| OBJECTIVEMultiple myeloma（MM）is the second most prevalent hematologic malignancy.Approval of bortezomib（BTZ）,the first in-class proteasome inhibitor,has significantly improved clinical outcomes of MM patients in the past decade;however,the almost inevitable refractory or resistance as well as the toxicities have made a cure of MM out of reach.Therefore,identification and validation of novel effective targeted agents are necessary.As one of the natural compounds isolated from Traditional Chinese Medicine gamboge,gambogenic acid（GNA）is a very promising anti-tumor agent and has been proved to have a more potent anticancer effect and less systemic toxicity according to early investigations.In this study,our aim was to investigate and explore the concrete apoptotic mechanisms of GNA and BTZ combination treatment against MM in pre-clinical models.METHODSThe Cell Counting Kit-8（CCK-8）assay was used to detect the proliferation inhibition of BTZ and GNA on myeloma MM.1S and RPMI 8226 cells,and the genomic and transcriptomic data of these two cell lines were further analyzed based on the results of proliferative inhibition results.The flow cytometry（FCM）was used to detect the cell cycle arrest of BTZ and GNA alone or synergistically with different concentrations for MM.1S cells.The levels of apoptosis-related proteins PARP,p53,Caspase-3,Bax and Bcl-2 were detected by Western blot.The xenograft tumor model was established to evaluate the in vivo efficacy of BTZ,GNA and the combination of BTZ and GNA.After average tumor volumes reached to 150200 mm3,the mice transplanted with 1×107 MM.1S cells were randomly divided into four groups and injected intravenously of BTZ(0.25mg kg-1 d1,4,8,11),GNA(2.0mg kg-1 per 2 days)and combination(0.25mg kg-1 BTZ d1,4,8,11 plus 2.0mg kg-1 GNA per 2 days)or vehicle dimethyl sulfoxide（DMSO）by the same doses and time for a period of 2weeks,respectively.After two weeks,all the mice were sacrificed.Terminal deoxynucleotidyl transferase dUTP nick end labeling（TUNEL）was used to detect apoptosis of tumor-bearing tissue cells.Immunohistochemistry（IHC）and Western blot were used to detect expression levels of cell growth-and apoptosis-related proteins Ki-67,PARP,p53,Caspase-3,Bax and Bcl-2.Furthermore,level of reactive oxygen species（ROS）was evaluated by ROS detection kit.The expressions of p38MAPK and p-p38 MAPK protein were detected by Western blot.FCM was used to detect apoptotic inhibition effect of GNA and BTZ combination therapy on MM.1S cells after adding thiol antioxidant N-acetyl cysteine（NAC）and p38 MAPK inhibitor SB203580.RESULTS1.After treatment with different concentrations of BTZ and GNA,MM.1S and RPMI8226 cells exhibited morphological changes.CCK-8 results showed that both BTZ and GNA alone could inhibit the proliferation of MM.1S and RPMI 8226 cells in a dose-dependent manner;however,synergistic effect of combined treatment of BTZ and GNA was only applied in MM.1S cells.MM.1S and RPMI 8226 cells exhibited different characteristics in both the genomic and transcriptomic levels.2.According to the EC50 value of BTZ and GNA for 48 h,we selected 2.3 nM BTZ and 0.51μM GNA,and 4.0 nM BTZ and 0.90μM GNA as combinations for further exploration.We found that the ratio of G2/M phase cells was 27.01±1.80%and31.09±2.16%after treatment with 2.3 nM and 4.0 nM BTZ for 48 h,respectively;whereas after 48 h,the proportion of G2/M phase cells was 23.19±1.44%and26.68±1.96%after treatment with 0.51μM and 0.90μM GNA respectively;the proportion of cells in G2/M phase was 31.34±1.81%and 19.88±1.89%respectively after combined treatment with 2.3 nM BTZ and 0.51μM GNA,4.0 nM BTZ and 0.90μM GNA for 48h;in the control group,only 17.23±1.65%cells were arrested in G2/M phase.After treatment with 0.90μM GNA and the combination of 4.0 nM BTZ and 0.90μM GNA for 48 h,the proportion of MM.1S cells in sub G1 phase was30.23±0.66%and 34.93±3.09%,respectively.After incubation of 2.3 nM BTZ,0.51μM GNA,the combination of 2.3 nM BTZ with 0.51μM GNA,4.0 nM BTZ,0.90μM GNA,and the combination of 4.0 nM BTZ with 0.90μM GNA for 48 h,both BTZ and GNA could up-regulate the expressions of Clv-PARP,p53,Clv-Caspase-3and Bax proteins,and down-regulate the expression of Bcl-2 protein（p<0.05）.Compared with the single-agent group,the combined groups could significantly change the expressions of apopotosis-related proteins（p<0.05）.3.In the MM.1S tumor-bearing mouse model,the tumor inhibition rate was 9.68%in the BTZ group,19.35%in the GNA group,and 41.94%in the combined group.The two agents had no apparent organ damage on nude mice.The rate of apoptotic cells was 2.78±1.93%in the control group,52.75±7.80%in the BTZ group,40.00±7.62%in the GNA group and 66.75±6.08%in the combined BTZ and GNA group by TUNEL.IHC and Western blot showed that the expression levels of apoptosis-related proteins were significantly different between the experimental groups and the control group（p<0.05）.Compared with the BTZ and GNA monotherapy groups,the combined group significantly increased the expression levels of Clv-PARP,p53,Clv-Caspase-3 and Bax proteins（p<0.01）,while the expression levels of Ki-67 and Bcl-2 proteins in the combined group were significantly decreased than those in the single group（p<0.01）.4.Reactive oxygen species（ROS）detection showed that ROS reached a peak at 12 h after 4.0 nM BTZ treatment on MM.1S cells,at 4 h after 0.90μM GNA treatment,and at 1 h after combined BTZ and GNA treatment to MM.1S cells.Western blot revealed up-regulation of p-p38 MAPK protein after 48 h treatment of 2.3 nM BTZ,0.51μM GNA,combined 2.3 nM BTZ with 0.51μM GNA,4.0 nM BTZ,0.90μM GNA,and combined 4.0 nM BTZ with 0.90μM GNA.The level of p-p38 MAPK protein was increased in a dose dependent manner and that of the combined group was superior to the single-agent group（p<0.05）.After pre-treatment with ROS inhibitor NAC and p38 MAPK inhibitor SB203580 for 6 h,the apoptotic rate of MM.1S cells induced by either 4.0 nM BTZ or 0.90μM GNA single agent as well as the combination group were decreased（p<0.05）.BTZ,GNA and BTZ combined with GNA could significantly up-regulate ROS level in MM.1S cells.NAC could inhibit the up-regulation of ROS induced by BTZ,GNA and BTZ combined with GNA（p<0.001）,but SB203580 failed to inhibit the up-regulation of ROS induced by BTZ,GNA and BTZ combined with GNA（p>0.05）.CONCLUSIONS1.Both myeloma MM.1S and RPMI 8226 cells were sensitive to BTZ and GNA single agent.Interestingly,GNA could only enhance the chemosensitivity of BTZ on MM.1S cell line.2.Both 0.51μM GNA combined with 2.3 nM BTZ and 0.90μM GNA combined with4.0 nM BTZ could arrest MM.1S cells in G2/M phase and induce early apoptosis of MM.Notably,increased apoptosis of MM.1S cells was found via the activations of PARP cleavage,p53,Caspase-3 cleavage and Bax proteins and inhibition of Bcl-2protein.3.An increased antitumor effect of combination therapy of GNA with BTZ on MM.1S xenograft models was also observed.4.Oxidative stress-mediated ROS/p38 MAPK signaling pathway was involved in the enhancement of chemosensitivity of GNA to BTZ on MM cells.5.The combination of BTZ and GNA-based therapeutic regimen could be a very promising option in the treatment of MM patients in the future. |
