Method for treating multiple myeloma in subcutaneous and in-situ tumor models through C3G

Abstract

The invention provides a method for treating multiple myeloma in subcutaneous and in-situ tumor models by using C3G. The method comprises the following steps: S1, testing the effect of C3G on tumor growth of an MM model mouse; s2, testing the effect of C3G on the activity, cell cycle and cell proliferation of an MM cell line; s3, testing the effect of C3G on MM cell apoptosis; s4, testing the effect of C3G on NAD < + > secretion and MM cell apoptosis; s5, the effect of C3G on Sirt1 expression and NF-kB acetylation of the MM cells is tested; s6, proving an acetylation regulation mechanism of the C3G to the NF-kappa B; the natural compound C3G is used as a novel therapy for clinically treating multiple myeloma, the C3G has the advantages of low cost, no side effect and the like, and researches prove that the C3G shows a remarkable inhibition effect on MM cells, which means that the C3G has clinical feasibility in the aspect of treating MM, and when the C3G is combined with a CD38 monoclonal antibody, such as dartumumab, not only is the curative effect of the monoclonal antibody not affected, but also the curative effect of the CD38 monoclonal antibody is not affected, and the C3G has a remarkable inhibition effect on the MM cells. And on the contrary, a synergistic effect is possibly generated, and the effect of treating the MM is remarkably enhanced.

Description

Technical Field

[0001] The present invention relates to a method for treating multiple myeloma, in particular to a method for treating multiple myeloma in subcutaneous and in situ tumor models using C3G, belonging to the technical field of clinical treatment of multiple myeloma. Background Art

[0002] Multiple myeloma (MM) is a malignant plasma cell tumor. Its histopathological manifestations are malignant proliferation and extensive infiltration of monoclonal plasma cells in the bone marrow, which inhibit the proliferation of polyclonal plasma cells and the secretion of normal immunoglobulins. A model-based estimate of cancer incidence and mortality shows that although advances in various treatments have improved the survival rate of MM patients, the incidence of MM ranks second only to non-Hodgkin's lymphoma among hematological malignancies.

[0003] Currently, monoclonal antibodies (mabs) targeting CD38 have become one of the important means of treating multiple myeloma (MM). They exert anti-tumor effects through various mechanisms, such as Fc-dependent immune effects, direct induction of apoptosis, inhibition of CD38 enzyme activity, and reduction of CD38+ immunosuppressive cells. Although anti-CD38 mabs have achieved significant clinical efficacy, they still face problems such as lymphopenia, increased risk of infection, and high treatment costs during use. Therefore, a C3G treatment method for multiple myeloma in subcutaneous and orthotopic tumor models is proposed. Summary of the invention

[0004] In view of this, the present invention provides a C3G treatment method for multiple myeloma in subcutaneous and orthotopic tumor models, using the naturally occurring compound C3G as a new therapy for the clinical treatment of multiple myeloma to reduce treatment costs, eliminate treatment side effects, and at least provide a beneficial option.

[0005] The technical solution of the present invention is implemented as follows: A C3G method for treating multiple myeloma in subcutaneous and orthotopic tumor models, comprising:

[0006] S1. Test the effect of C3G on tumor growth in MM model mice;

[0007] S2. Test the effect of C3G on the activity, cell cycle and cell proliferation of MM cell lines;

[0008] S3. Test the effect of C3G on MM cell apoptosis;

[0009] S4. Test the effect of C3G on NAD+ secretion and MM cell apoptosis;

[0010] S5. Test the effect of C3G on Sirt1 expression and NF-κB acetylation in MM cells;

[0011] S6. Prove the regulatory mechanism of C3G on NF-κB acetylation.

[0012] Further preferably, in S1, an SP2 / 0-luc cell line is established and injected subcutaneously into BALB / c mice to establish a subcutaneous tumor-bearing mouse model, the tumor size is measured with a vernier caliper every other day, and the volume is calculated using the formula V=long diameter×(short diameter)2 / 2.

[0013] Further preferably, in S1, the tumors of subcutaneous MM tumor model mice are stained and observed by histochemical method, and the MM orthotopic transplant tumor model is established by tail vein injection of SP2 / 0-luc cells, and in vivo imaging is performed every week.

[0014] Further preferably, in S2, the effective concentration range of C3G is determined, PBMCs from healthy volunteers are isolated and seeded in 96-well plates, treated with C3G at concentrations of 0 mM, 50 mM, 100 mM, 200 mM, 400 mM and 600 mM for 48 hours, and CCK-8 assay is used to evaluate cell viability.

[0015] Further preferably, in S2, the effect of C3G on the cell cycle of MM cells is evaluated. Before flow cytometry analysis, the cells are treated with 400 mM C3G for 48 hours, followed by flow cytometry analysis, and the SP2 / 0 colony formation images are processed using ImageJ software to quantitatively analyze the area of ​​colony formation.

[0016] Further preferably, in S3, flow cytometry is used to evaluate the effect of C3G on MM cell apoptosis, BMMCs from newly diagnosed or relapsed MM patients are treated with 400 mM C3G for 48 h, PE-labeled anti-human CD138 antibody is used to mark primary malignant plasma cells, incubated with annexin V-FITC staining solution, and cell apoptosis is detected by flow cytometry.

[0017] Further preferably, in S4, after treatment with 400 μM C3G for 48 hours, the expression of CD38 on the surface of MM cells was evaluated by flow cytometry, and the transcription level of CD38 mRNA in three MM cell lines was detected by real-time quantitative PCR, which demonstrated that there was no significant difference between the control group and the C3G group. The effect of C3G on the NAD+ level in MM cells was evaluated by colorimetric analysis. In order to determine whether NAD+ accumulation induces apoptosis in myeloma cells, the cells were treated with exogenous 1 mM NAD+ for 48 hours, and flow cytometry showed that NAD+ could significantly induce apoptosis of myeloma cells.

[0018] Further preferably, in S5, real-time quantitative PCR is used to evaluate the effect of C3G on the expression level of Sirt1 mRNA in MM cell lines, and Western blot is used to analyze the expression of Sirt1 protein in MM cell lines and tumor tissues of subcutaneous tumor-bearing mice.

[0019] Further preferably, in said S5, the MM cell lines were treated with 400 μM C3G for 48 hours, and PBS was used as a control. Western blot analysis was used to evaluate NF-κB protein expression, revealing that C3G had no significant effect on protein expression in the three cell lines. In order to re-determine whether inhibition of NF-κB has a therapeutic effect on MM, MM.1S cells were treated with the NF-κB inhibitor PDTC at 25 μM for 48 hours.

[0020] Further preferably, in the S6, it is evaluated whether inhibition of CD38 increases the expression of Sirt1 and decreases the expression of acetylated NF-κB, CD38+MM.1S are separated from MM.1S cells by magnetic bead sorting, and their purity is verified, and the protein levels of Sirt1, acetylated NF-κB and NF-κB in CD38+MM.1S cells are compared with those of unsorted MM.1S cells as a control to clarify the regulation of Sirt1 on NF-κB acetylation during C3G treatment of MM, and at the same time, the effects of C3G, NAM, C3G intervention after NAM pretreatment, and RSV on MM.1S cell apoptosis are detected.

[0021] The embodiment of the present invention has the following advantages due to the adoption of the above technical solution:

[0022] The present invention utilizes the naturally occurring compound C3G as a novel therapy for the clinical treatment of multiple myeloma. C3G has the advantages of low cost and no side effects, and studies have shown that it exhibits a significant inhibitory effect on MM cells, which means that C3G is clinically feasible in the treatment of MM. The combined use of C3G and CD38 monoclonal antibodies, such as daratumumab, will not only not affect the efficacy of the monoclonal antibodies, but is more likely to produce a synergistic effect, significantly enhancing the effect of MM treatment. The working principle of this combination therapy is that C3G directly inhibits the growth and survival of MM cells. At the same time, CD38 monoclonal antibodies can accurately identify and target CD38 proteins on the surface of MM cells, and effectively kill MM cells through various mechanisms such as activating the killing function of immune cells. The synergistic effect of the two can maximize the control of MM cell proliferation and spread, thereby effectively alleviating the condition of MM patients.

[0023] The above summary is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments and features described above, further aspects, embodiments and features of the present invention will be readily apparent by reference to the accompanying drawings and the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0025] Figure 1 is a flow chart of the method of the present invention;

[0026] Figure 2 This is a graph showing that C3G in the present invention inhibits the growth of subcutaneous MM tumors in mice;

[0027] Figure 3 This is a graph showing that C3G in the present invention inhibits the growth of mouse MM orthotopic tumors;

[0028] Figure 4 This is a graph showing the effect of C3G on the activity, cell cycle and proliferation of MM cell lines in the present invention;

[0029] Figure 5 This is a diagram showing the effect of C3G on apoptosis of multiple myeloma cells in the present invention;

[0030] Figure 6 This is a diagram showing that C3G in the present invention inhibits CD38 enzyme activity, leading to NAD+ accumulation;

[0031] Figure 7 This is a graph showing that C3G increases the expression of Sirt1 in the present invention;

[0032] Figure 8 This is a diagram showing the acetylation effect of C3G in the present invention on inhibiting NF-κB;

[0033] Fig. 9 This is a verification diagram of the CD38 / Sirt1 / NF-κB pathway of C3G in the present invention.

[0034] Figure 2Middle: A is the preparation and intervention plan of subcutaneous tumor-bearing mice, B is the statistical display of the changes in tumor volume of animal models in the control group and C3G treatment group at different observation time points, C is the photographing of tumor-bearing mice at the end of the observation time, D and F are the photographing of subcutaneous transplanted tumors in the control group and C3G group at the end of the observation time, and their weights are statistically analyzed, E and G are the in vivo fluorescence imaging of tumor-bearing mice and the statistical analysis of the number of collected photons, H is the body weights of the healthy control group, the control group and the C3G group were not significantly different, I is the chemical structure of C3G;

[0035] Figure 3 Middle: A is the preparation and intervention scheme of the orthotopic transplantation tumor model, B and C are the in vivo fluorescence imaging of tumor-bearing mice and the statistical analysis of the collected light intensity;

[0036] Figure 4 Middle: A is the effect of C3G on the activity of healthy human peripheral blood mononuclear cells, B is the effect of C3G on the viability of MM cell lines MM.1S, RPMI-8226 and SP2 / 0, CE is the effect of C3G on the cell cycle of MM.1S, RPMI-8226 and SP2 / 0, FH is the effect of C3G on the proliferation of MM.1S, RPMI-8226 and SP2 / 0 cells, I is the colony formation experiment of SP2 / 0;

[0037] Figure 5 Middle: A and B are MM.1S, RPMI8226, and SP2 / 0 cells treated with 400 μM C3G for 48 h, after which the percentage of apoptotic cells was determined by flow cytometry using Annexin V / PI double staining, C and D are representative graphs showing the gating strategy for flow cytometry detection of CD138+ MM cell apoptosis in MM bone marrow samples, and the percentage of Annexin-V expression in the control group and C3G-treated group was statistically analyzed;

[0038] Figure 6 Middle: A is the expression of CD38 on the surface of multiple myeloma cells detected by flow cytometry, B is the mRNA expression level of CD38 in bone marrow cells detected by real-time quantitative PCR, C and D are the changes of NAD+ and NADH in three MM cell lines after treatment with C3G400μm for 48h, E is that exogenous NAD+ can cause MM cell apoptosis;

[0039] Figure 7Middle: A and B are the detection of Sirt1 mRNA expression levels in MM cells or tumor-bearing mice by real-time quantitative PCR, C and D are the detection of Sirt1 protein expression in MM cell lines and tumor tissues of tumor-bearing mice by immunoblotting; β-actin expression was used as an internal reference to standardize the expression for statistical analysis, E and F are representative bands of Sirt1 expression in cells and tissues after PBS treatment and C3G treatment;

[0040] Figure 8 Middle: A is the detection of NF-κB mRNA expression level in MM cells by real-time quantitative PCR, B is the detection of acetylated NF-κB and NF-κB protein expression levels in MM cells by WB, C is the detection of NF-κB mRNA expression level in tumor tissues of tumor-bearing mice by real-time quantitative PCR, D is the detection of acetylated NF-κB and NF-κB protein expression in tumor tissues by Western blotting, and E is the promotion of MM.1S cell apoptosis by NF-κB inhibitor PDTC;

[0041] Fig. 9 Middle: A is the separation of CD38-MM.1S from MetenyiBiotec magnetic beads, and the unsorted MM.1S was used as a control. B is the Western blot detection of the changes in acetylated NF-κB and NF-κB expression in MM.1S cells after different treatments. C is the proportion of apoptotic cells caused by C3G intervention after pretreatment with NAM. DETAILED DESCRIPTION

[0042] In the following, only some exemplary embodiments are briefly described. As those skilled in the art will appreciate, the described embodiments may be modified in various ways without departing from the spirit or scope of the present invention. Therefore, the drawings and descriptions are considered to be exemplary and non-restrictive in nature.

[0043] The embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0044] Embodiment 1

[0045] like Figure 1-5 As shown, the embodiment of the present invention provides a method for treating multiple myeloma in subcutaneous and orthotopic tumor models using C3G, comprising:

[0046] S1. Test the effect of C3G on tumor growth in MM model mice. The SP2 / 0-luc cell line was established and injected subcutaneously into BALB / c mice to establish a subcutaneous tumor-bearing mouse model. The tumor size was measured with a vernier caliper every other day. The volume was calculated using the formula V = long diameter × (short diameter) 2 / 2 and plotted on Figure 2In B, the growth of subcutaneous tumors was significantly inhibited in the C3G-treated group compared with the control group ( Figure 2 From the 8th day after inoculation, a significant difference in subcutaneous tumor volume was observed between the two groups (P<0.001). By the 15th day after inoculation, the tumor volume of mice treated with C3G was significantly lower than that of the control group (1466±146.4mm3 vs. 587.0±113.2mm3, P<0.001, n=5). After the experiment, the mice were euthanized, and the subcutaneous tumors were photographed and weighed ( Figure 2 C, D), compared with the control group, the tumor weight of the C3G-treated group was significantly reduced (881.6±101.9 mg vs. 313.4±54.52 mg, P<0.01 Figure 2 F), using an in vivo luminescence imager to collect bioluminescent signals from tumor cells to quantitatively evaluate tumor growth in a tumor-bearing mouse model with higher precision. The results showed that the light intensity in the C3G group was significantly lower than that in the untreated control group (P<0.01, Figure 2 E, G), no significant differences were observed among the healthy control group, control group, and C3G group (19.6±0.1g, 18.72±0.38g, 19.09±0.54g; Figure 2 H), the chemical structure of C3G is as follows Figure 2 As shown in Figure 1, the tumors of subcutaneous MM tumor model mice were stained and observed by histochemical method. Compared with the C3G treatment group, the control group had more necrosis of tumor cells, nuclear condensation, deep staining, and fragmentation. The necrotic foci were eosinophils, indicating that the tumor cells proliferated vigorously. After C3G treatment, the necrosis of subcutaneous tumor cells decreased. The MM orthotopic transplantation tumor model was established by tail vein injection of SP2 / 0-luc cells. In vivo imaging was performed every week. The results showed that compared with the control group, the tumor growth of the C3G treatment group was significantly inhibited (P<0.05, Figure 3 B, C);

[0047] S2. The effect of C3G on the activity, cell cycle and cell proliferation of MM cell lines was tested. The effective concentration range of C3G was determined. PBMCs from healthy volunteers were isolated and seeded in 96-well plates. They were treated with C3G at concentrations of 0 mM, 50 mM, 100 mM, 200 mM, 400 mM and 600 mM for 48 hours. CCK-8 assay was used to evaluate cell viability. The results showed that the cell viability of healthy PBMCs was not affected when the final concentration of C3G was 400 mM or lower ( Figure 4A), human cell lines MM.1S, RPMI-8226 and mouse cell line SP2 / 0 were also treated with C3G at concentrations of 0 mM, 50 mM, 100 mM, 200 mM and 400 μM. The results showed that treatment with 400 mM C3G for 48 hours significantly reduced the viability of myeloma cell lines compared with the 0 mM group (71.97 ± 1.49%, P < 0.000170.53 ± 0.79%, P < 0.000174.35 ± 2.40%, P < 0.0001n = 3; Figure 4 B), to evaluate the effect of C3G on the cell cycle of MM cells, cells were treated with 400 mM C3G for 48 h before flow cytometry analysis. Figure 4 As shown in CE, compared with the PBS-treated control group, the proportion of the S phase of the cell cycle in MM.1S cells (28.51±2.57% vs. 13.65±0.08%, P<0.001, n=3), RPMI-8226 cells (62.60±0.43% vs. 50.38±1.16%, P<0.001, n=3) and SP2 / 0 cells (65.81±2.34%) was significantly reduced. C3G can inhibit the cell cycle of MM cells. In order to study the effect of C3G on the proliferation of MM cells, MM.1S, RPMI-8226 cells were labeled with CFSE. MI-8226 and SP2 / 0 cells and cultured in the presence of 400 mM C3G for 48 h, followed by flow cytometry analysis, the results showed that the proliferation of tumor cells in the C3G treatment group was inhibited (MM.1S: 26085±609.9 vs 43699±461.4, P<0.001, n=3; RPMI-8226: 15956595.3 vs 22011583.8, P<0.01, n=3; SP2 / 0: 687076.49 vs 836894.41, P<0.001, n=3; Figure 4 FH), among the three cell lines, only SP2 / 0 cells showed adherent growth, and the images of SP2 / 0 colony formation were processed using ImageJ software to quantitatively analyze the area of ​​colony formation. Compared with the PBS-treated control group, the colony area of ​​the C3G group was reduced (4.24±0.07vs3.56±0.05, P<0.01, n=3; Figure 4 I);

[0048] S3. The effect of C3G on MM cell apoptosis was tested. Flow cytometry was used to evaluate the effect of C3G on MM cell apoptosis. After 48 h of treatment with 400 mM C3G, the apoptosis rate of MM.1S cells increased from 3.46±0.08% in the control group to 17.02±0.31% (P<0.001, n=3). Similarly, the apoptosis rate in RPMI-8226 cells increased from 5.58±0.29% to 17.06±0.83% (P<0.001, n=3), and the apoptosis rate in SP2 / 0 cells increased from 9.05±0.61% to 17.63±2.90% (P<0.05, n=3). C3G can promote apoptosis of MM cell lines ( Figure 5 A, B), BMMCs from newly diagnosed or relapsed MM patients were treated with 400 mM C3G for 48 h, PE-labeled anti-human CD138 antibody was used to label primary malignant plasma cells, incubated with annexin V-FITC staining solution, and cell apoptosis was detected by flow cytometry. C3G significantly promoted the apoptosis of primary malignant plasma cells from patients in vitro (P < 0.05, n = 7; Figure 5 C, D);

[0049] S4. The effect of C3G on NAD+ secretion and MM cell apoptosis was tested. After treatment with 400 μM C3G for 48 hours, the expression of CD38 on the surface of MM cells was evaluated by flow cytometry. Compared with the control group, the CD38 level on the surface of MM.1S, RPMI-8226 and SP2 / 0 cells after C3G treatment did not change significantly ( Figure 6 A), Real-time quantitative PCR was used to detect the transcription level of CD38 mRNA in three MM cell lines, demonstrating that there was no significant difference between the control group and the C3G group ( Figure 6 B), Colorimetric analysis was used to evaluate the effect of C3G on NAD+ levels in MM cells ( Figure 6C, D), compared with the control group, C3G could significantly increase the NAD+ content (5.59±0.43 vs 7.74±0.10, P<0.012.6390.28 vs 4.700.40 P<0.05n=3) and NAD+ / NADH ratio (1.10±0.05 vs 1.50±0.05, P<0.010.58±0.07 vs 1.28±0.08, P<0.01n=3), but had no significant effect on the total NAD content. For RPMI-822 6 cells. Compared with the control group, C3G also tended to increase the NAD+ content (8.35±0.96 vs 10.95±0.46, P=0.07, n=3), and significantly increased the NAD+ / NADH ratio (0.98±0.15 vs 1.56±0.13, P<0.05, n=3). To determine whether NAD+ accumulation induces apoptosis in myeloma cells, cells were treated with exogenous 1mNAD+ for 48h. Flow cytometry showed that NAD+ could significantly induce apoptosis of myeloma cells ( Figure 6 E), C3G induced NAD+ accumulation in MM cell lines by inhibiting CD38 enzymatic activity, thereby promoting apoptosis without changing CD38 expression;

[0050] S5. The effect of C3G on Sirt1 expression and NF-κB acetylation in MM cells was tested. Real-time quantitative PCR was used to evaluate the effect of C3G on Sirt1 mRNA expression in MM cell lines. The results are shown in Figure 7 As shown in A, after 48 hours of treatment with 400 μM C3G, Sirt1 expression was significantly upregulated in MM.1S, RPMI-8226 and SP2 / 0 cells relative to the PBS-treated control group (1.07±0.24 vs. 2.45±0.21, P<0.05 0.83±0.18 vs. 2.32±0.44, P<0.05 1.01±0.09 vs. 2.08±0.15, P<0.01 n=3), and similar results were also detected by PCR in tumor tissues derived from SP2 / o-cell-bearing subcutaneous tumor-bearing mice ( Figure 7 B); Western blot was used to analyze the expression of Sirt1 protein in MM cell lines and tumor tissues of subcutaneous tumor-bearing mice. After C3G treatment, the level of Sirt1 protein in MM cells was significantly upregulated ( Figure 7 C, E). In addition, Sirt1 protein levels were significantly increased in tumor tissues of C3G-treated mice compared with control mice bearing subcutaneous tumors ( Figure 7D, F), whether C3G affects the expression or acetylation of NF-κB in MM was studied. MM cell lines were treated with 400 μM C3G for 48 hours, and PBS was used as a control. Real-time quantitative PCR results showed that there was no significant difference in NF-κB mRNA levels in C3G-treated MM.1S, RPMI-8226, and SP2 / 0 cells compared with control cells ( Figure 8 A), Western blot analysis to assess NF-κB protein expression revealed that C3G had no significant effect on protein expression in the three cell lines ( Figure 8 B), to determine whether inhibition of NF-κB has a therapeutic effect on MM, MM.1S cells were treated with NF-κB inhibitor PDTC at 25 μM for 48 hours. Compared with PBS control, PDTC significantly increased MM cell apoptosis (4.15±0.19% vs. 97.82±1.30%, P<0.001, n=3; Figure 8 E);

[0051] S6. To demonstrate the regulatory mechanism of C3G on NF-κB acetylation, and to evaluate whether inhibition of CD38 would increase the expression of Sirt1 and decrease the expression of acetylated NF-κB, CD38+MM.1S cells were isolated from MM.1S cells by magnetic bead sorting, and their purity was verified. The protein levels of Sirt1, acetylated NF-κB, and NF-κB in CD38+MM.1S cells were compared with those of unsorted MM.1S cells as a control. Compared with normal MM.1S cells, CD38+MM.1S cells showed a significant increase in Sirt1 and a decrease in acetylated NF-κB, while the protein level of NF-κB did not change significantly ( Fig. 9 A), to elucidate the regulation of Sirt1 on NF-κB acetylation during C3G treatment of MM, MM.1S cells treated with PBS were used as a control group. In one of the intervention groups, MM.1S cells were pretreated with the Sirt1 inhibitor NAM (100 μM) overnight, then washed out and subsequently treated with 400 μM C3G. The remaining intervention groups were treated with C3G (400 μM), NAM (100 μM), NAD+ (1 mM) or Sirt1 activator RSV (20 μM), respectively. After 48 h of culture, Western blot analysis was used to evaluate the changes in acetylated NF-κB and NF-κB protein expression ( Fig. 9 B); Compared with the control group, the acetylated NF-κB level was significantly decreased in the C3G and RSV groups, but increased in the NAM group. The acetylated NF-κB level was not significantly different from the control group when MM.1S cells were pretreated with NAM and then intervened with C3G. No significant difference in NF-κB level was observed between the groups ( Fig. 9B), at the same time, the effects of C3G, NAM, C3G intervention after NAM pretreatment and RSV on MM.1S cell apoptosis were detected. Both C3G and RSV can promote MM.1S cell apoptosis, while NAM has no significant effect. C3G intervention after NAM pretreatment leads to a significant increase in the proportion of apoptotic cells ( Fig. 9 C), C3G inhibits CD38 activity and thus upregulates Sirt1 expression, which in turn reduces NF-κB acetylation, resulting in a therapeutic effect in MM.

[0052] In one embodiment, in S3, the cytotoxicity of C3G on PBMCs of healthy volunteers was evaluated, and the measurement results showed that C3G had no toxicity to normal human PBMCs at a concentration of ≤400 μM. At the same time, CCK-8 was used to detect the cell activity of three MM cell lines, and it was found that C3G reduced cell activity at a concentration of ≥200 mM. C3G at a concentration of 400 mM was selected for subsequent experiments; in proliferation and cell cycle analysis, MM cell lines showed decreased proliferation after C3G treatment, and the proportion of cells entering the S phase of the cell cycle was reduced. Flow cytometric analysis further revealed that C3G treatment increased apoptosis of MM cell lines. BMMCs were collected from patients, and flow cytometry showed that the proportion of annexin V-positive apoptotic cells in primary myeloma cells increased after C3G treatment.

[0053] In one embodiment, in S5, it was found that C3G effectively inhibited NF-κB acetylation, because C3G significantly reduced the level of acetylated NF-κB protein ( Figure 8 B); Real-time PCR and Western blotting were also performed on MM tissue samples of subcutaneous tumor-bearing mice treated with C3G; the results also showed that there was no significant difference in NF-κB transcription level and protein after C3G treatment, while the expression of acetylated NF-κB protein was significantly reduced compared with the PBS-treated control group ( Figure 8 C, D).

[0054] Embodiment 2

[0055] A C3G treatment method for multiple myeloma in subcutaneous and orthotopic tumor models comprises the following steps:

[0056] S1. C3G inhibits tumor growth in MM model mice:

[0057] ①Prepare reagents and cell lines;

[0058] ②Establishment of stable transfected cell lines;

[0059] ③Construction of tumor-bearing mouse model and C3G treatment.

[0060] In one embodiment, in S1, when preparing reagents and cell lines, C3G, NAD+, nicotinamide, pyrrolidine dithiocarbamate and resveratrol reagents are dissolved in PBS and stored at 80°C, and mouse myeloma cell line SP2 / 0, human myeloma cell line MM.1S and RPMI-8226 are cultured in RPMI1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin solution at 37°C and 5% CO2.

[0061] In one embodiment, in S1, when establishing a stably transfected cell line, SP2 / 0 cells were transfected with a luciferase-expressing lentiviral vector GV260-Fluc-puro for 48 hours, followed by selection with 2 μg / ml puromycin for 7 days to establish a stable SP2 / 0-luc cell line, and D-luciferin potassium salt was added to the culture medium for 5 minutes before measuring cell luminescence using an in vivo imaging system.

[0062] In one embodiment, in S1, when constructing a tumor-bearing mouse model and treating it with C3G, female BALB / c mice aged 6-8 weeks were prepared. For the subcutaneous tumor-bearing model, the mice were randomly divided into a healthy control group (n=5) and a subcutaneous tumor-bearing group (n=10). The mice in the tumor-bearing group were injected with 2×10^5SP2 / 0-luc cells in the right axilla, and then randomly divided into a control group (n=5) and a C3G group (n=5). On the third day after inoculation, the tumor-bearing mice received PBS or 25 mg / kgC3G through tail vein injection every other day, for a total of six injections. For the orthotopic transplanted tumor model, 1×10^7SP2 / 0-luc cells were inoculated into the mice through the tail vein. Starting from one week after inoculation, PBS or 25 mg / kgC3G were given every other day for four weeks. The mice were injected with D-luciferin potassium salt intraperitoneally, anesthetized by inhalation of isoflurane, and then imaged using an in vivo imaging system, IndiGo software, and the above experiment was repeated 3 times.

[0063] S2 and C3G inhibited the proliferation and cell cycle of MM cell lines in vitro and promoted apoptosis of CD138+MM cells in MM cell lines and MM patient bone marrow samples:

[0064] ① Isolation of mononuclear cells;

[0065] ② Cell activity determination;

[0066] ③Cell cycle analysis;

[0067] ④Cell proliferation test;

[0068] ⑤ Colony formation test;

[0069] ⑥Cell apoptosis detection.

[0070] In one embodiment, during the isolation of mononuclear cells in S2, blood samples from 5 healthy volunteers and bone marrow samples from 7 MM patients are collected, and Ficoll density gradient centrifugation is used to separate peripheral blood mononuclear cells from healthy volunteers and bone marrow mononuclear cells from MM patients.

[0071] In one embodiment, in S2, during the cell activity assay, the cell counting kit-8 (CCK-8) reagent was used to evaluate the viability of MM.1S, RPMI-8226 and mouse myeloma cell line SP2 / 0 and PBMCs, and the cells were seeded into a 96-well plate at a density of 1×10^4 cells / 100 μl / well, and different concentration gradients of C3G were added to the cells. The CCK-8 reagent was added to each well and incubated in the dark at 37°C for 4 hours, and the absorbance at 450 nm was measured using a spectrophotometric plate reader.

[0072] In one embodiment, in S2, during cell cycle analysis, a cell cycle detection kit is used to evaluate cell cycle distribution by flow cytometry, MM.1S, RPMI-8226 and SP2 / 0 cells are treated with C3G or PBS at a final concentration of 400 μM for 48 hours, the cells are collected and fixed in 70% ethanol at -20°C for 2 hours, after washing with PBS, the cells are incubated with RNaseA reagent at 37°C for 30 minutes, propidium iodide reagent is used to label the cells, and then analyzed by flow cytometry.

[0073] In one embodiment, in S2, during the cell proliferation assay, the proliferation of MM.1S, RPMI-8226 and SP2 / 0 cells was quantitatively detected by carboxyfluorescein succinimidyl ester flow cytometry, the cells were labeled with 0.5 μl CFSE solution and incubated in the dark at 37°C for 10 minutes, the labeled cells were seeded in a 12-well plate, treated with C3G at a final concentration of 400 μM, and cultured in the dark for 48 hours, and the fluorescence intensity was measured by flow cytometry.

[0074] In one embodiment, in S2, for the adherent SP2 / 0 cells, a colony formation assay was performed to evaluate the proliferation ability, 1×10^3 SP2 / 0 cells per well were seeded in a 6-well plate, and C3G was added at a final concentration of 400 μM after a 24-hour adhesion period. After one week, the culture medium was aspirated, and the cells were fixed with 4% paraformaldehyde for 15 minutes and stained with 0.1% crystal violet and photographed.

[0075] In one embodiment, in S2, during apoptosis detection, apoptosis was detected by flow cytometry. MM.1S, RPMI-8226 and SP2 / 0 cells were seeded in 12-well plates at 5×10^4 cells / well and treated with C3G or PBS at a final concentration of 400 μM as a control. After 48 hours, the cells were harvested and analyzed using an Annexin V-FITC / PI kit. The cells were resuspended in 1× binding buffer, Annexin V-FITC and PI staining solutions were added, and then incubated at room temperature in the dark for 15 minutes before flow cytometric analysis using NovoCyte D2040R. Patient-derived BMMCs were seeded in 12-well plates and treated with C3G or PBS at a final concentration of 400 μM for 48 hours. The cells were incubated with PE anti-human CD138 antibody and Annexin V-FITC in the dark at room temperature for 15 minutes, and the level of apoptosis was determined by flow cytometry.

[0076] S3, C3G for MM treatment:

[0077] ① Detection of CD38 expression by flow cytometry;

[0078] ②NAD+ / NADH quantitative detection;

[0079] ③Magnetic adsorption cell sorting of CD38 cells;

[0080] ④Real-time quantitative PCR;

[0081] ⑤Protein blot analysis.

[0082] In one embodiment, in S3, when CD38 expression was detected by flow cytometry, the surface expression of CD38 on human myeloma cell lines MM.1S and RPMI-8226 was detected using APC-labeled anti-human CD38 antibody, while the mouse myeloma cell line SP2 / 0 was detected using FITC-labeled anti-mouse CD38 antibody, 5 μl of antibody was added to every 100 μl of cell suspension, and the mixture was incubated at 4°C in the dark for 30 minutes, followed by flow cytometry detection.

[0083] In one embodiment, in S3, during the quantitative detection of NAD+ / NADH, NAD+ and NADH levels were determined using a NAD+ / NADH detection kit, MM.1S, RPMI-8226 and SP2 / 0 cells were seeded in a 6-well plate, treated with 400 μM C3G or PBS for 48 hours, collected, the cells were lysed, and the lysate was obtained. After incubation with alcohol dehydrogenase, a colorimetric reagent was added, and the absorbance at 450 nm was measured using a spectrophotometric plate reader. The protein concentration of the lysate was measured using the BCA method, and the results were expressed as the NAD content per unit protein.

[0084] In one embodiment, in S3, during magnetic adsorption cell sorting of CD38 cells, a CD38 microbead kit is used to select CD38 cells from the MM.1S cell line, a suspension of MM.1S is incubated with a CD38-biotin antibody and anti-biotin microbeads to label CD38+MM.1S cells, and applied to an LS separation column, and unlabeled CD38-MM.1S cells pass through the column and are collected.

[0085] In one embodiment, in S3, during real-time quantitative PCR, real-time quantitative PCR is used to detect the mRNA expression levels of target genes including CD38, Sirt1 and NF-κB, total RNA is extracted from cells or animal tissues using FreeZol reagent, and then reverse transcribed into cDNA using HiScriptIIIRTSuperMix, and detected on real-time quantitative PCR using ChamQUniversalSYBRqPCRMasterMix, and the relative target gene expression levels are normalized with an internal reference, and 2 -ΔΔ Calculation by CT method.

[0086] In one embodiment, in S3, during western blot analysis, the expression level of the target protein was analyzed using western blot. MM.1S, RPMI-8226 and SP2 / 0 cell lines and tumor tissues from subcutaneous tumor-bearing mice were lysed in RIPA buffer, and the supernatant was collected after centrifugation to obtain total protein. The total protein samples were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane. The PVDF membrane was incubated with primary rabbit antibodies specific to Sirt1, NF-κB and acetylated NF-κB, respectively. β-actin was used as an internal control. After overnight incubation at 4°C, the PVDF membrane was incubated with goat anti-rabbit IgG and observed using the Hesper chemiluminescence imaging system. The relative expression level of the protein was quantified using ImageJ software.

[0087] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of various changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A method for treating multiple myeloma in subcutaneous and orthotopic tumor models using C3G, characterized in that: include: S1. Test the effect of C3G on tumor growth in MM model mice; S2. Test the effect of C3G on the activity, cell cycle and cell proliferation of MM cell lines; S3. Test the effect of C3G on MM cell apoptosis; S4. Test the effect of C3G on NAD+ secretion and MM cell apoptosis; S5. Test the effect of C3G on Sirt1 expression and NF-κB acetylation in MM cells; S6. Prove the regulatory mechanism of C3G on NF-κB acetylation.

2. The method of treating multiple myeloma in subcutaneous and orthotopic tumor models with C3G according to claim 1, characterized in that: In S1, the SP2 / 0-luc cell line was established and injected subcutaneously into BALB / c mice to establish a subcutaneous tumor-bearing mouse model. The tumor size was measured with a vernier caliper every other day, and the volume was calculated using the formula V=long diameter×(short diameter)2 / 2.

3. The method of treating multiple myeloma in subcutaneous and orthotopic tumor models with C3G according to claim 1, characterized in that: In S1, the tumors of subcutaneous MM tumor model mice were stained and observed by histochemistry, and the MM orthotopic transplantation tumor model was established by tail vein injection of SP2 / 0-luc cells, and in vivo imaging was performed every week.

4. The method of claim 1 for treating multiple myeloma in subcutaneous and orthotopic tumor models with C3G, characterized in that: In S2, the effective concentration range of C3G was determined, PBMCs from healthy volunteers were isolated and seeded in 96-well plates, treated with C3G at concentrations of 0 mM, 50 mM, 100 mM, 200 mM, 400 mM and 600 mM for 48 hours, and CCK-8 assay was used to evaluate cell viability.

5. The method of treating multiple myeloma in subcutaneous and orthotopic tumor models with C3G according to claim 1, characterized in that: In S2, the effect of C3G on the cell cycle of MM cells was evaluated. Cells were treated with 400 mM C3G for 48 hours before flow cytometry analysis. SP2 / 0 colony formation images were processed using ImageJ software to quantitatively analyze the area of ​​colony formation.

6. The method of claim 1 for treating multiple myeloma in subcutaneous and orthotopic tumor models with C3G, characterized in that: In S3, flow cytometry was used to evaluate the effect of C3G on MM cell apoptosis. BMMCs from newly diagnosed or relapsed MM patients were treated with 400 mM C3G for 48 h, PE-labeled anti-human CD138 antibody was used to label primary malignant plasma cells, incubated with annexin V-FITC staining solution, and cell apoptosis was detected by flow cytometry.

7. The method of claim 1 for treating multiple myeloma in subcutaneous and orthotopic tumor models with C3G, characterized in that: In S4, after treatment with 400 μM C3G for 48 hours, the expression of CD38 on the surface of MM cells was evaluated by flow cytometry, and the transcription level of CD38 mRNA in three MM cell lines was detected by real-time quantitative PCR, which demonstrated that there was no significant difference between the control group and the C3G group. The effect of C3G on the NAD+ level in MM cells was evaluated by colorimetric analysis. In order to determine whether NAD+ accumulation induces apoptosis in myeloma cells, the cells were treated with exogenous 1 mM NAD+ for 48 hours, and flow cytometry showed that NAD+ could significantly induce apoptosis of myeloma cells.

8. The method of claim 1 for treating multiple myeloma in subcutaneous and orthotopic tumor models with C3G, characterized in that: In S5, real-time quantitative PCR was used to evaluate the effect of C3G on the expression level of Sirt1 mRNA in MM cell lines, and Western blot was used to analyze the expression of Sirt1 protein in MM cell lines and tumor tissues of subcutaneous tumor-bearing mice.

9. The method of claim 1 for treating multiple myeloma in subcutaneous and orthotopic tumor models with C3G, characterized in that: In S5, MM cell lines were treated with 400 μM C3G for 48 hours, and PBS was used as a control. Western blot analysis was used to evaluate NF-κB protein expression, revealing that C3G had no significant effect on protein expression in the three cell lines. In order to determine again whether inhibition of NF-κB has a therapeutic effect on MM, MM.1S cells were treated with NF-κB inhibitor PDTC at 25 μM for 48 hours.

10. The method of claim 1 for treating multiple myeloma in subcutaneous and orthotopic tumor models with C3G, characterized in that: In S6, it was evaluated whether inhibition of CD38 would increase the expression of Sirt1 and reduce the expression of acetylated NF-κB. CD38+MM.1S were isolated from MM.1S cells by magnetic bead sorting and their purity was verified. The protein levels of Sirt1, acetylated NF-κB and NF-κB in CD38+MM.1S cells were compared with those of unsorted MM.1S cells as controls to elucidate the regulation of Sirt1 on NF-κB acetylation during C3G treatment of MM. At the same time, the effects of C3G, NAM, C3G intervention after NAM pretreatment and RSV on MM.1S cell apoptosis were detected.

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