Study On The Therapeutic Effect Of 10-hydroxycamptothecin On Neuroblastoma

Background:Neuroblastoma,the most common extracranial solid tumor in childhood,ischaracterized by high malignancy,strong invasiveness,earlier metastasis,moreadvanced stage cases and bad therapeutic efficacy.Due to the greater chemoresistenceto the first line drugs in patients with high-risk neuroblastoma,long-term survival rateof those patients remains very low.It has become the main impediment for theimprovement of survival in children with neuroblastoma and new effective drugs areurgently needed for high-risk patients.10-hydroxycamptothecin (HCPT) is a naturalcompound extracted from Comptotheca acuminate,a Chinese herbal medicine.It hasbeen shown to be very effective in treating some solid tumors and the toxicities aretolerable.Furthermore,the cost of HCPT is low.However,its efficacy in neuroblastomahas not been determined.In this study,we tried to evaluate the in vitro and in vivoeffectiveness of HCPT in the treatment of neuroblastoma and its mechanism. Part 1 Effects of HCPT on proliferation of human neuroblastoma cellline SMS-KCNR cellsObjective:To investigate the effects of HCPT on the proliferation and cell cycle progressionof cultured human neuroblastoma cell line SMS-KCNR cells.Methods:Using MTT assay,the cytotoxic effect of HCPT on human neuroblastoma cell lineSMS-KCNR cells was determined by treating the cells with varying concentrations ofHCPT treatment at 2.5nM,5nM,10nM and 20nM for 24,48,72 hours.Theanti-proliferative effect of HCPT on SMS-KCNR cells was determined byanchorage-dependent colony-forming assay,in which the cells were tested for theirpotential to proliferate and to form individual colonies.Varying concentrations ofHCPT was coincubated with SMS-KCNR cells for 24 and 48 hours to determine thecell cycle arrest effects using flow cytometry.Results:1.Using the SMS-KCNR cell line,we found that HCPT treatment resulted indose-dependent and time-dependent inhibition of cellular proliferation and cell viability.2.Reduction in cell viability with HCPT treatment at concentrations of 2.5-200nMafter 24 and 48 hours ranged from 3.6% to 64.7% and 12.5% to 91.7%,respectively,whereas treatment at concentrations of 2.5-20nM after 72 hours ranged from 36.7% to97%.3.There was a marked decrease in the ability of the SMS-KCNR cells to formcolonies with increasing doses of HCPT.HCPT at dosages of 10nM and 20nMcompletely inhibited the proliferation of the cells with no colonies formed by the end of14 days' culture. 4.As shown by flow cytometry,HCPT treatment at 2.5nM,5nM,10riM and 20nMof the SMS-KCNR cells resulted in a significant s-phase arrest of the cell cycle after 24hours and G2/M-phase arrest after 48 hours.Conclusions:Our data demonstrate that HCPT treatment results in a significant dose-andtime-dependent inhibition in the growth and colonogenic survival of SMS-KCNR cells.Our study also indicates that HCPT causes an s-phase arrest after 24 hours andG2/M-phase arrest after 48 hours.Part 2 Effects of HCPT on apoptosis of human neuroblastoma cellline SMS-KCNR cellsObjectives:The aim of the present study was to investigate the effects of HCPT on apoptosisof human neuroblastoma SMS-KCNR cells and to identify the altered signalingpathway involving in response to HCPT exposure.We also wanted to confirm thatHCPT-mediated loss of SMS-KCNR cell viability was the result of the induction ofapoptosis.Methods:The apoptosis-inducing effect of HCPT at 2.5nM,5nM,10nM and 20nM for 48hours's coulture was demonstrated by morphological observation under microscopeafter DAPI fluorescent staining and agarose gel electrophoresis.The extent of apoptosiswas quantified by flow-cytometric analysis of HCPT-treated cells labeled with AnnexinV and propidium iodide (PI).Alterations in mitochondrial signaling pathway weredetermined by the expression levels of P53,Bcl-2,Bax,cytochrome c,caspase-3 andPARP 1,based on the results from western blotting. Results:1.Using the SMS-KCNR cell line,we found that HCPT treatment at 2.5nM,5nM,10nM and 20nM increased the rate of apoptosis in a dose-dependent manner.2.Phase-contrast photomicrographs taken at 48h after HCPT treatment revealed adose-dependent decrease in cell density.The fragmentation of the induced cell nucleiwas showed with DAPI.DNA ladder typical for apoptosis was showed on agarose gelelectrophoresis.3.As shown by the Annexin V and PI staining,we found that HCPT caused adosage dependent apoptosis in SMS-KCNR cells.It was observed that treatment ofSMS-KCNR cells with 2.5-20nM of HCPT for 48 hours increased the number of earlyapoptotic cells (LR) from 12.22% to 50.78%,in a dose-dependent manner compared to4.15% in untreated control cells.The number of late apoptotic cells (UR) increased from8.67% to 67.32% compared with 0.13% in non-HCPT treated cells.The total percent ofapoptotic cells (UR + LR) increased from 4.28% in untreated SMS-KCNR cells to97.66% with 20nM of HCPT treatment for 48 hours.4.Westem blot analysis showed that treating SMS-KCNR with HCPT resulted in adose-dependent increase in P53 protein levels with no effect on Bcl-2,Bax and the ratioof Bax/Bcl-2.5.HCPT treatment of SMS-KCNR cell line resulted in a increase in cytoplasmiccytochrome c protein levels and a concomitant decrease in mitochondrial cytochrome cprotein levels.6.HCPT treatment of SMS-KCNR cell line resulted in activation of caspase-3,theprocaspase-3 protein levels was decreased and the actived 17kDa subunit was increasedin a dose-dependent manner.7.Western blot analysis indicated that treating SMS-KCNR with HCPT resulted inactivation of poly (ADP-ribose) polymerase (PARP1),the cleaved 89kDa subunit was increased in a dose-dependent manner.Conclusions:Our data suggested that HCPT treatment resulted in induction of apoptosis indose-dependent manner.HCPT induces cytochrome c release via the mitochondriaapoptosis pathway mediated by p53.By activating caspase-3 and PARP1,HCPT finallyleads to cell apoptosis.Based on above-mentioned studies,HCPT may be accepted as adrug for chemotherapy of neuroblastoma.Part 3 Pilot study on the efficacy of chemotherapy using HCPT onrecurrent or refractory neuroblastomaObjectives:Patients with recurrent or refractory neuroblastoma are very difficult to treat withvery poor prognosis and high mortality.HCPT is a natural compound extracted fromComptotheca acuminate,a native plant of China.It has been shown to be very effectivein some solid tumors such as gastric and colon cancers,lung cancers and ovary cancersetc.However,its efficacy in neuroblastoma has not been determined.Based on thepreliminary experimental studies in vitro,we aimed to investigate the therapeutic effectsof HCPT in the treatment of recurrent or refractory neuroblastoma in children.Methods:Ten children with recurrent neuroblastoma and two with refractory neuroblastomawere treated with HCPT.Of them,5 children with recurrent neuroblastoma were treatedwith HCPT alone,and the other 7 patients received combination chemotherapy ofHCPT plus other agents.The HCPT alone treatment group was injected with HCPT(7.5mg/m2 daily) for 14 consecutive days.The combination chemotherapy group wasalternately treated with the modified new protocol A1 (cyclophosphamide 1200mg/m~2 on day 1,etoposide 100mg/m~2 on days 1-5,HCPT 5mg/m~2 on days 1-3,cisplatin90mg/m~2 on day 4) and the modified protocol B (ifosfamide 1.5g/m~2 on days 1-5,HCPT 5mg/m~2 on days 1-3,carboplatin 450mg/m~2 on days 2).Results:Four patients (33.3%) achieved partial remission and 8 patients (66.7%) had stabledisease.The median remission time was 3.5 months (2-5months).HCPT treatment assingle agent resulted in mild side effects.Myelosuppression and digestive disorder werefound as the main adverse events in the combined chemotherapy group and clinicallymanageable.No chemotherapy related death was found.Conclusion:The HCPT is safe and effective in the treatment of recurrent or refractoryneuroblastoma.The toxicities are tolerable.But the long-term efficacy warrants furtherexploration.