Acupuncture Synergized With Bortezomib Improves Survival of Multiple Myeloma Mice via Decreasing Metabolic Ornithine

Abstract

Multiple myeloma (MM) is a hematological malignancy worldwide in urgent need for novel therapeutic strategies. Since Velcade (bortezomib) was approved for the treatment of relapsed/refractory MM in 2003, we have seen considerable improvement in extending MM patient survival. However, most patients are fraught with high recurrence rate and incurability. Acupuncture is known for alleviating patient symptoms and improving the quality of life, but it is not well investigated in MM, especially in combination with bortezomib. In this study, we employed LC-MS and UHPLC-MS together with bioinformatics methods to test serum samples from 5TMM3VT MM murine model mice with four different treatments [control (C) group, bortezomib (V) treatment group, acupuncture (A) group, and combined (VA) group]. MM mice in group VA had longer survival time than mice in group A or group V. Joint pathway analysis indicated the underlying arginine and proline metabolism pathway among the 32 significantly decreased metabolites in group VA. CCK-8 assay and in vivo experiments validated that ornithine, the metabolite of arginine, promoted MM cell proliferation. In addition, gene expression omnibus (GEO) database analysis suggested that MM patients with higher ornithine decarboxylase 1 (ODC1) expression were evidently associated with poor overall survival. In summary, this study demonstrates the synergistic effects of acupuncture and bortezomib on extending the survival of MM model mice and provides potential therapeutic targets in the treatment of MM.

Keywords: ODC1; acupuncture; metabolomics; multiple myeloma; ornithine.

Figure 1

Efficacy evaluation of VA treatment in 5TMM3VT myeloma mice. (A) Animal model and blood collection. (B) Survival curve of group C, V, A, and VA. (C) Survival curve of group C vs VA.

Figure 2

Typical chromatograms of TIC in serum samples. TIC of group C (A), V (B), A (C), and VA (D) in ESI⁺ mode. TIC of group C (E), V (F), A (G), and VA (H) in ESI⁻ mode.

Figure 3

PCA score plot based on the data of ESI^+/− modes. (A) PCA score plot of all groups in ESI⁺ mode. (B) PCA score plot of all groups in ESI⁻ mode. (C) 3D scatter plot of all groups in ESI⁺ mode. (D) 3D scatter plot of all groups in ESI^- mode.

Figure 4

OPLS-DA score plot based on the data of ESI^+/− modes and validations of OPLS-DA models by 200 permutation tests. In ESI⁺ mode: (A, B) group C vs group V, (C, D) group C vs group A, (E, F) group C vs group VA. In ESI⁻ mode: (G, H) group C vs group V, (I, J) group C vs group A, (K, L) group C vs group VA.

Figure 5

Ornithine is a therapeutic target of VA treatment in MM mice. (A) Venn diagram displaying the 20 upregulated distinct metabolites in the serum of Group VA. (B) Venn diagram displaying the 32 downregulated distinct metabolites in the serum of Group VA. (C) Summary of joint pathway analysis in group VA with MetaboAnalyst 5.0. (D) Summary of joint pathway analysis in group VA with MetaboAnalyst 5.0. (E) Heatmap showing arginine and ornithine were downregulated metabolites in group VA.

Figure 6

Ornithine concentration is decreased in serum samples of group VA. (A) Characteristic chromatographic peak of ornithine standard. (B) Standard curve of ornithine in targeted metabolomics. (C) Ornithine concentration in serum samples of group C and VA. (D) The possible mechanism involved in VA treatment.

Figure 7

Increased ODC1 expression is associated with poor prognosis in MM. (A–D) Arginine and its metabolite promoted ARP1, H929, OCI, and 5TMM3VT cell proliferation. *P < 0.05. (E, F) High ODC1 expression in MM patients was correlated with poor OS in TT2 cohort, and APEX phase III clinical trial by log-rank test. (G, H) The mRNA level of ODC1 from NP, MGUS, SMM, and MM was significantly increased in MM samples by ordinary one-way ANOVA test.