Knockdown of cyclin D1 inhibits proliferation, induces apoptosis, and attenuates the invasive capacity of human glioblastoma cells

Abstract

Elevated cyclin D1 (CCND1) in human glioblastoma correlates with poor clinical prognosis. In this study, the human glioblastoma cell lines SHG-44 and U251 were stably transfected with short hairpin RNA (shRNA) targeting cyclin D1 or with ectogenic cyclin D1 by lentivirus-mediated transfection. Glioblastoma cells overexpressing or underexpressing cyclin D1 were then examined by in vitro growth assays, apoptosis assays, cell cycle analysis, and invasion assays. Cyclin D1 knockdown in SHG-44 cells inhibited cell proliferation, induced apoptosis, and attenuated migration across Matrigel, a model of invasive capacity. Western blot analysis and quantitative reverse-transcription polymerase chain reaction (RT-PCR) revealed that cells underexpressing CCND1 exhibited decreased multidrug resistance protein 1 (MDR1) and B-cell lymphoma-2 (Bcl-2) expression, but enhanced apoptosis effector caspase-3 expression. In contrast, cyclin D1 overexpression promoted cell proliferation, attenuated apoptosis, and enhanced invasive capacity. Furthermore, cyclin D1 overexpression was associated with increased expression of MDR1 and Bcl-2, and decreased caspase-3 expression. Results using the U251 cell line confirmed the effects of CCND1-targeted shRNA and lentivirus-mediated overexpression on proliferation and apoptosis of glioblastoma cells. Overexpression of cyclin D1 enhanced the proliferation and invasive potential of human glioblastoma cells, while reducing apoptosis. The ability to suppress the malignant phenotype by downregulating cyclin D1 expression may provide a new gene therapy approach for patients with malignant glioma.

Coexpression of cyclin D1 and alpha-internexin in oligodendroglial tumors

“…In brain tumors, the expression of cyclin D1 has been reported in medulloblastomas [31]. Overexpression of cyclin D1 in glioblastomas has also been indicated and it has been related with its aggressive behavior [32,33]. At present, it is uncertain whether cyclin D1?…”

Overexpression of cyclin D1 in meningioma is associated with malignancy grade and causes abnormalities in apoptosis, invasion and cell cycle progression

“…The synthetic interfering RNA (siRNA) sequences of cyclin D1 were carried out using pLVTHM vector as described previously, and purity was confirmed by Western blot analyses [7]. The IOMM-Lee and CH157-MN cells were stably transfected with GFP siRNA or cyclin D1-siRNA according to the manufacturer's protocol (Invitrogen).…”

“…Apoptosis assay was performed as reported previously [7]. Apoptosis was measured using an annexin V/fluorescein isothiocyanate (FITC) apoptosis detection kit (Bender Med System, CA).…”

“…The most frequent abnormalities relate to cyclin D1 [4]. This protein can stimulate gene transcription and tumor growth, inhibit apoptosis and cause resistance to chemotherapy [7][8][9][10][11]. Indeed, amplification or increased expression of cyclin D1 has been reported in several types of human cancer, including human breast, colon, lung, esophageal and glioma [6,7,[12][13][14].…”

Inhibition of Cyclin D1 Expression in Human Glioblastoma Cells is Associated with Increased Temozolomide Chemosensitivity

“…Then we demonstrated CCND1 overexpression could upregulate p-gp expression and contribute to temozolomide chemoresistance [21]. Herein, we studied the dysregulation of CCND1 in human astrocytoma tissues by immunohistochemical and Western blot analysis.…”