Lymphoma Immunochemotherapy: Targeted Delivery of Doxorubicin via a Dual Functional Nanocarrier.

Chemotherapy drug (paclitaxel, PTX) incorporated in a dual functional polymeric nanocarrier, PEG-Fmoc-NLG, has shown promise as an immunochemotherapy in a murine breast cancer model, 4T1.2. The formulation is composed of an amphiphilic polymer with a built-in immunotherapy drug NLG919 that exhibits the immunostimulatory ability through the inhibition of indoleamine 2,3-dioxygenase 1 (IDO-1) in cancer cells. This work evaluates whether the PEG-derivatized NLG polymer can also be used for delivery of doxorubicin (Dox) in treatment of leukemia. The Dox-loaded micelles were self-assembled from PEG-Fmoc-NLG conjugate, which have a spherical shape with a uniform size of ∼120 nm. In cultured murine lymphocytic leukemia cells (A20), Dox-loaded PEG-Fmoc-NLG micelles showed a cytotoxicity that was comparable to that of free Dox. For in vivo studies, significantly improved antitumor activity was observed for the Dox/PEG-Fmoc-NLG group compared to Doxil or the free Dox group in an A20 lymphoma mouse model. Flow cytometric analysis showed that treatment with Dox/PEG-Fmoc-NLG micelles led to significant increases in the numbers of both total CD4+/CD8+ T cells and the functional CD4+/CD8+ T cells with concomitant decreases in the numbers of myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Treg). Dox/PEG-Fmoc-NLG may represent a promising immunochemotherapy for lymphoma, which warrants more studies in the future.

Insight into mechanisms of cellular uptake of lipid nanoparticles and intracellular release of small RNAs.

PURPOSE: Understanding mechanisms of cellular uptake and intracellular release would enable better design of nanocarriers for delivery of nucleic acids such as siRNA and microRNA (miRNA). METHOD: In this study, we investigated cellular pharmacokinetics of siRNA by co-encapsulating fluorescently labeled siRNA and molecular beacon (MB) in four different formulations of cationic lipid nanoparticles (LNPs). A miRNA mimic was also used as a probe for investigating cellular pharmacokinetics, which correlated well with RNAi activities. RESULTS: We tried to find the best LNP formulation based on the combination of DOTMA and DODMA. When the DOTMA/DODMA ratio was at 5/40, the LNP containing a luciferase siRNA produced the highest gene silencing activity. The superior potency of DOTMA/DODMA could be attributed to higher uptake and improved ability to facilitate siRNA release from endosomes subsequent to uptake. CONCLUSIONS: Our findings may provide new insights into RNAi transfection pathways and have implications on cationic LNP design.

[A review on treatment of high-risk neuroblastoma].

So far treatment of advanced neuroblastoma is still difficult, due to its high malignancy. Currently comprehensive therapies, including high-dose multi-drug chemotherapy, surgery, stem cell transplantation, radiation, biological therapy and immune therapy as well as target therapy dominant the treatment of this disease, and we hereby introduce the latest development of treatment protocols for this disease.

[Treatment of endodermal sinus tumor in children].

OBJECTIVE: To study the treatment and outcome of childhood endodermal sinus tumor. METHODS: The clinical data of twelve children with endodermal sinus tumor between April 2000 and July 2013 were reviewed. The basic demographics, stages of the lesion and the treatment outcome were analyzed. Of the twelve patients, seven were boys and five were girls. The age of the disease onset was between 1 and 3.3 years, except one in 11 years. Two patients were in Brodeur Stage I, four in Stage II, two in Stage III, and four in Stage IV. One patient underwent surgery alone, one underwent surgery plus a combination therapy with vincristine, actinomycin and cyclophosphamide (VAC), and the other ten were treated by surgery with the use of cisplatin, etoposide and bleomycin (PEB) before or after the operation. RESULTS: Eleven patients were successfully followed up and ten were alive. The length of survival was 4.5 to 66 months in the 10 patients. In the 10 patients treated with PEB before or after surgery, 8 achieved complete remission, one achieved partial remission and one was not followed up. The major complications associated with the PEB regimen included myelosuppression and gastrointestinal upset symptoms and no late toxicity was observed. CONCLUSIONS: Preoperative or postoperative administration of PEB may be an effective and safe management modality for childhood endodermal sinus tumor. Nevertheless, further validation is warranted in prospective studies involving a larger sample size.

[Clinical efficacy of CCG7942/POG9354 protocol in treatment of peripheral primitive neuroectodermal tumor in children].

OBJECTIVE: To investigate the clinical manifestations, diagnosis, and treatment of peripheral primitive neuroectodermal tumor (pPNET) in children and the survival of patients treated with the CCG7942/POG9354 protocol. METHODS: A retrospective analysis was performed on the clinical data of 10 patients with pPNET admitted from October 2008 to October 2013. Of the 10 patients, 3 had metastasis, while others had no metastasis. The 7 patients without metastasis were treated with the Children's Cancer Study Group 7942 (CCG7942) protocol, and the other 3 patients with metastasis with the Pediatric Oncology Group 9354 (POG9354) protocol. The therapeutic response and chemotherapy-related toxicities were evaluated by WHO criteria and Common Terminology Criteria for Adverse Events (version 4.0). RESULTS: In the 7 patients treated with the CCG7942 protocol, 4 achieved a complete remission (CR), 1 had stable disease, 2 developed progressive disease (PD), and 2 had recurrence. In the 3 patients treated with the POG9354 protocol, 1 achieved a CR, 2 developed PD, 2 had recurrence, and 2 died. For the 7 patients without metastasis, the survival time was 5-60 months, and the event-free survival rate was 71%. For the 3 patients with metastasis, the survival time was 13-25 months, and the event-free survival rate was 33%. All patients developed grade 4 bone marrow suppression, and other grade 1-2 toxicities, including gastrointestinal reactions, liver function impairment, and renal function impairment, were also seen. CONCLUSIONS: CCG7942 protocol is effective and safe for children with non-metastatic pPNET. However, POG9354 protocol has unsatisfactory efficacy in children with metastatic pPNET, so further studies are needed to improve the therapy for this disease.

Target delivery of MYCN siRNA by folate-nanoliposomes delivery system in a metastatic neuroblastoma model.

BACKGROUND: Folate-nanoliposomes delivery system has emerged recently as a specific and safety delivery method and gradually used as the carrier of a variety kinds of drugs including compounds, plasmids and siRNAs. METHODS: In this study, we established a bone marrow and bone metastasis xenograft mouse model by injecting the LA-N-5 cell into the bone marrow cavity. Fluorescence microscopy, TUNEL Assay, Quantitative RT-PCR and western blot were conducted to analysis the distribution of folate-nanoliposomes entrapped MYCN (V-myc myelocytomatosis viral related oncogene) siRNA in mice and the relevant suppression effect. RESULTS: The folate-nanoliposomes entrapped MYCN siRNA can be specifically distributed in tumor tissues. Further study shows that folate-nanoliposomes entrapped MYCN siRNA lead to MYCN mRNA expression significantly down-regulated (>50%, and p < 0.05) compared with negative control siRNA treatment. MYCN protein expression was inhibited about 60% in vivo, thus induced tumor cell apoptosis markedly. CONCLUSION: This study point to a new way for treatment of metastatic neuroblastoma and could widen the application of folate-nanoliposomes delivery system in tumor therapy.

[Treatment of nasopharyngeal carcinoma in children].

OBJECTIVE: To evaluate the clinical efficacy of the ARAR0331 protocol for treatment of childhood nasopharyngeal carcinoma. METHODS: The clinical data of eight children with nasopharyngeal carcinoma between May 2004 and May 2012 were retrospectively studied. The eight patients included six boys and two girls, and the onset age was between 3 and 13 years. Six patients were in AJCC Stage Ⅲ, one was in StageⅡA and one was in Stage ⅣA. One patient had been treated with combined radiotherapy and chemotherapy which mainly included EAP, BEP and EA. The other seven patients had been treated with the ARAR0331 protocol provided by the America Children's Oncology Group (COG). RESULTS: The patient who had been treated with combined radiotherapy and chemotherapy developed multiple bony metastasis during the chemotherapeutic period. Four out of seven patients who had been treated with ARAR0331 protocol achieved complete remission, and two achieved partial remission. The seven patients were followed-up from 8 to 75 months and the survival rate was 100%. The ARAR0331 protocol treatment-related complications included radiodermatitis, mucocitis and nausea. Late toxicity was not found. CONCLUSIONS: Based on the limited cases, ARAR0331 protocol appears to be effective and safe for childhood nasopharyngeal carcinoma.

[Childhood rhabdomyosarcoma: a retrospective review of 23 cases].

OBJECTIVE: To study the clinical characteristics, treatment and outcome of childhood rhabdomyosarcoma. METHODS: The clinical data of 23 children with rhabdomyosarcoma from January, 1998 to October, 2008 were retrospectively reviewed. RESULTS: Of the 23 cases, 15 were male and 8 were female, with a mean age of 5 years old (7 months to 12 years old). Based on the American IRS staging system, 2 cases were in stage I, 4 cases in stage II, 8 cases in stage III, and 9 were in stage IV. The primary sites were found in head and neck (14 cases), extremities (4 cases), bladder (2 cases), kidney (1 case), post-peritoneum (1 case) and bile duct (1 case). All of the children were confirmed with rhabdomyosarcoma by biopsy and immunohistochemistry. The clinical manifestations were related to the tumor tissues-induced space occupying, compression and erosion and were aspecific. The patients in different IRS stages were given different treatment regimens. The chemotherapy regimens VDCA, VAC or VadrC were used before 2002. After 2002, the Children's Oncology Group (COG) protocol was employed. The two-year survival rate was 63% in 19 patients who received a combination of surgery, chemotherapy and radiotherapy, but none of 4 patients who received a surgery alone or a combination of surgery and chemotherapy or radiotherapy survived more than two years. CONCLUSIONS: The clinical manifestations of childhood rhabdomyosarcoma are not specific. A combination therapy including surgery, chemotherapy and radiation is effective to the improvement of the survival rate in children with rhabdomyosarcoma.

All-trans retinoic acid is capable of inducing folate receptor ß expression in KG-1 cells.

The high expression of folate receptor (FR) on cancer cells might be a potential target for cancer therapy. In this study, the FR-ß expression and the modulation effect of all-trans retinoic acid (ATRA) in a number of cancer cell lines were analyzed. The gateway of ATRA activity on FR-ß expression was further studied by a panel of retinoid activators and inhibitors. The results revealed that ATRA was capable of upregulating the expression of FR-ß protein in KG-1 cells in a dosage-dependent manner, not in KG-1a, NB4, HL60, 293, L1210, JAR, and Hela cells. FR-ß mRNA expression in KG-1 cells was higher when ATRA was present in culture medium at 10⁻6 mol/L for 5 days, and it went down to baseline when ATRA was removed from the medium, vice versa. The upregulation of FR-ß expression in KG-1 cells by ATRA was not associated with cell proliferation and differentiation. In addition, activators of retinoid acid receptor (RAR)α and RARγ, CD336, and CD2781 also induced FR-ß expression. The induction of FR-ß expression by CD336 could be inhibited by RARγ antagonist CD2665; RARß agonist CD-417 and CD-2314 as well as retinoid X receptor (RXR) agonist LG100364 could not induce FR-ß expression. These results indicate that ATRA within a certain range of concentration could reversibly induce the expression of FR-ß in a dosage- and cell type-dependent manner, and its action in KG-1 cells might be associated with the signal transduction of retinoid receptor RARα and RARγ, rather than RARß and RXRs.

Silencing of the MYCN gene by siRNA delivered by folate receptor-targeted liposomes in LA-N-5 cells.

INTRODUCTION: MYCN amplification is highly associated with malignancy and correlates with poor prognosis in patients with neuroblastoma. MATERIALS AND METHODS: We developed a novel liposome-MYCN siRNA-folic acid complex, and the transfection efficacy was measured in LA-N-5 cells by cy-3 fluorescence density in each microgram of protein from the transfected cell lysate. MYCN expression and cell growth were studied with quantitative RT-PCR and MTT assays, and the expression of MYCN protein was studied with Western blot, respectively. An SCID mouse model with subcutaneous LA-N-5 xenografted tumor was established. The animals were divided into four groups (n = 5) and they were peritoneally injected with liposome-encapsulated MYCN siRNA (siRNA 125 µg/kg/day), lipid-encapsulated control siRNA, MYCN siRNA, or liposome only, respectively, for 5 consecutive days. The animals were killed 24 h after the last injection, and the expression of MYCN mRNA in tumor tissue was detected by RT-PCR. RESULTS: Our results are as follows: the transfect efficacy reached 1808.5 ± 140.2 pg siRNA/µg protein in LA-N-5 lysates after treatment with 100 nmol/L MYCN siRNA encapsulated with lipid, and fluorescence could be visualized in 92% of LA-N-5 cells after transfection. At 72 h post-transfection, MYCN mRNA expression in LA-N-5 cells was downregulated by 79.2%, MYCN protein was downregulated by 71.3% and cell growth was inhibited by 66.2%, as measured by MTT assay. In the in vivo study, MYCN mRNA expression was knocked down 53.1% in tumor tissues with injection of liposome-encapsulated MYCN siRNA as compared to control siRNA. CONCLUSION: These results suggest that targeted delivery of MYCN siRNA by folate receptor-targeted lipid vesicles into LA-N-5 cells is efficacious and capable of suppressing MYCN mRNA expression both in vitro and in vivo.

[Neuroblastoma LA-N-6 cells express Foxp3 which can be suppressed by chemotherapeutic agents].

OBJECTIVE: To investigate whether neuroblastoma cells LA-N-6 express Foxp3 and whether the expression of Foxp3 is sensitive to chemotherapy by cyclophosvnamide (CTX)and pirarubicin (THP). METHODS: Expression of Foxp3 on LA-N-6 cells was examined by flow cytometry analysis. The dose-effects of chemotherapy drugs including CTX and THP on LA-N-6 cells were investigated by MTT assay. The effects of CTX and THP on Foxp3 expression were examined by flow cytometry and real-time PCR assays. RESULTS: Flow cytometry analysis showed that LA-N-6 cells expressed Foxp3 at a high level. At sub-optimal concentration, chemotherapy drugs CTX and THP significantly down-regulated expression of Foxp3 on LA-N-6 cells at protein level (P<0.05). CTX also decreased the expression of Foxp3 at mRNA level (P<0.05). CONCLSUSIONS: Neuroblastoma cells LA-N-6 express Foxp3 at a high level, which can be suppressed by chemotherapy drugs CTX and THP. These data suggest that chemotherapy might suppress the growth and metastasis of tumor cells partially through inhibiting the expression of Foxp3.

[Effect of topotecan on MAGE gene expression of HPB-AM cells].

OBJECTIVE: To investigate whether topotecan, a novel anti-tumor agent, down-regulates gene expression of melanoma antigen-encoding (MAGE) in HPB-AM cells. METHODS: MAGE mRNA expression of HPB-AM cells was detected by RT-PCR 4, 8, 12 and 16 hrs after different concentrations (0.05, 0.10, 0.15 and 0.20 micromol/L) of topotecan treatment. RESULTS: MAGE mRNA expression of HPB-AM cells decreased with increasing concentrations of topotecan 12 hrs after treatment. The MAGE mRNA expression of HPB-AM cells treated by 0.10, 0.15 and 0.20 micromol/L of topotecan was significantly lower than that in the blank control group (P<0.05). MAGE mRNA expression of HPB-AM cells was significantly reduced in a time-dependent manner after 0.10 micromol/L of topotecan treatment. The MAGE mRNA expression of HPB-AM cells treated by 0.10 micromol/L of topotecan was significantly lower than that in the blank control group 12 and 16 hrs after treatment (P<0.05). CONCLUSIONS: Topotecan is capable of inhibiting the expression of MAGE mRNA of HPB-AM cells in a time- and dose-dependent manner.

[Curative effects of the protocol of CDV combined with CiE as pre-operative chemotherapy in high-risk childhood neuroblastoma].

OBJECTIVE: To evaluate the effects and the toxicity of the protocol of CDV combined with CiE as pre-operative chemotherapy in childhood stage IV neuroblastoma. METHODS: The clinical data of 27 children aged from 1.2 to 8 years with neuroblastoma in stage IV was retrospectively studied. The primary sites of the diseases were abdomen (n = 21), posterior mediastinum (n = 4) and pelvic cavity (n = 2). Twenty three patients had bone marrow metastasis. Twelve patients had bone metastasis. All patients were treated with the CDV protocol (cyclophosphamide + doxorubicin + vincristine) for 3 cycles and the CiE protocol (cisplatin + etoposide) for 2 cycles. Neuroblastoma therapeutic response evaluation criterion and common terminology criteria for adverse events of National Cancer Institute were used to evaluate effects and chemotherapy related toxicity. RESULTS: All patients received the pre-operative chemotherapy. The overall response rate was 82%. After chemotherapy, 24 patients received operations. Total resection of primary tumor was found in 14 patients (58%) and part resection in 10 patients (42%). The most common chemotherapy related toxicity was bone marrow suppression: grade IV suppression of neutrophils (n = 27), reduction in hemoglobin (III grade, n = 7; IV grade, n = 20) and reduction in platelet (III grade, n = 2; IV grade, n = 25). Infection was found in all patients and was controlled with antibiotics. I or II grade lesions of digestive, liver and kidney were found and could be recovered after therapy. Grade I neurotoxicity occurred in 2 patients (7%). The heart function damage was not found in any of patients. CONCLUSIONS: The protocol of CDV combined with CiE as pre-operative chemotherapy might be effective in children with stage IV neuroblastoma.

[Treatment of acute leukemia complicated by invasive aspergillosis in children].

OBJECTIVE: To study the antifungal treatment and intensive chemotherapy in children with acute leukemia and invasive aspergillosis. METHODS: The diagnosis and treatment of 4 cases of childhood acute leukemia complicated by invasive aspergillosis between July 2007 and July 2008 were studied retrospectively. RESULTS: Three children who underwent remission induction chemotherapy for ALL and one who underwent consolidation chemotherapy for AML developed invasive aspergillosis. One child with proven aspergillosis and 3 with possible aspergillosis all had halo sign on CT at diagnosis. Voriconazole or amphotericin B was given as primary therapy. Improvements of fungal lesions were shown by CT after two to four weeks of antifungal therapy. Complete radiologic remissions were achieved between 4 months and one year. The intensive chemotherapy schedule was continued in all of 4 cases. The median time from fungal infection to the continuation of chemotherapy was 35 days. None showed recurrence of fungal infection. CONCLUSIONS: The halo sign on CT may be a reliable indicator for the early diagnosis of invasive aspergillosis. The preemptive antifungal therapy on the basis of the identification of a halo sign and the reversal of immunosuppression may improve the outcome of invasive aspergillosis. Prolonged antifungal treatment during subsequent cycles of chemotherapy permits completion of scheduled intensive chemotherapy without fungal recurrence.

[Response to therapy of 13 children with rhabdomyosarcoma].

OBJECTIVE: To study the clinical response to comprehensive therapy in children with rhabdomyosarcoma. METHODS: Clinical data of 13 children (8 males and 5 females) with rhabdomyosarcoma from January 1998 through October 2005 were retrospectively studied. Their ages ranged from 7 months to 12 years. The 13 cases of rhabdomyosarcoma consisted of 2 cases in stage I, 2 cases in stage II, 3 cases in stage III, and 6 cases in stage IV. Rhabdomyosarcoma was confirmed by biopsy, 12 cases (92.3%) presenting as embryonal type and 1 as alveolar type in histology. One patient underwent surgery treatment alone, one patient received surgery plus local radiation treatment, one patient received surgery plus chemotherapy and 10 patients were administered with a combination of surgery, local radiation treatment and chemotherapy. The chemotherapy protocol before 2002 was VDCA, VAC or VadrC. After 2002, the COG protocol was employed, with CDV+IE for stage III, and CT+VAC or CT+VAC+VCT for stage IV patients. RESULTS: The 2-year overall survival was 60% in the 10 patients who received a combination of surgery, local radiation treatment and chemotherapy, but the three patients died without receiving combination therapy. The 2-year overall survival in the 13 patients was 46.2%. The 2-year overall survival of the patients after 2002 (60%, 3/5) was higher than that before 2002 (37.5%, 3/8). CONCLUSIONS: Embryonal rhabdomyosarcoma dominates the histology type in children, which is highly malignant. A combination therapy of surgery, local radiation and chemotherapy can result in a satisfactory therapeutic effect in children with rhabdomyosarcoma.

[Matrine inhibits the proliferation of neuroblastoma LA-N-5 cell and MYCN mRNA expression].

OBJECTIVE: Neuroblastoma is the most common malignant solid tumor in children under 4 years. Amplification of MYCN oncogene is associated with advanced-stage disease, rapid tumor progression, resistance to treatment, and poor outcome. Matirne has the anti-tumor activity. This study was designed to investigate the effects of matrine on LA-N-5 cell line proliferation and MYCN gene mRNA expression. METHODS: Neuroblastoma LA-N-5 cells were treated by 0.25, 0.50, 0.75 or 1.00 mg/mL matrine. MTT was used to measure the levels of the proliferation of LA-N-5 cells cultured with different concentrations of matrine. MYCN gene mRNA expression in LA-N-5 cells was measured using real time RT-PCR with SYBR GREEN I fluorescence. RESULTS: The proliferation of LA-N-5 cells was obviously inhibited by matrine in a dose- and time- dependent manner. Matrine of 1.00 mg/mL treatment for 72 hrs produced a best effect, with an inhibitory rate of LA-N-5 cell proliferation of 36.3% and an inhibitory rate of MYCN gene mRNA expression of 44.6%. CONCLUSIONS: Matrine may inhibit the growth of neuroblastoma cells and down-regulate MYCN mRNA expression. It may be promising as a new drug for treatment of neuroblastoma.

All-trans retinoic acid induces autophagic degradation of ubiquitin-like modifier activating enzyme 3 in acute promyelocytic leukemia cells.

All-trans retinoic acid (ATRA) has demonstrated notable success in the treatment of acute promyelocytic leukemia (APL) by inducing granulocytic differentiation. The underlying mechanisms of ATRA therapeutic effects have not been entirely clarified. Here, we reported that the regulation of neddylation, a ubiquitination-like post-translational modification, was involved in the treatment of ATRA on APL. Treating APL cells with ATRA led to the degradation of UBA3, a subunit of neddylation E1. Lysosome-autophagy pathway but not proteasome pathway was responsible for the degradation of UBA3. Neddylation suppression in APL cells was capable of inducing apoptosis, differentiation and proliferation inhibition, suggesting a pivotal role of neddylation in APL cells. ATRA treatment also led to UBA3 degradation in primary APL cells. Taken together, our findings indicated that neddylation was important to maintain the malignant features of APL cells, and suppression of neddylation was involved in the effects of ATRA on APL cells.

[Clinical features and prognosis of advanced neuroblastoma in children].

OBJECTIVE: To investigate the clinical features, treatment modalities and the prognosis of advanced neuroblastoma in children. METHODS: The medical records of 63 children with stage III or IV neuroblastoma from January 1996 to December 2005 were retrospectively reviewed. Sixty patients were treated by tumor resection and (or) chemotherapy and (or) radiation. Fourteen out of the 60 patients received another autologous peripheral blood stem cell transplantation. RESULTS: Of the 63 patients with advanced neuroblastoma, the male/female ratio was 2.7:1 and the median age at diagnosis was 4 years old. Most of the initial symptoms included pyrexia, abdominal pain, abdominal mass, and leg or articular pain. Primary tumor sites were adrenal (38%), retroperitoneal (35%), mediastinal (17%), pelvic (6%) and cervical (2%). The sites of metastasis at diagnosis included local (41%) and (or) distant (37%) lymph nodes, bone marrow (60%), bone (46%) and liver (16%). The median survival time of the 63 patients was 32.7 months. The 2-year survival rate was 44.3%. Statistical analysis demonstrated that unfavorable survival prognostic factors were the following: age > 1 year at diagnosis (P < 0.05); serum neuro-specific enolase > 100 mg/L (P < 0.05); serum lactic dehydrogenase > 1500 U/L (P < 0.01); serum ferritin >150 mg/L (P < 0.05). The overall survival period of the patients was prolonged through total resection of the primary tumor (P < 0.05). Intensive chemotherapy in combination with autologous peripheral blood stem cell transplantation could also result in a prolonged overall survival period (P < 0.01). CONCLUSIONS: Neuroblastoma with advanced stages often presents with various clinical manifestations and has a poor prognosis. It is beneficial to improve the prognosis of neuroblastoma through an early diagnosis and a comprehensive therapy including total resection of the primary tumor, autologous peripheral blood stem cell transplantation and intensive chemotherapy.

[Detection of MYCN mRNA in neuroblastoma cell lines by quantitative RT-PCR].

OBJECTIVE: To examine the feasibility and practicability of quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) with SYBR GREEN I fluorescence for detecting the MYCN mRNA expression in neuroblastoma cell line LA-N-5. METHODS: MYCN mRNA expression in LA-N-5 cells was measured using real time RT-PCR with SYBR GREEN I. Glyceraldehyde phosphate dehydrogenase (GAPDH) was used as internal control. The level of the MYCN mRNA was calculated as MYCN copies/GAPDH copies. RESULTS: Standard curves were linear and showed high correlations (R2>0.99). The ratio of MYCN mRNA copies to GAPDH mRNA copies was calculated based on specific PCR products. The MYCN mRNA level in LA-N-5 cells was obtained (17.4 +/- 1.2). CONCLUSIONS: Quantitative RT-PCR with SYBR GREEN I fluorescence may be a sensitive and reliable method for detecting the MYCN mRNA expression. It may also be potential applicable for detecting the MYCN mRNA expression in the small amount neuroblastoma tissues.