Autophagy Inhibition And Its Potential Significance In Childhood Nephroblastoma

ObjectivesNephroblastoma, also known as Wilm’s tumor, is the most common abdominal malignant neoplasms and has the highest mobidity (6%) in pediatric abdominal tumors. Autophagy is a bulk degradation process whereby senile organelle and cytoplasmic contents are engulfed and targeted to lysosomes. Moreover, it has been proved to participate in the onset and progression of various malignant tumors. Associated with a range of illnesses, autophagic dysfunction has been regarded as a novel hallmark of cancer. As a protective mechanism to maintain cellular homeostasis, autophagy plays dual role on cell death. Investigations of autophagy in nephroblastoma have not been reported before. It is known that in nephroblastoma, there are abnormal expressions of Bcl-2, β-catenin and p53, which play key roles in tumorigenesis as well as autophagy regulation. Based on the previous findings, we hypothesize that there could be autophagy deregulation in nephroblastoma. Our study attempts to explore the relationship between autophagy and nephroblastoma with the purpose to find optimized protocol to treat highly malignant nephroblastoma by autophagy regulation as an adjuvant therapeutic strategy.Methods1. Determination of the autophagy level in nephroblastoma.Tissue level:Total RNA was extracted from nephroblastoma tissues (post-therapy) and the surrounding normal kidney tissues by using Trizol reagent, mRNA level of LC3, BECLIN1, P62and ATG5genes was detected by Q-PCR assay. The expression of those genes was assayed by immunohistochemical staining in nephroblastoma tissues (before/after chemotherapy) and surrounding normal kidney tissues as well as tissues from chemoresistant cases. Total proteins of the refered tissues, nephroblastoma tissues (post-therapy) and the surrounding normal kidney tissues were harvest by RIPA lysate, autopahgy related proteins including p62, Beclin1, LC3levels were analyzed by Western-blotting subsequently.2. Clarify if autophagy regulation can efficiently improve inhibition rate of common cancer chemotherapeutics (VCR) in G401or not.3-MA and CQ were applied to block autophagy while rapamycin was used to activate autophagy. Choose nephroblastoma cell G401, set up drug concentration gradient to treat G401, MTT assay was used to detect cell viability thus to find out dose-effect relationship between G401growth and the autophagy-regulatory drugs, and inhibition rate of each drug in G401cell were determined. Inhibition rates of VCR on G401cells before and after combination of autophagy regulation drugs were compared to tell either drug has synergistic or antagonistic or additive effect with VCR.3. Basing on MTT assays, suitable concentration of each autopahgy regulator was used to treat cell, then RIPA lysate was applied to harvest total protein. Western-blotting assay was applied likewise to detect levels of autopahgy related proteins including p62，Beclin1and LC3,thus effective doses of each autopahgy regulator in G401were determined.4. Key autophagy gene (ATG7) was downregulated by using lentivirus to further verify whether autophagy-induction has growth inhibition effect in G401and whether the efficacy of the multi-targeted rapamycin is related to autophagy regulation. Key autophagy-related gene (ATG7) was knocked-down (KD) by lentivirus expressing GFP-SiRNA-ATG7in G401.Different concentration of rapamycin (autophagy inducer), and same drug settings were applied in ATG7normal G401and ATG7KD G401cells. Growth inhibition rates between two groups are compared.Results1. According to Q-PCR result, BECLIN1/P62/ATG5/LC3both had higher expression in nephroblastoma tissues (atfer chemotherapy) on mRNA level.(ACT is presented as mean±SE of3repeated experiments.*P<0.05).Western-blot suggested that:nephroblastoma tissues (atfer chemotherapy),Beclin1level showed no obvious change,while p62accumulated,LC3I and LC3Ⅱ increased.Immunohistochemical staining results:nephroblastoma tissues before chemotherapy, comprared to surrounding normal kidney tissues, presented lower Beclin-1,p62and total LC3levels,particularly Beclin-1.While after chemotherapy Beclin-1, p62, total LC3levels both increased.Moreover, chemoresistance tissues had higer Beclin-1,p62, LC3levels as well.2. Growth inhibition could reach to50%when G401was treated with20nM of VCR according to MTT result.There was no obvious increase or even blockage to growth inhibition of VCR after combination of autophagy inhibitors (CQ/3-MA). After combining rapamycin,a autophagy inducer,however,growth inhibition rised dramatically.Western-blotting results suggested that:in G401,0.5mM3-MA could block (upstream) autophagy effectively. lOuM CQ(downstream) had remarkable inhibition effect,while the effect saturated soon.1μM rapamycin (upstream) can activate autophagy evidently.Meanwhile (0-50nM)VCR itself had no edvident autophagy inhibition effect.3. Knocking-down of autophagy related gene-ATG7in G401:Through MTT assay,found out that when the drug(rapamycin) used alone had linear inhibition trend in small dose scope (1-5μM).Despite lentivirus(transfection required) itself had a great suppression on G401growth itself, after ATG7gene had been knocked-down,the linear inhibition effect on ATG7downregulated G401attenuates evidently.Conclusion1. Overall autophagy inhibition is likely to present in nephroblastoma. Chemotherapy can activate autophagy in nephroblastoma, but whether autophagy activation contributes to tumorigenesis or anticancer effect remains unkown. Autophagy activation may be an underlying mechanism of chemoresistance in nephroblastoma.2. Both autophagy inhibition (by CQ/3-MA or by siRNA of ATG7) and autophagy promotion (by rapamycin) suppresses nephroblastoma cell line G401. The cells are more sensitive to autophagy inhibition.3. Autophagy inhibitory drugs (CQ/3-MA) exhibit no synergistic effect with VCR to inhibit G401cell growth, the effect of co-administration is even antagonistic. However, co-administration of the autophagy-inducer rapamycin with VCR has notable synergistic effect.4. By specifically blocking autophagy by knocking-down of ATG7, it was confirmed that rapamycin inhibits G401cells through regulation of the autophagy pathway.