| Background and objective:The emergence of multiple drug resistance (MDR) exists as a major challenge in the treatment of leukemia at present. Aberrant activation in signal transduction pathway, defective regulation of apoptotic gene expression, imbalance of cell signaling network, and overexpression of membrane drug transport proteins including P-glycoprotein (P-gp) and multidrug resistance related protein (MRP) are all contributing mechanisms giving rise to MDR. Lots of solid tumors, such as glioblastoma, breast cancer, colon cancer, pancreatic cancer, hepatocellular carcinoma all have been identified to be related to the activated Hedgehog (Hh) signaling. There were three Hh homologues have been reported in mammals:Sonic hedgehog (Shh), Indian hedgehog (Ihh) and Desert hedgehog (Dhh), with Shh being most extensively studied. The signaling pathway is composed of signaling molecule shh, the transmembrane receptor Patched (Ptch) and Smoothened (Smo) as well as a family of zinc finger transcription factors, such as the glioma-associated oncogenes Gli-1, Gli-2, and Gli-3. In the normal circumstances, Ptch was activated to restrain activity of Smo and the downstream signaling Gli in proteasome (lysosomal) was truncated. Gli went into the nucleus in the form of carboxyl end truncated, which could inhibit the transcription of downstream target genes. When the pathway was activated, the inhibition of the Smo was released through the combination of Ptch and Hh, prompting the formation of macromolecular compound with Gli protein and the protein kinase A. Then span Gli protein went into the nucleus to activate downstream target genes, resulting in the chemotherapy drug resistance.NVP-LDE225 is a new and selective Smo antagonist, which has entered drug resistance evaluation in patients with solid tumors. While the Hh pathway in the in vivo study of leukemia was still at the preliminary stage. Hitherto, only one Tokyo experience published by Seiichiro in 2013 has been reported. He has demonstrated that Hh pathway is activated in chronic myeloid leukemia (CML) stem cells via up-regulation of Smo. Compared with single agents, co-treatment of Smo inhibitor and ABL1 tyrosine kinase inhibitors may help eliminate therapy-resistant T315I BCR-ABL1-positive leukemia cells, which resulted in prolong survival and reduce the in vivo growth in the mice model of CML and Ph-positive acute lymphocytic leukemia.But the study of combination with a Hh pathway inhibitor and chemotherapy drug in leukemia in vivo still remained rare about drug resistance. It is well known that HL60/ADM is an acute myeloid leukemia cell line with multiple drug resistance. Our preliminary results showed that, compared to the non-resistant cells and non refractory primary AML cells, a significant upregulation of the Hh signaling pathway expression was observed in the drug-resistant cell lines and refractory primary cells. Single drug NVP-LDE225 can reverse the drug resistance to ADM, DNR, HHT, Ara-C in HL60/ADM cell line, with increasing Adriamycin (ADM) uptake rate. The mechanism may be achieved by inducing specific down-regulation of Gli-1, IRS-1, p-Akt and MRP1 protein. Whether the Hh pathway has a similar role in vivo remains elusive, and this study therefore aimed at evaluating the antitumor efficacy, toxicity, mechanism and optimal schedule of NVP-LDE225 alone or in combination with ADM in the MDR human leukemia cell line (HL60/ADM) xenograft model.Methods:1. 1×107 HL60/ADM cells in 200μl RPMI medium were injected subcutaneously into the right flank of nude mice. Following the development of palpable tumor (2 mm×2 mm minimum 5 days post engraftment) animals were divided into cohorts (n=7) and treated with different drugs.2. Grouping scheme was implemented as follows:(a) vehicle (0.5% methyl cellulose/0.5% Tween 80 in phosphate-buffered saline), (b) ADM alone (3mg/kg/d, d1;4;7) (c) NVP-LDE225 alone (80mg/kg/d, d1～10), (d) NVP-LDE225 combined with ADM (NVP-LDE225 80mg/kg/d, dl～10+ADM 3mg/kg/d, d1;4;7), (e) NVP-LDE225 treated for 2days sequentially combined with ADM (NVP-LDE225 80mg/kg/d, d-2～10+ADM 3mg/kg/d, d1;4;7), (f) NVP-LDE225 treated for 5days sequentially combined with ADM (NVP-LDE225 80mg/kg/d, d-5～10+ADM 3mg/kg/d, d1;4;7). Vehicle and LDE225 were in oral administration once a day and ADM was administered i.p. q3d for three injections.3. Tumor sizes (V) were measured every day using the caliper. It was calculated according to the formula:V= 1/2×a×b2, where a was the longest and b was the shortest diameter of the tumor mass (in mm), respectively. The rate of tumor inhibition under each treatment group was expressed as relative tumor volumes calculated as:(1-Vt/V0)×100%, where Vt and Vo was the average tumor volume of treatment and control group respectively. Moreover, the survival of the mice was followed.4. To monitor the toxicity of the treatment, general condition of the mice (such as spirit, diet, activities, urine, body weights) were weighted and recorded every 3 days. Complete blood count (CBC) analysis was obtained from tail bleed samples before and after treatment every 3 days. White blood cell (WBC), red blood cell (RBC) and platelet (PLT) counts were used to calculate myelosuppression.5. When the end of medication and deathbed, organs (including heart, liver, spleen, lung, and kidney), femurs and tumors of the mice in each group were harvested. Tumor and major organ tissues were stained with hematoxylin and eosin (H&E). Bone marrow aspirate smears was stained by Wright-Giemsa for the examination of cytomorphology.6. Upon immunohistochemistry analysis, after antigen retrieval, the tumor histological sections were stained with Gli-1, IRS-1, p-AKT and MRP1.7. The statistical significances of differences between different groups in tumor volume and survival time as well as complete blood counts were assessed by a one-way analysis of variance (ANOVA) using SPSS 13.0 program. The differences of the tumor volume and the body weight before and after administration in each group were tested by repeated measure ANOVA. Bonferroni method was used for comparison between two means in regular variance, Dunnett’s T3 was used for comparison between two means in irregular variance. P values<0.05 were regarded to be significantly different.Results:1. Inoculated with HL60/ADM cells culturing in vitro, tumor growth were found in 33.3% of nude mice. While when inoculated with HL60/ADM cells culturing in vivo, the xenograft tumor mice were presented at the rate of 81.8%. Tumor grew only at the sites of cells injection. There were no tumor metastases to be found in organs. No leukemic infiltrations were detected in the liver, spleen, lungs, kidneys or bone marrow. Smears of transplantation tumor were tested by cell morphology, the tumor cells were large, myeloid and primitive. Meanwhile it had a few promyelocytes.2. There were significant differences in transplantation tumor volumes before or after drug administration (F=659.38, P<0.001). And it also had significant differences between different groups (F=69.09, P<0.001). The tumor volume in LDE225 combined with ADM, LDE225 treated for 2days sequentially combined with ADM and LDE225 treated for 5days sequentially combined with ADM grew the slowest, followed by LDE225 alone and ADM alone. And the tumor in control group grew the fastest. The combination of ADM and LDE225 inhibited tumor growth to a significantly greater extent than control group since the 8th day after treatment. Identically, LDE225, treated for 5 or 2 days ahead of schedule, significantly inhibited tumor growth since the 7th or 8th days after combined with ADM compared with the control group. There were no significant differences between either vehicle-or ADM-or LDE225-treated mice. The different was more and more obvious to the 15th days. The tumor inhibiting ratios were 17.1%,10.6%,69.0%,73.7% and 76.7% in Group b, c, d, e and f.3. In this in vivo animal study, no significant survival differences were found among all the groups (F=16.97, P<0.001). The survival time was significantly longer in LDE225-5d sequentially combined with ADM group (36.67±0.577days), LDE225-2d sequentially combined with ADM group (35.33±1.528days) and LDE225 combined with ADM group (35.00±2.000days) than that of the control group (P=0.006, P=0.003 and P<0.001) or ADM alone group (P=0.011, P=0.006 and P=0.001) or LDE225 alone group (P=0.02, P=0.011 and P=0.001). Moreover, in this in vivo animal study, there was no survival difference between control (30.33±0.577), NVP-LDE225 (31.00±1.00) and ADM (30.67±0.577) group.4. During the period of drug delivery, the general status including mental status, food and drink intake as well as body weight in NVP-LDE225-treated mice were no significant differences with the control group and there was an increase in the body weight along with time went on. There were significant differences in the body weight of each group before or after administration (F=60.316, P=0.000). In ADM-treated mice, measurable weight loss occurred on the 5th day after treatment initiation and rebounded to near starting level or higher on day 11. In addition, combination of NVP-LDE225 simultaneously or post 2days or 5days with ADM mice exhibited a drop on the 3rd day in body weight after initiation of ADM exposure, with a gradual recovery to near starting levels within 13 days.5. Serial CBC analysis was performed both in the control and treatment groups. Combination of NVP-LDE225 simultaneously or post 2days or 5days with ADM and ADM-treated mice exhibited a significant drop in WBC and PLT counts by day 10 after initiation of exposure, all of which rebounded to near starting level on day 15. On the 10th day, WBC (109/l) and PLT (109/1) in each group was as follows: 2.58±0.30 and 449.25±77.45 (ADM alone),1.28±0.63 and 243.25±89.14 (LDE225+ADM),1.88±0.75 and 335.50±115.14 (LDE225-2d+ADM),2.03±0.10 and 218.75±65.78 (LDE225-5d+ADM). We did not observe a significant drop in WBC and PLT upon exposure to NVP-LDE225 alone. In addition, RBC was stable in all mice from the beginning of treatment to the 15th day post treatment.6. Within the control group, tumor cells were close-packed and comprised of pleomorphic cells which were small-to medium-sized cells with coarse nuclear chromatin, large nuclear, distinct nucleoli, and basophilic cytoplasm. Within the NVP-LDE225-or ADM-treated group, distribution density of tumor cells were slightly reduced than the control group. In addition, within the combination group, tumor cells were populated at the least density with apoptosis cells and apoptotic bodies increasing. In the transplantation tumors from dying experimental mice, there were multifocal areas of necrosis that comprised the tumor mass. And there were a handful of apoptosis cells and apoptotic bodies.7. To determine whether LDE225 or ADM or combination treatment related to toxicity in an in vivo model, the histopathology of five organs were measured, concluding heart, liver, spleen, lung, and kidney. No significant differences were found in the above organ histology in different groups. Morphological examination of bone marrow in all treatment mice presented that nucleated cells were active proliferation with a normal ratio of granulocytes and erythrocytes, revealed that these drugs showed no myelosuppression compared to the control group. Moreover, there were no leukemia cells infiltrating within the bone marrow tissues8. At the end of the medication, xenograft tumor tissue immunohistochemical examination of each group showed:The Gli-1, IR.S-1, p-AKT and MRPlwere mainly localized to the cytoplasm with some localized in the nucleus. In the control and ADM-treated group, these proteins were strong expression (+++). While in the three groups of combination treatment, the expression strength of Gli-1 decreased to negative (-), and those of IRS-1, p-AKT and MRP I were weak expression (+). The results indicated that the expression rate and strength was significantly decreased after treatment of LDE225.Conclusions:1. Combination of LDE225 and ADM significantly enhanced the antitumor activity compared to the control mice.2. Combination with LDE225 reduced drug resistance of HL60/ADM xenograft tumors to ADM, which was likely to be mediated via inhibition of IGF-1R/Akt/MRP1 pathway.3. After administration of NVP-LDE225 alone or combined with ADM, the myelosuppression and toxicity could be well tolerated in the xenograft mice. |
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