羟基喜树碱治疗神经母细胞瘤的研究
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摘要
研究背景：
NB是儿童期最常见的颅外实体瘤，它不但恶性程度高、侵袭力强，而且早期转移率高、晚期病例多、治疗效果差。尤其是晚期高危病例，常因对一线药物不敏感导致耐药复发，长期生存率很低，是制约NB患儿生存率提高的主要原因。因此积极寻找有效的二线化疗药物是提高NB患儿生存率的重要手段。HCPT是我国利用天然资源优势研制出的纯天然生物碱抗肿瘤药，对多种肿瘤具有较好的疗效，且毒副作用较低、价格低廉。但其在小儿NB的治疗作用国内外尚未见报道。因此本研究通过体外实验结合临床治疗来探讨HCPT对NB的疗效，为探索NB新的、有效的化疗方案，提高生存率，提供理论基础与临床经验。
第一部分羟基喜树碱对人神经母细胞瘤SMS-KCNR细胞增殖的影响
目的：
研究HCPT在体外对人NB细胞系SMS-KCNR细胞增殖和细胞周期的影响。
方法：
采用MTT法观察不同浓度的HCPT(2．5～200nM)作用不同时间(24h、48h、72h)后对SMS-KCNR细胞增殖的影响。同时应用平板集落形成试验观察不同浓度的HCPT(2．5nM、5nM、7．5nM、10nM、20nM)对SMS-KCNR细胞集落形成的影响。用不同浓度的HCPT(2．5nM、5nM、10nM、20nM)作用SMS-KCNR细胞24h及48h，碘化丙锭(PI)染色后用流式细胞仪检测并分析细胞周期的改变。
结果：
1．HCPT能显著抑制SMS-KCNR细胞增殖，并随药物浓度的升高和作用时间的延长而抑制作用增强，呈明显浓度和时间依赖性。
2．在HCPT(2．5～200nM)作用24h及48h后SMS-KCNR细胞增殖抑制率分别为3．6～64．7%、12．5～91．7%，而2．5～20nM浓度范围HCPT作用72h的抑制率为36．7～97%。
3．HCPT能明显抑制SMS-KCNR细胞集落形成，浓度为10nM和20nM时能完全抑制其集落形成。
4．经流式细胞仪检测，不同浓度HCPT(2．5nM、5nM、10nM、20nM)作用SMS-KCNR细胞24h，使细胞周期阻滞于S期；作用48h使细胞周期阻滞于G2/M期。
结论：
HCPT能显著抑制SMS-KCNR细胞增殖和集落形成，其抑制作用呈时间和剂量依赖性。HCPT作用SMS-KCNR细胞24h及48h，分别使细胞周期阻滞于S期及G2/M期。
第二部分羟基喜树碱对人神经母细胞瘤SMS-KCNR细胞凋亡的影响
目的：
研究HCPT在体外对人NB细胞系SMS-KCNR细胞凋亡的诱导作用，并探讨其诱导凋亡的信号转导机制，证实HCPT对SMS-KCNR增殖抑制作用是诱导细胞凋亡的结果。
方法：
用光学显微镜及DAPI染色荧光显微镜观察HCPT作用48h后SMS-KCNR细胞出现的凋亡形态学改变。用琼脂糖凝胶电泳检测DNA Ladder的形成。用流式细胞仪(Annexin V和PI双染法)检测细胞凋亡率的变化。采用Western blot检测不同浓度HCPT(2．5nM、5nM、10nM、20nM)作用SMS-KCNR细胞48h后线粒体凋亡途径中P53、Bcl-2、Bax、cytochrome c、caspase-3及PARP蛋白表达水平的变化。
结果：
1．不同浓度HCPT(2．5nM、5nM、10nM、20nM)作用SMS-KCNR细胞48h后能明显诱导细胞凋亡，凋亡的诱导作用呈剂量依赖性。
2．光学显微镜显示不同浓度HCPT(2．5nM、5nM、10nM、20nM)作用SMS-KCNR细胞48h后使细胞密度呈剂量依赖性降低。DAPI染色荧光显微镜下观察到典型的凋亡小体形成。琼脂糖凝胶电泳检测到明显DNA梯形条带(DNA ladder)。
3．流式细胞仪(AnnexinV和PI双染法)检测显示HCPT作用SMS-KCNR细胞48h后细胞凋亡率呈剂量依赖性增加。在2．5～20nM的HCPT作用48h后，早期凋亡细胞分别为12．22～50．78%，晚期凋亡细胞分别为8．67～67．32%，总凋亡细胞分别为20．89～97．66%，明显高于对照组(分别为4．15%、0．13%、4．28%)。
4．HCPT(2．5～20nM)作用于SMS-KCNR细胞48h后，随作用浓度的增加，P53表达逐渐增高，呈显著的浓度依赖性。
5．HCPT(2．5～20nM)作用于SMS-KCNR细胞48h后，Bcl-2、Bax表达水平、Bax/Bcl-2比值未见改变。
6．HCPT(2．5～20nM)作用于SMS-KCNR细胞48h后，随作用浓度的增加，胞质cytochrome c表达逐渐增高，线粒体cytochrome c表达逐渐降低，呈显著的浓度依赖性。
7．HCPT(2．5～20nM)作用于SMS-KCNR细胞48h后，caspase-3被激活，procaspase-3表达逐渐减少，有活性的17kDa亚基表达逐渐增加，呈显著的浓度依赖性。
8．HCPT(2．5～20nM)作用于SMS-KCNR细胞48h后，PARP被激活，89kDa的裂解片段表达逐渐增加，呈显著的浓度依赖性。
结论：
HCPT能诱导SMS-KCNR细胞凋亡，凋亡的诱导作用呈显著剂量依赖性。HCPT通过P53介导的线粒体凋亡途径，诱导cytochrome c释放，并最终通过激活caspase-3及PARP促使细胞凋亡。HCPT有临床应用治疗NB的可行性。
第三部分羟基喜树碱治疗复发和难治性神经母细胞瘤的疗效观察
目的：
复发和难治性NB疗效差，生存时间短，死亡率高。HCPT是纯天然生物碱抗肿瘤药，对多种肿瘤有较好疗效，但它在NB的治疗作用国内外尚未见报道。本部分在体外预实验的基础上应用HCPT治疗复发和难治性NB，探讨其疗效。
方法：
10例复发和2例难治性NB患儿接受HCPT化疗，其中5例复发患儿接受HCPT单药治疗，余7例患儿接受HCPT联合治疗。HCPT单药治疗用法：7．5mg/m~2，静脉滴注，d1～14；HCPT联合化疗采用改良新A1与改良B方案交替应用。改良新A1方案：CTX 1200mg/m~2 d1、VP16 100mg/m~2 d1～5、HCPT 5mg/m~2 d1～3、CDDP90mg/m~2 d4；改良B方案：IFO 1．5g/m~2 d1～5、HCPT5mg/m~2 d1～3、CBP450mg/m~2d2，均采用静脉滴注。
结果：
部分缓解4例(33．3%)，疾病稳定8例(66．7%)。有效患者中位缓解时间3．5(2～5)个月。HCPT单药治疗不良反应轻微，HCPT联合化疗的主要不良反应为骨髓抑制及消化道反应，无化疗相关的死亡。
结论：
HCPT治疗复发和难治性NB有一定的近期疗效，毒性可耐受，远期效果需进一步研究。
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 cells
Objective:
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 cells
Objectives:
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 neuroblastoma
Objectives:
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.
NB是儿童期最常见的颅外实体瘤，它不但恶性程度高、侵袭力强，而且早期转移率高、晚期病例多、治疗效果差。尤其是晚期高危病例，常因对一线药物不敏感导致耐药复发，长期生存率很低，是制约NB患儿生存率提高的主要原因。因此积极寻找有效的二线化疗药物是提高NB患儿生存率的重要手段。HCPT是我国利用天然资源优势研制出的纯天然生物碱抗肿瘤药，对多种肿瘤具有较好的疗效，且毒副作用较低、价格低廉。但其在小儿NB的治疗作用国内外尚未见报道。因此本研究通过体外实验结合临床治疗来探讨HCPT对NB的疗效，为探索NB新的、有效的化疗方案，提高生存率，提供理论基础与临床经验。
第一部分羟基喜树碱对人神经母细胞瘤SMS-KCNR细胞增殖的影响
目的：
研究HCPT在体外对人NB细胞系SMS-KCNR细胞增殖和细胞周期的影响。
方法：
采用MTT法观察不同浓度的HCPT(2．5～200nM)作用不同时间(24h、48h、72h)后对SMS-KCNR细胞增殖的影响。同时应用平板集落形成试验观察不同浓度的HCPT(2．5nM、5nM、7．5nM、10nM、20nM)对SMS-KCNR细胞集落形成的影响。用不同浓度的HCPT(2．5nM、5nM、10nM、20nM)作用SMS-KCNR细胞24h及48h，碘化丙锭(PI)染色后用流式细胞仪检测并分析细胞周期的改变。
结果：
1．HCPT能显著抑制SMS-KCNR细胞增殖，并随药物浓度的升高和作用时间的延长而抑制作用增强，呈明显浓度和时间依赖性。
2．在HCPT(2．5～200nM)作用24h及48h后SMS-KCNR细胞增殖抑制率分别为3．6～64．7%、12．5～91．7%，而2．5～20nM浓度范围HCPT作用72h的抑制率为36．7～97%。
3．HCPT能明显抑制SMS-KCNR细胞集落形成，浓度为10nM和20nM时能完全抑制其集落形成。
4．经流式细胞仪检测，不同浓度HCPT(2．5nM、5nM、10nM、20nM)作用SMS-KCNR细胞24h，使细胞周期阻滞于S期；作用48h使细胞周期阻滞于G2/M期。
结论：
HCPT能显著抑制SMS-KCNR细胞增殖和集落形成，其抑制作用呈时间和剂量依赖性。HCPT作用SMS-KCNR细胞24h及48h，分别使细胞周期阻滞于S期及G2/M期。
第二部分羟基喜树碱对人神经母细胞瘤SMS-KCNR细胞凋亡的影响
目的：
研究HCPT在体外对人NB细胞系SMS-KCNR细胞凋亡的诱导作用，并探讨其诱导凋亡的信号转导机制，证实HCPT对SMS-KCNR增殖抑制作用是诱导细胞凋亡的结果。
方法：
用光学显微镜及DAPI染色荧光显微镜观察HCPT作用48h后SMS-KCNR细胞出现的凋亡形态学改变。用琼脂糖凝胶电泳检测DNA Ladder的形成。用流式细胞仪(Annexin V和PI双染法)检测细胞凋亡率的变化。采用Western blot检测不同浓度HCPT(2．5nM、5nM、10nM、20nM)作用SMS-KCNR细胞48h后线粒体凋亡途径中P53、Bcl-2、Bax、cytochrome c、caspase-3及PARP蛋白表达水平的变化。
结果：
1．不同浓度HCPT(2．5nM、5nM、10nM、20nM)作用SMS-KCNR细胞48h后能明显诱导细胞凋亡，凋亡的诱导作用呈剂量依赖性。
2．光学显微镜显示不同浓度HCPT(2．5nM、5nM、10nM、20nM)作用SMS-KCNR细胞48h后使细胞密度呈剂量依赖性降低。DAPI染色荧光显微镜下观察到典型的凋亡小体形成。琼脂糖凝胶电泳检测到明显DNA梯形条带(DNA ladder)。
3．流式细胞仪(AnnexinV和PI双染法)检测显示HCPT作用SMS-KCNR细胞48h后细胞凋亡率呈剂量依赖性增加。在2．5～20nM的HCPT作用48h后，早期凋亡细胞分别为12．22～50．78%，晚期凋亡细胞分别为8．67～67．32%，总凋亡细胞分别为20．89～97．66%，明显高于对照组(分别为4．15%、0．13%、4．28%)。
4．HCPT(2．5～20nM)作用于SMS-KCNR细胞48h后，随作用浓度的增加，P53表达逐渐增高，呈显著的浓度依赖性。
5．HCPT(2．5～20nM)作用于SMS-KCNR细胞48h后，Bcl-2、Bax表达水平、Bax/Bcl-2比值未见改变。
6．HCPT(2．5～20nM)作用于SMS-KCNR细胞48h后，随作用浓度的增加，胞质cytochrome c表达逐渐增高，线粒体cytochrome c表达逐渐降低，呈显著的浓度依赖性。
7．HCPT(2．5～20nM)作用于SMS-KCNR细胞48h后，caspase-3被激活，procaspase-3表达逐渐减少，有活性的17kDa亚基表达逐渐增加，呈显著的浓度依赖性。
8．HCPT(2．5～20nM)作用于SMS-KCNR细胞48h后，PARP被激活，89kDa的裂解片段表达逐渐增加，呈显著的浓度依赖性。
结论：
HCPT能诱导SMS-KCNR细胞凋亡，凋亡的诱导作用呈显著剂量依赖性。HCPT通过P53介导的线粒体凋亡途径，诱导cytochrome c释放，并最终通过激活caspase-3及PARP促使细胞凋亡。HCPT有临床应用治疗NB的可行性。
第三部分羟基喜树碱治疗复发和难治性神经母细胞瘤的疗效观察
目的：
复发和难治性NB疗效差，生存时间短，死亡率高。HCPT是纯天然生物碱抗肿瘤药，对多种肿瘤有较好疗效，但它在NB的治疗作用国内外尚未见报道。本部分在体外预实验的基础上应用HCPT治疗复发和难治性NB，探讨其疗效。
方法：
10例复发和2例难治性NB患儿接受HCPT化疗，其中5例复发患儿接受HCPT单药治疗，余7例患儿接受HCPT联合治疗。HCPT单药治疗用法：7．5mg/m~2，静脉滴注，d1～14；HCPT联合化疗采用改良新A1与改良B方案交替应用。改良新A1方案：CTX 1200mg/m~2 d1、VP16 100mg/m~2 d1～5、HCPT 5mg/m~2 d1～3、CDDP90mg/m~2 d4；改良B方案：IFO 1．5g/m~2 d1～5、HCPT5mg/m~2 d1～3、CBP450mg/m~2d2，均采用静脉滴注。
结果：
部分缓解4例(33．3%)，疾病稳定8例(66．7%)。有效患者中位缓解时间3．5(2～5)个月。HCPT单药治疗不良反应轻微，HCPT联合化疗的主要不良反应为骨髓抑制及消化道反应，无化疗相关的死亡。
结论：
HCPT治疗复发和难治性NB有一定的近期疗效，毒性可耐受，远期效果需进一步研究。
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 cells
Objective:
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 cells
Objectives:
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 neuroblastoma
Objectives:
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.
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