1.9 nm nanosilver and DMF in a xenograft model; (A) Tumor volume of human U266MM xenografts in mice treated twice weekly with control solvent vehicle, nanosilver (5 mg / kg body weight) and / or DMF (10 mg / kg body weight) for 11 days. (BC) Representative images (B) and weight quantification (C) of tumor xenografts. (D) Kaplan-Meier survival analysis of mice treated with control solvent vehicle, nanosilver, and nanosilver + DMF. Statistical significance was calculated using the log-rank test. n = 8 per group. (E) Hematological analysis of mice treated with control solvent vehicle, nanosilver, and nanosilver + DMF. Data represent mean ± SD. n = 8 per group. ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001.

[0021] Fig.11 This is a graph showing the effect of 5nm nanosilver and 5nm nanosilver Ag+DMF on the proliferation of U266 cells detected by CCK-8.

[0022] Fig.12 This is a graph showing the effect of 30nm nanosilver and 30nm nanosilver Ag+DMF on the proliferation of U266 cells detected by CCK-8.

[0023] Fig.13 This is a graph showing the expression of ROS in U266 multiple myeloma cells after treatment with DMF, nanosilver, and nanosilver + DMF.

[0024] Fig.14 These are the results of HE staining of liver, kidney and spleen sections. DETAILED DESCRIPTION

[0025] In order to allow those skilled in the art to understand the present invention more clearly and intuitively, the present invention will be further described below in conjunction with the accompanying drawings.

[0026] Experimental animals

[0027] 40 5-week-old newborn severely immunodeficient mice were purchased from Yuslake Jingda Biological Co., Ltd. The experimental mice were kept in the animal room of Changsha Medical College, and the environment was disinfected regularly during the experiment. The bedding and feed used in the experiment were all treated with ultraviolet disinfection, and the drinking water was single distilled water. The mice were able to drink water and eat freely.

[0028] Cell culture

[0029] Human MM cell lines MM1S (provided by the laboratory of Central South University), H929, ARP-1 (provided by the laboratory of Central South University), RPMI8226 (CL-0546, Procell) and U266 (CL-0510, Procell) were cultured in RPMI-1640 medium (01-100-1A, Biological Industries) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin antibiotics (P / S).

[0030] Sources

[0031] 1.9nm nanosilver is retained in the laboratory. The particle size distribution of 1.9nm nanosilver is shown in the figure below. Figure 1 shown.

[0032] 5nm and 30nm nanosilver were purchased from Beijing Biotech Biotechnology Co., Ltd. The electron microscope image and particle size distribution diagram of 5nm nanosilver are shown in Figure 2 and Figure 3 As shown; the electron microscope image and particle size distribution diagram of 30nm nanosilver are shown as Figure 4 and Figure 5 shown.

[0033] N-acetyl-L-cysteine ​​(NAC; A9165) was purchased from Sigma, and dimethyl fumarate (DMF; HY-17363) and NK252 (HY-19734) were purchased from MedChemExpress.

[0034] Preparation of dimethyl fumarate

[0035] Weigh an appropriate amount of dimethyl fumarate drug powder, and fully dissolve the drug powder with an appropriate amount of DMSO to make the concentration of the mother solution 200 mMol, and store it in a refrigerator at -80°C.

[0036] Cell viability assay

[0037] Cell viability was assessed using a cell counting kit-8 (CCK-8; C008-3, 7Sea Biotech) according to the manufacturer's instructions. 4 Cells / well were seeded into 96-well culture plates and treated with different concentrations of nanosilver and / or other compounds for 24 h.

[0038] Apoptosis assay

[0039] MM cells were treated with a series of concentrations of nanosilver and / or other compounds for 24 h, and then apoptosis was detected using the Annexin V-FITC / PI kit (40306ES60, Yeasen Biotech) according to the manufacturer's instructions. Apoptotic cells were analyzed using flow cytometry.

[0040] Measurement of ROS generation

[0041] Intracellular ROS production was assessed using a DCFH-DA probe (D6883, Sigma). Cells were treated with 0-4 μg / mL of nanosilver and / or other compounds for 6 hours, then washed three times with PBS and incubated with 10 μM DCFH-DA at 37°C for 20 minutes. Afterwards, cells were washed twice with PBS and analyzed by fluorescence microscopy (Apotome, Zeiss) or microplate reader (Varioskan LUX, Thermo Fisher Scientific).

[0042] Western blot analysis

[0043] MM cells were treated with 0-4 μg / mL of nanosilver for 6 hours, then collected and lysed in RIPA lysis buffer (CW2333S, CWBio) for 30 minutes. Protein concentration was determined using the BCA protein assay (P0012, Beyotime). Equal amounts of protein were separated by SDS-PAGE and transferred to PVDF membranes. The membrane was blocked with 5% skim milk and incubated with primary antibodies: anti-β-Actin (#3700, Cell Signaling Technology) and anti-NRF2 (A11159, ABclonal). HRP-conjugated secondary antibodies were used for detection, and the bands were visualized by the ECL detection system (Bio-Rad).

[0044] Quantitative RT-PCR

[0045] Total RNA was extracted using TRIzol reagent (Invitrogen) and cDNA was synthesized using a reverse transcription kit (Takara). The sequences of the primers used are shown in Table 1. 18S RNA was used as a reference for mRNA normalization. qRT-PCR was performed on an ABI 7500 real-time PCR system using SYBR Green PCR Master Mix (Applied Biosystems).

[0046] Table 1List of primer sequences.

[0047]

[0048] Animals and handling

[0049] This study was approved by the Ethics Committee of Changsha Medical College (approval number: 2021050) and was performed in accordance with institutional guidelines. Five-week-old male NOD / SCID mice (SJA Laboratory) were used in this study. Approximately 2 × 10 6 U266 cells were used to establish tumor xenograft models. Tumor volume was calculated as volume (mm 3 )=(length×width 2 ) / 2. When the tumor volume reaches about 100mm 3 At 4 hr, mice were randomly assigned to different treatment groups (n=8 per group). Treatment included tail vein injection of vehicle, nanosilver (5 mg / kg body weight) and / or DMF (10 mg / kg body weight) twice a week. Mice were sacrificed when the largest tumor reached 2 cm.

[0050] Histology and immunohistochemical staining

[0051] After the mice were anesthetized, the tissues were dissected and fixed in 4% formaldehyde. The samples were processed for paraffin embedding and cut into 5-10 μm thick sections. The sections of each group were stained with hematoxylin and eosin (H&E) for histological analysis. Immunohistochemistry (IHC) staining was performed using anti-NRF2 antibody (A11159, 1:400, ABclonal) to detect NRF2 accumulation in tumor tissues.

[0052] Statistical analysis

[0053] Data are presented as mean ± SD and analyzed using GraphPad Prism 10. Statistical significance was determined using unpaired two-tailed Student t test or one-way analysis of variance (ANOVA). Mouse survival was measured using the Kaplan-Meier method, and groups were compared using the log-rank test. P values ​​< 0.05 were considered statistically significant.

[0054] result

[0055] Low concentrations of 1.9 nm silver nanoparticles induce cytotoxicity in multiple myeloma cells

[0056] We performed cell viability assays on five human MM cell lines (MM1S, RPMI8226, ARP-1, U266, and H929) using the Cell Counting Kit-8 (CCK-8). Cells were treated with different concentrations of 1.9 nm silver nanoparticles (1 μg / mL). Our results showed that in all tested MM cell lines, cell viability decreased in a dose-dependent manner ( Figure 6 A). In particular, at a concentration of 4 μg / mL, nanosilver induced significant cytotoxicity, reducing the cell viability of the ARP-1 cell line by up to 80% ( Figure 6 A). In contrast, the same concentration of nanosilver did not significantly affect the viability of other cancer cell lines, including A549 (lung cancer), HepG2 (liver cancer), MG63 (osteosarcoma), and GC7901 (gastric cancer) ( Figure 6 B). This suggests that MM cells have a higher sensitivity to AgNPs, possibly due to differences in the levels of intrinsic oxidative stress.

[0057] To further elucidate the mechanism of silver nanoparticle-induced cytotoxicity on MM cells, we used Annexin V / PI staining and flow cytometry to evaluate the apoptosis of MM1S and U266 cells. After treatment with 2 μg / mL silver nanoparticles, apoptosis of both cell lines increased significantly ( Figure 6 C). In MM1S cells, the proportion of early apoptotic cells (AnnexinV+ / PI-) increased from 14.1% in the control group to 33.7%, and the proportion of late apoptotic cells (AnnexinV+ / PI+) increased from 10.1% to 47.9% ( Figure 6 C). Similarly, in U266 cells, the proportion of early apoptotic cells increased from 7.65% to 38.5%, and the proportion of late apoptotic cells increased from 3.75% to 25.6% ( Figure 6 C). Microscopic observation showed that after 12 hours of nanosilver treatment, MM cells showed obvious morphological damage, including cell shrinkage and membrane blebbing ( Figure 6 D).

[0058] These results collectively indicate that low concentrations of nanosilver induce significant cytotoxic effects on MM cells, primarily through induction of apoptosis. The enhanced sensitivity of MM cells to nanosilver highlights its potential as a targeted therapeutic strategy, which warrants further investigation of its underlying mechanisms and possible clinical applications.

[0059] ROS activation-mediated 1.9 nm silver nanoparticles induce MM cell death

[0060] We first evaluated the basal ROS levels of various cancer cell lines using DCFH-DA dye. Our results showed that the baseline ROS levels of MM cells (ARP-1, MM1S, and U266) were higher than those of other cancer cells (MG63, A549, and HepG2) ( Figure 7 A). To further explore the role of ROS in nanosilver-induced cytotoxicity, we used a multifunctional microplate reader to measure changes in intracellular ROS levels after MM cell treatment. Nanosilver significantly increased the ROS levels of MM1S and U266 cells ( Figure 7 B). This increase in ROS levels completely disappeared after the addition of N-acetyl-l-cysteine ​​(NAC), a potent ROS scavenger ( Figure 7 B).

[0061] Next, we studied the protective effect of NAC on the cytotoxicity induced by nanosilver. MM cells were pretreated with NAC before nanosilver treatment. CCK-8 experiments showed that NAC pretreatment rescued most MM cells from the cytotoxic effects of nanosilver, as shown by a significant increase in cell survival ( Figure 7 C). Flow cytometry analysis by Annexin V / PI staining showed that the level of apoptosis was significantly reduced ( Figure 7 D). In particular, in MM1S cells, late apoptosis was reduced from 63.8% to 27.6%, and in U266 cells, late apoptosis was reduced from 20.7% to 11.4% ( Figure 7 D). In addition, microscopic morphological observations showed that NAC pretreatment alleviated the morphological damage induced by nanosilver. Compared with cells treated with nanosilver alone, MM cells treated with NAC+nanosilver showed less cell shrinkage and membrane blebbing ( Figure 7 E).

[0062] These findings suggest that ROS play a key role in AgNP-induced MM cytotoxicity. The protective effect of NAC highlights the importance of oxidative stress in mediating the cytotoxic effects of AgNPs.

[0063] NRF2 accumulation mediates 1.9 nm silver nanoparticle-induced MM cytotoxicity

[0064] To elucidate the potential mechanism of nanosilver-induced MM cell toxicity, we investigated the role of nuclear factor E2-related factor 2 (NRF2), a key regulator of oxidative stress response. NRF2 activates the transcription of multiple antioxidant enzymes to alleviate stress-related toxicity. We hypothesized that NRF2 may play a key role in the toxic effects of nanosilver on MM cells.

[0065] In preliminary studies, we detected the level of NRF2 protein in multiple myeloma (MM) cells after treatment with nanosilver. Western blot analysis showed that nanosilver treatment led to a dose-dependent accumulation of NRF2 in U266 cells (see Figure 8 A). This accumulation indicated that NRF2 was activated in response to nanosilver-induced oxidative stress. Next, we evaluated the expression levels of NRF2 downstream target genes, particularly heme oxygenase-1 (HO-1) and NADPH quinone oxidoreductase-1 (NQO-1). Quantitative polymerase chain reaction (qPCR) analysis revealed that HO-1 and NQO-1 mRNA levels were significantly upregulated in nanosilver-treated U266 cells, indicating that the activation of NRF2 led to an enhanced antioxidant response (see Figure 8 B).

[0066] These results indicate that the increased cytotoxicity observed in NRF2-deficient cells highlights the importance of NRF2 in regulating the cellular response to silver nanoparticles. This suggests that by targeting the NRF2 pathway, it may be possible to enhance the therapeutic efficacy of silver nanoparticles, thereby providing a more effective treatment option for multiple myeloma cells.

[0067] NRF2 activation enhances the cytotoxicity of 1.9 nm silver nanoparticles in MM cells

[0068] To further investigate the role of NRF2 in mediating the cytotoxic effects of AgNPs, we aimed to activate NRF2 and evaluate its effect on the sensitivity of MM cells to AgNPs. Dimethyl fumarate (DMF), a known NRF2 activator approved by the FDA for the treatment of multiple sclerosis, was used to enhance the expression of NRF2 in MM cells.

[0069] We treated MM1S and U266 cells with DMF and evaluated the combined effects of DMF and nanosilver on cell viability. Contrary to expectations, cell viability experiments showed that the addition of DMF significantly increased the sensitivity of MM cells to nanosilver-induced cytotoxicity ( Fig. 9 A). In contrast, DMF did not enhance the cytotoxicity of AgNPs in A549 (lung cancer) or HepG2 (liver cancer) cells ( Fig. 9 B), showing a selective effect in MM cells.

[0070] To quantify the extent of apoptosis induced by the combined treatment, we performed flow cytometric analysis on U266 cells. The results showed that late apoptosis was significantly increased when cells were co-treated with nanosilver and DMF compared to treatment with nanosilver alone ( Fig. 9 C). Specifically, after adding DMF, the percentage of late apoptotic cells increased from 11.9% to 29.9% ( Fig. 9C). To confirm these findings, we also treated MM cells with another potent NRF2 inducer, NK-252, and the results showed that, similar to DMF, NK-252 also enhanced the cytotoxic effect of nanosilver in MM cells, further validating the role of NRF2 activation in enhancing nanosilver-induced cell death ( Fig. 9 D).

[0071] Furthermore, by measuring the fluorescence increase intensity using the DCFH-DA probe, we observed that DMF treatment promoted the generation of ROS in Ag-treated MM cells ( Fig. 9 E). This suggests that the enhanced cytotoxicity following NRF2 activation is associated with increased oxidative stress.

[0072] Together, these results suggest that NRF2 activation amplifies the cytotoxic effects of silver nanoparticles in myeloma cells. Therefore, the selective sensitization of MM cells to silver nanoparticles by NRF2 activators highlights a potential therapeutic strategy to combine NRF2 activation with silver nanoparticle therapy to effectively target MM.

[0073] 1.9 nm silver nanoparticles and DMF synergistically inhibit MM growth in a xenograft model

[0074] To further explore the efficacy of DMF and nanosilver combination therapy, we evaluated their effects in vivo using a human myeloma xenograft mouse model. U266 cells were transplanted under the abdominal skin of NOD / SCID mice (18-22 grams). After the xenograft model was established, the mice were treated with a control solvent (Vehicle), nanosilver alone, DMF alone, or a combination of the two.

[0075] Treatment with nanosilver alone or in combination with DMF significantly reduced tumor growth ( Fig.10 AB) and promoted mouse survival ( Fig.10 C). The combination of nanosilver and DMF synergistically reduced tumor burden, whereas DMF alone failed to inhibit tumor growth ( Fig.10 AB). In addition, we observed increased accumulation of NRF2 in the single or combined treatment groups ( Fig.10 D), while no accumulation of NRF2 was detected in control mice. DMF supplementation significantly increased Nrf2 protein levels. We then tested the toxic effects of nanosilver and DMF treatment on the liver, kidneys, and other major organs of mice.

[0076] We also evaluated the potential toxicity of treatment on major organs, including the liver, kidneys, and spleen, using hematoxylin-eosin (HE) staining. Fig.14HE staining results showed no obvious toxic effects on the liver, kidneys and spleen. Hematological parameter results also showed no abnormalities after nanosilver and DMF treatment ( Fig.10 E). These results suggest that nanosilver alone or in combination with DMF can effectively inhibit the growth of MM cells in vivo without causing obvious toxic effects.

[0077] These in vivo results suggest that silver nanoparticles, especially when combined with DMF, can effectively inhibit myeloma tumor growth without significant toxicity to the host. The enhanced antitumor activity observed with activation of NRF2 using DMF suggests a promising therapeutic strategy for multiple myeloma.

[0078] In this paper, we demonstrated for the first time that 1.9nm AgNPS can inhibit the growth of MM cells in vitro and in vivo and induce apoptosis. 1.9nm AgNPS can be toxic to MM cells at very low concentrations by inducing oxidative stress. In addition, we found that the NRF2 inducers DMF and NK-252 can aggravate the cytotoxicity of MM cells induced by 1.9nm AgNPS.

[0079] In the study, we found that myeloma cells were particularly sensitive to 1.9nm AgNPS and had higher basal levels of ROS compared to other cancer cells. Myeloma cells are derived from plasma cells and have the unique ability to synthesize and secrete antibodies. Due to the metabolic demands of malignant proliferation, myeloma cells have higher protein synthesis rates and ROS levels. Moderate elevations in ROS may be beneficial for cell survival and cell growth signaling, while overproduction of ROS is thought to inhibit tumor cell proliferation.

[0080] Cancer cells can use multiple mechanisms to combat oxidative stress and eliminate excess ROS. In our study, NRF2 knockdown made MM cells more sensitive to silver nanoparticles. However, the addition of DMF and NK-252 (a strong inducer of NRF2) did not reduce but increased the sensitivity of MM cells to AgNPS. DMF exacerbated the increase of ROS in AgNPS-treated MM cells.

[0081] Finally, we tested the inhibitory effect of AgNPS on myeloma cell growth in vivo. At the same time, we found that DMF, an FDA-approved drug for the treatment of multiple sclerosis and psoriasis, can synergize with AgNPS to inhibit myeloma growth in xenograft models. Existing studies have confirmed that DMF can inhibit the growth of cancer cells in vitro at higher concentrations (50–200 μM), but is not toxic to cancer cells at low concentrations (<20 μM), which is consistent with our results. However, low levels of DMF are able to amplify the toxicity of AgNPS in MM cells. Therefore, when used in combination with DMF in in vivo experiments, this strategy allows us to reduce the concentration of AgNPS to a relatively safe range. In fact, in recent years, the toxic side effects of nanosilver particles on human health have attracted increasing attention. This combined approach will help overcome some of the limitations of silver particles in clinical applications and reopen the door to silver particles for cancer treatment.

[0082] Validation test of AgNPs with different particle sizes

[0083] Combination of 5nm and 30nm nanosilver and dimethyl fumarate further inhibits the proliferation of multiple myeloma cells

[0084] In order to study the effect of the combined use of nanosilver and dimethyl fumarate on the cell proliferation rate, we selected U266 myeloma cells in good growth state, added 100ul to each well of a 96-well plate, and added the drug. A blank group, a nanosilver group (two different particle sizes of nanosilver were selected, 5nm and 30nm, with concentrations of 0μg / ml, 2μg / ml, and 4μg / ml), and a nanosilver + dimethyl fumarate group (the nanosilver concentration was the same as above, and the dimethyl fumarate concentration was 20μmol) were set up. After 24 hours of incubation, the absorbance (OD value) of CCK-8 at a wavelength of 450nm was used to detect the proliferation of U266 myeloma cells. The experimental results are as follows: Fig.11 and Fig.12 As shown, the combination of 5nm nanosilver and dimethyl fumarate has a stronger inhibitory effect on multiple myeloma cells than nanosilver alone.

[0085] The combined use of 5nm nanosilver and dimethyl fumarate will increase the level of ROS

[0086] In order to study the effect of nanosilver and dimethyl fumarate on ROS in multiple myeloma cells, this experiment used the DCFH-DA fluorescent probe method to label U266 myeloma cells and detected the fluorescence intensity in an ELISA instrument. We set up a blank group, a dimethyl fumarate group, a nanosilver group, and a nanosilver plus dimethyl fumarate group. Fig.13As shown in the experiment, it was found that the ROS level in the cells after the combination of the two drugs was higher than that of the single drug group. That is, the combined use of nanosilver + dimethyl fumarate can induce an increase in ROS levels and promote the apoptosis of U266 myeloma cells.

[0087] Nanosilver increases the level of ROS in cells, which, combined with the high background level of ROS in multiple myeloma cells, provides an environment of excessive ROS. Dimethyl fumarate (DMF) is an agonist of Nrf2. Under conditions of excessive ROS, the continuous activation of Nrf2 leads to its accumulation in the nucleus and subsequent binding to the Klf9 promoter. DMF further increases ROS through Klf9, leading to the death of tumor cells.

[0088] The above description of the embodiments is to facilitate the understanding and application of the present invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the embodiments herein, and improvements and modifications made to the present invention by those skilled in the art based on the disclosure of the present invention should be within the scope of protection of the present invention.

Claims

1. Application of nanosilver combined with NRF2 inducer in the treatment of multiple myeloma.

2. The use according to claim 1, characterized in that: The NRF2 inducer is selected from DMF and / or NK-252.

3. The use according to claim 1 or 2, characterized in that: The average particle size of the nano silver is 1 to 100 nm.

4. The use according to claim 1 or 2, characterized in that: The average particle size of the nano silver is 1.9-30 nm.

5. The use according to claim 1 or 2, characterized in that: The average particle size of the nano silver is 1.9-5 nm.

6. The use according to claim 1 or 2, characterized in that: The concentration of the nanosilver is 2-4 ug / mL.

7. The use according to claim 2, characterized in that: The NRF2 inducer is DMF.

8. The use according to claim 7, characterized in that: The concentration of DMF was 20 μM.

9. A drug for treating multiple myeloma, characterized in that: Including NRF2 inducers and silver nanoparticles.

10. The drug according to claim 9, characterized in that The NRF2 inducer is selected from DMF and / or NK-252, preferably DMF; The average particle size of the nanosilver is 1.9 nm to 30 nm, preferably 1.9 nm to 5 nm, and more preferably 1.9 nm.

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