Radiosensitization of Human Melanoma Cells by Ribozyme-Mediated Inhibition of Survivin Expression

Abstract

Survivin is a structurally unique member of the inhibitors of apoptosis protein family and is involved in the control of cell division and inhibition of apoptosis. The notion that survivin is overexpressed in most human tumors but absent in normal adult tissues with only a few exceptions has led to the proposal of survivin as a promising therapeutic target for novel anticancer therapies. In this context, we generated a hammerhead ribozyme targeting the 3' end of the CUA110 triplet in the survivin mRNA. Two human melanoma cell lines (JR8 and M14) overexpressing survivin were stably transfected with the pRc/CMV vector carrying the ribozyme sequence. Two polyclonal cell populations proven to endogenously express ribozyme and characterized by a markedly lower survivin protein level (-60% and -50%, respectively) than JR8 and M14 parental cells were selected for the study. Ribozyme-expressing cells showed a significantly (p<0.01) increased sensitivity to gamma-irradiation (as detected by clonogenic cell survival) compared to JR8 and M14 cells. Moreover, in the JR8 cell line, the extent of radiation-induced apoptosis (in terms of percentage of apoptotic nuclei in cells stained with propidium iodide and level of caspase-3 catalytic activity) was markedly greater in ribozyme-expressing cells than in parental cells. These results demonstrate for the first time that attenuation of survivin expression renders human melanoma cells more susceptible to gamma-irradiation.

Knockdown of survivin expression by small interfering RNA reduces the clonogenic survival of human sarcoma cell lines independently of p53

“…8,10,12,23,25,26 However, the fate of these polyploid cells is not completely known. Chen and co-workers suggested that many of the polyploid cells undergo apoptosis; however, we and others 12,13 did not observe a large number of these cells undergoing apoptosis. Interestingly, in the clonogenic survival assay, polyploid cells were alive after 10-14 days.…”

“…9,11,13,34,35 In a preliminary investigation, we found that an exogenous apoptotic stimulus, for example, radiation, in combination with the knockdown of survivin expression increased the caspase-3 and caspase-7 activity 2.5-fold in the sarcoma cell line A-204, which expresses wild-type p53 (unpublished results). However, even after an apoptotic stimulus is applied, only a small percentage of cells from sarcoma cell lines undergoes apoptosis.…”

“…Furthermore, clonogenic survival of cells from each line was reduced by 65-86% (Fig 1c, Table 1). By using antisense oligonucleotides, 8,[10][11][12][34][35][36] ribozymes, 9,13,37 and siRNA (in a colon cancer cell line) 23 to knock down expression of survivin mRNA and protein, other investigators have observed a remarkable reduction in survivin mRNA that ranged from 70 to 90% and a reduction in survivin protein that ranged from 60 to 90%. The study conducted by Williams et al 23 has described the application of survivin-specific siRNA, which differs from the construct applied in our study, resulting in a reduction of survivin protein in colon cancer.…”

“…Treatment with either survivin-specific antisense oligonucleotides or ribozymes has resulted in reduced survivin expression, which has been correlated with a decrease in tumor growth in mice, with an increase in caspasedependent cell death (apoptosis), and with the formation of polyploid cells in vitro. [8][9][10][11][12][13][14][15][16] In addition to the use of antisense oligonucleotides and ribozymes, the application of small interfering RNA (siRNA) is an effective way to silence gene transcription. 17,18 Transcription silencing is thought to be a defense strategy that preserves genomes through evolution from attack by double-stranded RNA viruses and transposons.…”

The effects of knockdown of wild-type survivin, survivin-2B or survivin-Δ3 on the radiosensitization in a soft tissue sarcoma cells in vitro under different oxygen conditions

“…5,[16][17][18][19][20] Furthermore, survivin overexpression was associated with radioresistance in tumor cell lines. [21][22][23][24][25][26] The knockdown of survivin by ribozymes resulted in an increase of sensitivity of mt-p53 melanoma cells to irradiation. 22 Moreover, transfection of survivin made radiosensitive pancreatic cancer cells more radioresistant and inhibited the activity of caspase-3.…”