GLUCOSYLCERAMIDE SYNTHASE IN A NOVEL TARGET FOR CANCER TREATMENT

这个子项目是许多利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 研究耐药基因作为一种新的治疗靶点，可以显著提高肿瘤的治疗效果。葡萄糖神经酰胺合酶（GCS）是神经酰胺糖基化的关键酶，在多种耐药癌细胞系和转移性肿瘤中过表达。高GCS活性通过破坏神经酰胺诱导的细胞凋亡而赋予细胞抗性，所述细胞凋亡与蒽环类、长春花生物碱和紫杉烷类的效率紧密相关。为了确定GCS是否是逆转耐药的靶点，我们评估了体外和体内针对GCS的反义方法的效果。设计MBO-asGCS（mixed-backbone oligonucleotide，20-mer）沉默人GCS基因。MBO-asGCS使耐药人（MCF-7-AdrR）和鼠乳腺癌细胞（EMT 6/AR 1）中的阿霉素敏感性分别显著增加83倍和43倍。相比之下，MBO-asGCS在药物敏感细胞中仅使阿霉素细胞毒性增加两倍，并且不影响人骨髓单核细胞中的细胞毒性。在耐药乳腺癌（MCF-7-AdrR）的原位肿瘤模型中，我们发现MBO-asGCS显著抑制肿瘤生长并使肿瘤对化疗敏感。与单独的MBO-乱序和MBO-乱序与多柔比星的组合（2 mg/kg/周）相比，施用MBO-asGCS（1 mg/kg/3天，42天）抑制肿瘤生长超过64%（356 vs 983 mm 3，N = 10），并使多柔比星敏感性增加58%（187 vs 432 mm 3，N=10）。GCS基因表达水平、神经酰胺糖基化和细胞凋亡的进一步评估表明，MBO-asGCS在体内的作用依赖于内源性GCS过表达的抑制和神经酰胺诱导的细胞凋亡的增强。这些证据表明GCS是逆转癌症耐药性的新靶点。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Characterizing gene underlying drug resistance, as a novel therapeutic target can significantly improve cancer treatment. Glucosylceramide synthase (GCS), a key enzyme for ceramide glycosylation, is overexpressed in various drug-resistant cancer cell lines and in metastatic tumors. High GCS activity confers cell resistance by disrupting ceramide-induced apoptosis that is tightly associated with the efficiency of anthracycline, vinca alkaloids and taxanes. To determine whether GCS is a target for the reversal of drug resistance, we evaluated the effects of antisense approach against GCS in vitro and in vivo. MBO-asGCS (mixed-backbone oligonucleotide, 20-mer) was designed to silence human GCS gene. MBO-asGCS substantially increased doxorubicin sensitivity in drug-resistant human (MCF-7-AdrR) and murine breast cancer cells (EMT6/AR1) by 83-fold and 43-fold, respectively. In contrast, MBO-asGCS only increased the doxorubicin cytotoxicity by twofold in drug-sensitive cells and did not affect that cytotoxicity in human bone marrow mononuclear cells. In the orthotopic tumor model for drug-resistant breast cancer (MCF-7-AdrR), we found that MBO-asGCS significantly inhibited tumor growth and sensitized tumors to chemotherapy. The administration of MBO-asGCS (1 mg/kg/3-day, 42 days) inhibited tumor growth more than 64% (356 vs 983 mm3, N=10) and increased doxorubicin-sensitivity by 58% (187 vs. 432 mm3, N=10), compared to MBO-scrambled alone and the combination of MBO-scrambled with doxorubicin (2 mg/kg/week), respectively. Further assessments of GCS gene expression levels, ceramide glycosylation, and apoptosis demonstrated that the effects of MBO-asGCS in vivo rely on the suppression of endogenous GCS overexpressed and the enhancement of ceramide-induced apoptosis. These evidences pinpoint that GCS is a novel target for the reversal of drug resistance in cancers.