Antimyeloma Activity of Norcantharidin in Nude Mouse Xenografts
|School||Hebei Medical University|
|Keywords||Animal experiment Multiple Myeloma Bortezomib Norcantharidin NF-κB TUNEL assay Immunohistochemistry|
Objective: As the second most common hematological malignancy,multiple myeloma (MM) is characterized by the accumulation of monoclonalplasma cells in patient’s bone marrow, and chemotherapy is the mainlymedical treatment. Bortezomib(BTZ) is the basic medication in the first-linetreatment of patients with multiple myeloma, and its curative effect has beenwidely recognised. However, nearly30%of patients have no response to BTZ,and37–44%of patients are compelled to discontinue their treatment due tothe serious adverse reaction. Therefore, there is an imperative need for somenew chemical entities, which have high performance but low toxicity, or couldenhance the anti-myeloma activity of BTZ.Norcantharidin (NCTD), the demethylated analog of cantharidin, retainsthe antitumor activity with reduced nephrotoxicity and recognised by manyresearchers. In previous studies, we have demonstrated that NCTD inducedapoptosis in U266cells and enhance the anti-myeloma activity of BTZthrough down-regulate NF-kB and NF-kB downstream molecules. Therefore,in this study, we investigated the anti-myeloma activity of NCTD in vivo byestablishing the animal models of multiple myeloma, and then provide moreexact theoretical basis for clinical application of NCTD against MM.Methods:1The toxicity test of NCTD: The maximum and minimum lethaldose(250and102.4mg/kg) of NCTD were defected by trial test. Then50micewere randomized into5groups(n=10/group) and treated with differentconcentrations of NCTD(250,200,160,128and102.4mg/kg, IP). Record thedeath distribution of each mice group for7days, and compute the LD50concentration of NCTD with the modified Karber formula.2Nude mouse xenograft assay:2×106U266cells were subcutaneously injected into the subscapularis of each6-week-old male nu/nu nude mice. Thetumor volume (V) was calculated with the following formula: V=[tumorlength in mm×(tumor width2in mm)]/2. When the tumor volume reached100mm3in size, the mice were randomly assigned into four groups (n=8/group).The control group was intraperitoneally (IP) treated with100mL normalsaline3/week for4weeks. The NCTD group was administered NCTD (20mg/kg, IP)3/week for4weeks. The BTZ group received BTZ (0.25mg/kg, IP)once per week for4weeks. The combination group was treated with acombination of NCTD (20mg/kg, IP) and BTZ (0.25mg/kg, IP). Tumorgrowth and animal body weights were monitored twice a week. The mice wereeuthanized4h after the last dose, and the tumor xenografts were removed forimmunohistochemical (IHC) analysis.3Pathology and apoptosis, immunohistochemistry analysis: The tumorxenografts were parrffin-embedded and sliced into5μm per section. Thesections were stained by HaematoxylinEosin and then observed with lightmicroscope. Apoptosis in tumors was determined by TUNEL assay and theexpression of NF-kB p65, PCNA was detected through Immunohistochemicalanalysis.Results：1LD50concentration of NCTD:According to the results of the toxicitytest, Σp=1.0+0.9+0.7+0.6+0.2=3.4, i=lg250/200=lg1.25=0.1, Xm=lg250=2.4, the LD50of NCTD was determined with the modified Karber formula:LD50=log-1[Xm–i (Σp–0.5)]=128mg/kg。2The tumor growth observation: There was no difference betweenaverage volumes of nude mice xenograft in both4groups before treatment(P>0.05). However, tumor volumes in3treatment groups were much smaller thanthe untreated control group after4weeks(P<0.05). The tumor volumereduction rates were respectively31.83,36.32%in NCTD, BTZ groups, whilein the combination group, the reduction rate reached64.49%, significantlygreater than BTZ-alone group(P<0.05).3The general condition and biochemical indexes comparison: There was no apparently panic, anxiety, or antifeeding and other phenomena throughoutthe whole intervention process but only limited mobility in the control group.Biochemical indexes detection after4weeks showed that in the3treatmentgroups, there was no significant renal impairment(average serum creatininelevels<0.5mg/dL) neither difference in alanine aminotransferase levels(P<0.05).4Apoptosis detection by TUNEL assay: Obviously apoptosis cells wereobserved in both the four treatment groups, characterized by karyopyknosisand brown nucleus after stained with diaminobenzidine. The apoptosis rateswere respectively29.91%,42.06%in NCTD, BTZ groups, with respect to14.95%in the control group. While in the NCTD-BTZ combination group, theapoptosis rate reached81.80%, much greater than BTZ-alone group(P<0.05).5The protein expression of PCNA in nude mice xenografts: As acommon index for evaluation of cell proliferation, the positive expression ofPCNA is located in nucleus and appears as a diffuse brown particles. OurImmunohistochemical tests showed that the PCNA positive expression rate upto (89.91±13.59)%, presented much lower in BTZ and NCTD group,respectively (71.85±8.93)%and (58.39±9.43)%. While in the NCTD-BTZcombination group, the positive expression rate was only (11.61±2.94)%,there was a significant difference compared with BTZ-alone group(P<0.05).6The protein expression of NF-κB P65in xenografts: The positiveexpression is located mostly in cytoplasm and appears as a diffuse brownparticles. Our Immunohistochemical tests showed that the NF-κB P65positiveexpression rate was up to (85.23±16.62)%, presented much lower in BTZ andNCTD group, respectively (71.51±9.86)%and (64.80±11.73)%. While in theNCTD-BTZ combination group, the positive expression rate was only(11.95±3.13)%, there was a significant difference compared with BTZ-alonegroup(P<0.05).Conclusions：1NCTD could significantly inhibit the proliferation and induce theapoptosis of myeloma cells in vivo. 2NCTD could potentiate the proliferate-inhibitor and apoptosis-induction effects of BTZ, both in vitro and in vivo.3With the same mechanism in vitro, NCTD could potentiate theanti-tumor effect of BTZ in vivo through down-regulating the NF-κB/IκBαsignaling pathway and can be administered in clinic.