JaponiconeA induces apoptosis of bortezomib-sensitive and -resistant myeloma cells in vitro and in vivo by targeting IKKβ

JaponiconeA (JA) inhibited MM tumor growth in a xenograft mice model. (A) Schematic diagram of the in vivo experiments. (B, C) JA (30 mg/kg) was intraperitoneally administrated daily for 10 days, and the tumor volumes were measured each day during the treatment period. On day 10, mice were sacrificed. Tumors were removed and the tumors of each group were placed together for the photographs. (D) The body weight of mice was recorded everyday 2 days during the treatment period. (E) Ki67 and TUNEL were detected using immunohistochemical analyses of the xenograft tumors in each group (200 x, *P < 0.05; **P < 0.01 vs. the control).

JaponiconeA (JA) potentiated the cytotoxic effect of bortezomib and partially overcame drug resistance to bortezomib. (A) MM cells were treated with bortezomib, JA separately, or together for 24 h, and the number of apoptotic cells was determined using flow cytometry with Annexin V/propidium iodide (PI). (B) MM cells were treated with bortezomib, JA separately, or together for 24 h, and the inhibition was determined using the CCK8 assay. The combination index (CI) was then analyzed using CompuSyn software. (C) H929-BR or NCI-H929 cells were treated with bortezomib for 24 h, and the IC₅₀ was calculated using Graphpad prism software. (D) H929-BR cells were treated with JA for 24 h, and the IC₅₀ was calculated using Graphpad prism software. (E) H929-BR cells were treated with JA for 24 h and apoptotic cells were detected by flow cytometry with Annexin V/PI. (F) JA (30 mg/kg) was intraperitoneally administrated daily for 11 days, and tumor volumes were measured. On day 11, the mice were sacrificed. Tumors were removed and tumors of each group were placed together and photographed. (G) The body weights of mice were recorded every 2 days during the treatment period. (H) H929-BR cells were treated with bortezomib/JA for 24 h to calculate the CI of JA and bortezomib. (I) After exposure to JA/bortezomib for 24 h, the numbers of apoptotic cells were determined by flow cytometry with Annexin V/PI (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 vs. the control).

JaponiconeA (JA) inhibited the activation of the NF-κB pathway. (A) MM1.S cells were treated with JA or dimethyl sulfoxide for 24 h, and then the cells were harvested and subjected to next-generation sequencing. A clustering heat map was shown as above. (B) A volcano map was used to indicate differentially expressed genes (DEGs). A total of 1,001 DEGs were selected based on the following criteria: adjusted P-value < 0.05 and FC > 2. (C) GO and KEGG enrichment analyses was performed on downregulated DEGs. (D) A brief description of the mechanism of c-MAP. (E) The components of the NF-κB pathway of MM cells were detected after treatment with bortezomib for 24 h. (F) The mRNA levels of NF-κB target genes including BAX, BCL-xl, c-Myc, and ICAM1 were quantified using q-PCR after treatment with JA for 12 h (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 vs. the control).

JaponiconeA (JA) inhibited the activation of the NF-κB pathway induced by bortezomib. (A) The components of the NF-κB pathway of MM cells were detected after treatment with bortezomib for 24 h. (B) NCI-H929 cells were treated by single or both drugs, followed by Western blot to determine the expressions of the indicated proteins. (C) NCI-H929 cells were treated with the indicated drugs for 24 h, and p65 was detected by immunofluorescence. (D) The expressions of p-IKKβ, p-p65, and p-IκBα in NCI-H929 and H929-BR cells were determined by Western blot. (E) H929-BR cells were treated by single or both drugs, followed by Western blot to detect specific proteins. (F) H929-BR cells were treated with JA for 24 h, and p65 was detected by immunofluorescence. (G) The detection of proteins of the NF-κB pathway in the NCI-H929 mouse tumors (left) and H929-BR mouse tumors (right) using Western blot.

JaponiconeA (JA) exerted its effect by binding NF-κB IKKβ. (A, B) The thermal stabilization of IKKβ when incubated with JA using various dosages and temperatures. (C) The DARTS assay showed direct binding of JA to IKKβ. (D) IKKβ kinase activity was detected using a quantitative detection kit. (E) The overexpression of IKKβ in NCI-H929 or MM1.S cells and its effect on downstream targets of NF-κB were verified by Western blot. (F) MM cells transfected with IKKβ and control cells were treated with JA for 24 h, and cell apoptosis was determined by flow cytometry (*P < 0.05; **P < 0.01; vs. the control).