ADCK1 is a potential therapeutic target of osteosarcoma.

We here showed that ADCK1 (AarF domain-containing kinase 1), a mitochondrial protein, is upregulated in human osteosarcoma (OS) tissues and OS cells. In primary and established OS cells, ADCK1 shRNA or CRISPR/Cas9-induced ADCK1 knockout (KO) remarkably inhibited cell viability, proliferation and migration, and provoked apoptosis activation. Conversely, ectopic ADCK1 overexpression exerted pro-cancerous activity by promoting OS cell proliferation and migration. ADCK1 depletion disrupted mitochondrial functions in OS cells and induced mitochondrial membrane potential reduction, ATP depletion, reactive oxygen species production. Significantly, ADCK1 silencing augmented doxorubicin-induced apoptosis in primary OS cells. mTOR activation is important for ADCK1 expression in OS cells. The mTOR inhibitors, rapamycin and AZD2014, as well as mTOR shRNA, potently decreased ADCK1 expression in primary OS cells. In nude mice, the growth of subcutaneous pOS-1 xenografts was largely inhibited when bearing ADCK1 shRNA or ADCK1 KO construct. Moreover, ADCK1 KO largely inhibited pOS-1 xenograft in situ growth in proximal tibia of nude mice. ADCK1 depletion, apoptosis activation and ATP reduction were detected in pOS-1 xenografts bearing ADCK1 shRNA or ADCK1 KO construct. Together, the mitochondrial protein ADCK1 is required for OS cell growth and is a novel therapeutic target of OS.

Interleukin-24 inhibits the phenotype and tumorigenicity of cancer stem cell in osteosarcoma via downregulation Notch and Wnt/ß-catenin signaling.

Osteosarcoma frequently presents as recurrence and metastasis, even if the primary lesion was eradicated and/or radiotherapy and chemotherapy were administered. Osteosarcoma cancer stem cells (CSCs) are one of the key factors for the recurrence and metastasis of osteosarcoma. We have shown that interleukin-24 (IL-24) inhibits osteosarcoma cell proliferation, migration and invasion in vitro. In the current study, we investigated the role of IL-24 in inhibiting the growth of osteosarcoma CSCs. IL-24 inhibited proliferation and invasion and decreased the stemness of osteosarcoma CSCs in vitro. In a nude mouse xenograft model, IL-24 significantly inhibited the growth of tumors originating from osteosarcoma CSCs. Moreover, we found that IL-24 was able to inactivate both Notch and Wnt/ß-Catenin signaling, which are important for the development of the biological characteristics of CSCs. These data demonstrate that IL-24 is able to kill not only cancer cells but also CSCs in osteosarcoma, suggesting that IL-24 might eradicate osteosarcoma and enhance long-term cure rates in patients with osteosarcoma.

Ubiquitin ligase RNF5 serves an important role in the development of human glioma.

The ubiquitin ligase ring finger protein 5 (RNF5) has previously been associated with the development of breast cancer. Patients with breast cancer and high RNF5 expression have been demonstrated to have a shorter survival time compared with patients with low RNF5 expression. However, the role of RNF5 in human glioma has not been determined. The present study analyzed the role of RNF5 in gliomas using bioinformatics analysis. The results revealed that RNF5 was differentially expressed in non-cancerous brain tissues and different grades of glioma. Furthermore, a high RNF5 expression in patients with glioma was associated with an improved prognosis compared with patients with low expression. Gene Set Enrichment Analysis revealed that RNF5 was particularly associated with 'Wnt signaling pathway', 'apoptosis', 'focal adhesion' and 'cytokine-cytokine receptor interaction' in patients with glioma. Additionally, 4 potential ubiquitination substrates for RNF5 were predicted, including sorting nexin 10, proprotein convertase subtilisin/kexin type 1, leucine rich glioma inactivated 1 and solute carrier family 39 member 12. These findings provided the basis for further investigation on the role of RNF5 in tumors.

Overexpression of CD155 relates to metastasis and invasion in osteosarcoma.

The rapid development of metastatic lesions remains the leading cause of mortality for patients with osteosarcoma. CD155 serves a key role in cancer cell migration, invasion and metastasis. However, the function and mechanism of CD155 has not been explored in osteosarcoma metastasis. In the present study, we found that CD155 was significantly upregulated in lung metastatic tissue and the highly metastatic cell line K7M2-WT (K7M2) of osteosarcoma. Overexpression of CD155 in K7M2 cells enhanced lung metastasis, while inhibition of CD155 by an anti-CD155 monoclonal antibody reduced metastasis. Blocking of CD155 also decreased migration and invasion of K7M2 cells in vitro. A western blot analysis revealed that blocking of CD155 inhibits metastasis by downregulating focal adhesion kinase (FAK) and phosphorylated FAK (pFAK) in osteosarcoma. The results revealed that CD155 serves a crucial role in the metastasis of osteosarcoma by regulating FAK and may provide a novel molecular target for therapeutic intervention in metastatic osteosarcoma.

Anti-cancer effect of oncolytic adenovirus-armed shRNA targeting MYCN gene on doxorubicin-resistant neuroblastoma cells.

Chemotherapy is one of the few effective choices for patients with neuroblastoma. However, the development of muti-drug resistance (MDR) to chemotherapy is a major obstacle to the effective treatment of advanced or recurrent neuroblastoma. The muti-drug resistance-associated protein (MRP), which encodes a transmembrane glycoprotein, is a key regulator of MDR. The expression of MRP is a close correlation with MYCN oncogene in neuroblastoma. We have recently shown ZD55-shMYCN (oncolytic virus armed with shRNA against MYCN) can down-regulate MYCN to inhibit tumor cells proliferation and induce apoptosis in neuroblastoma. Here we further report ZD55-shMYCN re-sensitized doxorubicin-resistant cells to doxorubicin (as shown by reduced proliferation, increased apoptosis, and inhibited cell migration), and reduced the in vivo growth rate of neuroblastoma xenografts by down-regulation of MRP expression. Sequential therapy with doxorubicin did not affect the replication of ZD55-shMYCN in doxorubicin-resistant neuroblastoma cells, but decreased the expression of Bcl-2, Bcl-XL, MMP-1. Thus, this synergistic effect of ZD55-shMYCN in combination with doxorubicin provides a novel therapy strategy for doxorubicin-resistant neuroblastoma, and is a promising approach for further clinical development.

Interleukin-24 inhibits osteosarcoma cell migration and invasion via the JNK/c-Jun signaling pathways.

Approximately 25% of osteosarcoma patients present with clinically detectable metastatic disease at the time of initial diagnosis. High-dose chemotherapy and/or surgery for the treatment of primary metastatic osteosarcoma is ineffective, and <20% of patients will survive 5 years from diagnosis. Therefore, the treatment of metastases is critical for the improvement of the prognosis of primary metastatic osteosarcoma patients. We have previously observed that overexpression of interleukin-24 (IL-24) inhibits neuroblastoma cell proliferation, migration and invasion in vitro. The present study investigated whether IL-24 may be a novel agent for osteosarcoma metastasis-suppressive treatment. It was observed that IL-24 is able to inhibit migration and invasion in spontaneously metastasizing human 143B osteosarcoma cells via the c-Jun N-terminal kinase (JNK)/c-Jun signaling pathway. IL-24 was effective in inhibiting JNK and c-Jun phosphorylation to downregulate matrix metalloproteinase (MMP)-2 and MMP-9, which contributed to the suppression of cell migration and invasion. It was concluded that IL-24 may be a potent agent in the inhibition of highly metastatic 143B osteosarcoma cells, and IL-24 may have translational potential as an effective therapeutic agent for the treatment of metastatic osteosarcoma.

microRNA-135b expression silences Ppm1e to provoke AMPK activation and inhibit osteoblastoma cell proliferation.

Forced-activation of AMP-activated protein kinase (AMPK) can possibly inhibit osteoblastoma cells. Here, we aim to provoke AMPK activation via microRNA silencing its phosphatase Ppm1e (protein phosphatase Mg2+/Mn2+-dependent 1e). We showed that microRNA-135b-5p ("miR-135b-5p"), the anti-Ppm1e microRNA, was significantly downregulated in human osteoblastoma tissues. It was correlated with Ppm1e upregulation and AMPKα1 de-phosphorylation. Forced-expression of miR-135b-5p in human osteoblastoma cells (MG-63 and U2OS lines) silenced Ppm1e, and induced a profound AMPKα1 phosphorylation (at Thr-172). Osteoblastoma cell proliferation was inhibited after miR-135b-5p expression. Intriguingly, Ppm1e shRNA knockdown similarly induced AMPKα1 phosphorylation, causing osteoblastoma cell proliferation. Reversely, AMPKα1 shRNA knockdown or dominant negative mutation almost abolished miR-135b-5p's actions in osteoblastoma cells. Further in vivo studies demonstrated that U2OS tumor growth in mice was dramatically inhibited after expressing miR-135b-5p or Ppm1e shRNA. Together, our results suggest that miR-135b-induced Ppm1e silence induces AMPK activation to inhibit osteoblastoma cell proliferation.

PI3K/Akt signaling mediated Hexokinase-2 expression inhibits cell apoptosis and promotes tumor growth in pediatric osteosarcoma.

Accumulating evidence has shown that PI3K/Akt pathway is frequently hyperactivated in osteosarcoma (OS) and contributes to tumor initiation and progression. Altered phenotype of glucose metabolism is a key hallmark of cancer cells including OS. However, the relationship between PI3K/Akt pathway and glucose metabolism in OS remains largely unexplored. In this study, we showed that elevated Hexokinase-2 (HK2) expression, which catalyzes the first essential step of glucose metabolism by conversion of glucose into glucose-6-phosphate, was induced by activated PI3K/Akt signaling. Immunohistochemical analysis showed that HK2 was overexpressed in 83.3% (25/30) specimens detected and was closely correlated with Ki67, a cell proliferation index. Silencing of endogenous HK2 resulted in decreased aerobic glycolysis as demonstrated by reduced glucose consumption and lactate production. Inhibition of PI3K/Akt signaling also suppressed aerobic glycolysis and this effect can be reversed by reintroduction of HK2. Furthermore, knockdown of HK2 led to increased cell apoptosis and reduced ability of colony formation; meanwhile, these effects were blocked by 2-Deoxy-d-glucose (2-DG), a glycolysis inhibitor through its actions on hexokinase, indicating that HK2 functions in cell apoptosis and growth were mediated by altered aerobic glycolysis. Taken together, our study reveals a novel relationship between PI3K/Akt signaling and aerobic glycolysis and indicates that PI3K/Akt/HK2 might be potential therapeutic approaches for OS.

Oncolytic adenovirus-mediated short hairpin RNA targeting MYCN gene induces apoptosis by upregulating RKIP in neuroblastoma.

The amplification of MYCN is a typical characteristic of aggressive neuroblastomas, whereas acquired mutations of p53 lead to refractory and relapsed cases. We had previously examined the applicability of the replication-competent oncolytic adenovirus, ZD55-shMYCN, to deliver a short hairpin RNA targeting MYCN gene for p53-null and MYCN-amplified neuroblastoma cell line LA1-55N. Our data have shown that ZD55-shMYCN has an additive tumor growth inhibitory response through shRNA-mediated MYCN knockdown and ZD55-mediated cancer cell lysis. In this regard, ZD55-shMYCN can downregulate MYCN and perform anticancer effects, thereby acquiring significance in the administration of MYCN-amplified and p53-null neuroblastomas. Hence, we further investigated the anticancer properties of ZD55-shMYCN in neuroblastomas. Our data showed that ZD55-shMYCN induced G2/M arrest via decreasing the levels of cyclin D1 and cyclin B1 irrespective of p53 status. ZD55-shMYCN effectively induced apoptosis in neuroblastomas through activation of caspase-3 and enhancing PARP cleavage. Furthermore, ZD55-shMYCN could downregulate phosphoinositide 3-kinase and pAkt and upregulate RKIP levels. Similarly, pro-apoptosis was revealed by the histopathologic examination of paraffin-embedded section of resected tumors of mice xenograft. In vitro and in vivo studies, we elucidate the apoptosis properties and mechanisms of action of ZD55-shMYCN, which provide a promising approach for further clinical development.

[Rectal mucosa stripping and pull-through from rectal muscle sheath of blind pouch in the treatment of congenital high anal atresia in the newborn].

OBJECTIVE: To explore the value of rectal mucosa stripping and pull-through from rectal muscle sheath of blind pouch in the treatment of congenital high anal atresia in the newborn. METHODS: Clinical data of 232 newborns diagnosed as congenital high anal atresia undergoing operation from January 2001 to December 2010 were retrospectively analyzed. Among these patients, 168 underwent rectal mucosa stripping and pull-through from rectal muscle sheath of blind pouch through the previous of sagittal approach (intrathecal pull-through group), and 64 cases underwent the Pena procedure (Pena group). Patients were followed up for two years. Kelly score was used to estimate postoperative anorectal function. Defecography was used to examine the morphology of anorectum. Rectal pressure was measured as well. RESULTS: Two years after operation, Kelly score revealed that 126 (75.0%) cases in the intrathecal pull-through group and 54 cases (84.4%) in the Pena group had good control defecation (P>0.05), while constipation rate was significantly lower in intrathecal pull-through group [8.3% (14/168) vs. 21.9% (14/64), P<0.05]. Postoperative barium defecography showed that defecation rectum maximum diameter was (2.2±0.3) cm in intrathecal pull-through group and (2.3±0.8) cm in the Pena group (P>0.05). Anorectal manometry showed rectal maximum capacity threshold value was (91.4±15.2) ml in the intrathecal pull-through group and (95.1±18.6) ml in the Pena group (P>0.05). There were no significant differences in defecography, anal bowel function and anorectal manometry between the two groups postoperatively (all P>0.05). CONCLUSIONS: Rectal mucosa stripping and pull-through from rectal muscle sheath of blind pouch through the former sagittal can be completed with one-stage operation in newborn for the treatment of congenital high anal atresia, the efficacy of which is similar to the classic Pena operation. This procedure can avoid other operations, ameliorate the pains of newborns, decrease the burden of family, and has lower constipation rate, therefore it is a valid surgical option.

Interleukin-24 inhibits cell migration and invasion in the neuroblastoma cell line SH-SY5Y.

Neuroblastomas are common pediatric solid tumors with a variable clinical course; approximately 50% of patients present with metastatic disease at diagnosis. The development of metastatic lesions often causes a fatal outcome. Therefore, the prevention of metastases during the early stage of tumor development is critical for the improvement of the prognosis of neuroblastoma patients. We previously observed the suppression of neuroblastoma growth in response to overexpression of interleukin-24 (IL-24) in vitro and in vivo. IL-24 exerts its tumor-suppressive effects by multiple mechanisms, including the balance of Bcl-2 family proteins toward the pro-apoptotic pathway and the activation of the caspase cascade. In this study, we used adenovirus-mediated IL-24 (Ad-IL24) to examine the effect of the ectopic production of IL-24 on cell migration and invasion in human neuroblastoma cells. We found that IL-24 effectively inhibits SH-SY5Y neuroblastoma cell migration and invasion by changing subcellular localization and cellular levels of ß-catenin and regulating the levels of proteins associated with cell migration and invasion. Thus, IL-24 should be considered a therapeutic agent that can inhibit primary neuroblastoma growth and that may prevent metastasis.

Adenovirus arming human IL-24 inhibits neuroblastoma cell proliferation in vitro and xenograft tumor growth in vivo.

Data have increasingly shown that interlukin-24 (IL-24) has growth suppression activity and can induce apoptosis in a broad spectrum of tumor cells. However, the therapeutic effect of IL-24 on human neuroblastoma has rarely been explored. In this study, we used a human neuroblastoma cell line (SH-SY5Y) to reveal the effect of adenovirus-mediated IL-24 (Ad-IL24) gene therapy for neuroblastoma. We showed that Ad-IL24 effectively inhibited the proliferation of SH-SY5Y cells in vitro by conspicuously inducing apoptosis. To further explore the molecular mechanism by which Ad-IL24 induced apoptosis in SH-SY5Y tumor cells, we found that Ad-IL24 increased the expression of Bax and promoted the activation of caspase-3, while decreasing Bcl-2 levels. We also demonstrated that Ad-IL24 significantly inhibited tumor growth in vivo in a xenograft neuroblastoma tumor in athymic nude mice. In summary, Ad-IL24 overexpression exerted potent antitumor activity via inducing apoptosis in neuroblastoma cells. Therefore, IL-24 has the potential to serve as an agent for gene therapy in the treatment of neuroblastoma.

Oncolytic adenovirus armed with shRNA targeting MYCN gene inhibits neuroblastoma cell proliferation and in vivo xenograft tumor growth.

PURPOSE: MYCN amplification and p53 inactivation are two typical characteristics of aggressive neuroblastomas and are strongly associated with cancer progression and treatment failure. In an effort to develop new therapeutic agents to treat the aggressive neuroblastomas, we constructed ZD55-shMYCN, an oncolytic adenovirus ZD55 carrying short hairpin RNA (shRNA) targeting MYCN gene, and investigated the effects on proliferation of the p53-null and MYCN-amplified neuroblastoma cell line LA1-55N in vitro and in vivo by ZD55-shMYCN. METHODS: In this study, we used ZD55-shMYCN to treat p53-null and MYCN-amplified neuroblastoma cells. To confirm the ability of selective replication of the ZD55-shMYCN, we examined the expression of E1A protein by western blotting. We used quantitative real-time PCR analysis and western blotting analysis to determine the inhibitory effect of ZD55-shMYCN on MYCN expression. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] cell proliferation assay and xenograft mouse model were used to test the antigrowth efficacy of ZD55-shMYCN. RESULTS: The results showed that ZD55-shMYCN selectively replicated and significantly downregulated the MYCN expression in LA1-55N cells. ZD55-shMYCN effectively inhibited the proliferation in LA1-55N cells in vitro and significantly inhibited tumor growth in vivo xenograft tumor in nude mice. CONCLUSIONS: ZD55-shMYCN provides a novel agent for treating MYCN-amplified and p53-inactive aggressive neuroblastoma, representing a promising approach for further clinical development.

[Modified Mollard procedure for congenital high anorectal malformations via rectal muscle sheath of blind bag out of previous sagittal approach].

OBJECTIVE: To explore the efficacies of treating infants with congenital anorectal malformation by drawing from rectal muscle sheath of blind bag out of previous sagittal approach (modified Mollard procedure). METHODS: Retrospective analyses of postoperative anus control and bowel movements were conducted for 172 patients with high anorectal malformation. The procedures included modified Mollard (n = 68, modified group), Pena (n = 64, Pena group) and abdominal perineal anus forming (n = 40, abdominoperineal group). The tensions of external sphincter and puborectalis were gauged by digital rectal examination and the perianal degree of fecal pollution was assessed by defecography. RESULTS: Among them, 28 boys and 18 girls had a good postoperative control of defecation in the modified group (P = 0.004). The ratios of postoperative external anal sphincter was strong were 73.5% (50/68) and 85.9% (55/64) respectively in the modified and Pena groups and they were higher than that of abdominal perineal group at 55.0% (22/40) (both P < 0.05). The difference in the former two groups was not statistically significant (P = 0.196). The incidence of constipation in the modified group was less than that in the Pena group (13.2% (9/68) vs 31.3% (20/64), P = 0.012). CONCLUSION: Modified Mollard procedure may avoid repeated operations, offer a better control of bowel function, ease patient suffering and improve their postoperative quality-of-life.