We examined whether survivin acts as a constitutive and inducible radioresistance factor in pancreatic cancer cells. Using a quantitative TaqMan reverse transcription-polymerase chain reaction for survivin mRNA in five pancreatic cancer cell lines, we found an inverse relationship between survivin mRNA expression and radiosensitivity. PANC-1 cells, which had the highest survivin mRNA levels, were most resistant to X-irradiation; MIAPaCa-2 cells, which showed the least survivin mRNA expression, were the most sensitive to X-irradiation. Our results suggested that survivin could act as a constitutive radioresistance factor in pancreatic cancer cells. To determine whether radioresistance is enhanced by induction of survivin expression by irradiation, PANC-1 and MIAPaCa-2 cells were subjected to sublethal doses of X-irradiation followed by a lethal dose. Survivin mRNA expression was increased significantly in both PANC-1 and MIAPaCa-2 cell lines by pretreatment with a sublethal dose of X-irradiation, as was cell survival after exposure to the lethal dose. In this system, enzymatic caspase-3 activity was significantly suppressed in cells with acquired resistance. These results suggest that survivin also acts as an inducible radioresistance factor in pancreatic cancer cells. Survivin, then, appears to enhance radioresistance in pancreatic cancer cells; inhibition of survivin mRNA expression may improve the effectiveness of radiotherapy.
3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (HRIs) are widely used to reduce serum cholesterol in patients with hypercholesterolemia. Previous studies have shown that HRIs can induce apoptosis in colon cancer cells. In this study, we investigated the mechanisms underlying the apoptosis-inducing effect of HRIs in greater detail. The HRI lovastatin induced apoptosis in the human colon cancer cell line SW480 by blocking the cholesterol synthesis pathway. Immunoblot analysis of antiapoptotic molecules, including survivin, XIAP, cIAP-1, cIAP-2, Bcl-2, and Bcl-X L , revealed that only survivin expression was decreased by lovastatin. Survivin downregulation by RNA interference induced apoptosis, and survivin overexpression rendered the cells resistant to lovastatin-induced growth inhibition. These results indicate that survivin downregulation contributes substantially to the proapoptotic properties of lovastatin. Farnesyl pyrophosphate and geranylgeranyl pyrophosphate, two downstream intermediates in the cholesterol synthesis pathway, simultaneously reversed survivin down-regulation and the blocking of Ras isoprenylation by lovastatin. Ras isoprenylation is important for the activation of Ras-mediated signaling, including the activation of the phosphatidylinositol 3-kinase (PI3-kinase)/Akt pathway. The PI3-kinase inhibitor down-regulated survivin in SW480 cells. In addition, lovastatin blocked Ras activation and Akt phosphorylation. We conclude that survivin down-regulation is crucial in lovastatin-induced apoptosis in cancer cells and that lovastatin decreases survivin expression by inhibiting Ras-mediated PI3-kinase activation via the blocking of Ras isoprenylation.3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) 3 reductase inhibitors (HRIs) are widely used to reduce serum cholesterol and are well tolerated by patients with hypercholesterolemia (1). HRIs prevent the formation of mevalonate from HMG-CoA by inhibiting the enzyme HMG-CoA reductase, thereby inhibiting cholesterol synthesis (2). In large clinical trials designed to study the changes in coronary events in coronary heart disease patients receiving HRIs, the number of newly diagnosed colon cancer cases showed a reduction of between 43% (3) and 19% (4) during a 5-year follow-up period. It has been reported that HRIs could induce apoptosis in colon cancer cells (5-7). Farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP), two downstream intermediates in the cholesterol synthesis pathway, belong to a class of compounds named isoprenoids. They are bound to several cellular proteins, including small GTPases such as Ras, Rho, and Rac, by a posttranslational modification known as isoprenylation. This process involves the addition of a 15-carbon farnesyl chain in FPP (farnesylation) or a 20-carbon geranylgeranyl chain in GGPP (geranylgeranylation) to a cysteine sulfhydryl group near the carboxyl terminus. Isoprenylation of these proteins is crucial for membrane attachment (8, 9) and the subsequent acquisition of biological activity (10)...
mL, and mean arterial pressure was significantly correlated with PHF concentrations [ Fig. 1C; slope, 0.008014 Ϯ 0.003734; y-intercept, Ϫ0.6246 Ϯ 0.3908 units/mL; R ϭ 0.394, P ϭ 0.0417 (significant); n ϭ 27].Our goal was to develop MAbs with high affinities to both the rat and human PHF antigens. The antibodies produced were selective for PHF, as demonstrated in the bioassay and cross-reactivity studies. Results generated with the PHF ELISA to detect PHF in biological samples correlated well with results obtained for PHF with the bioassay. The ELISA offers a distinct advantage over the bioassay in that it is faster, less expensive, and quantitative, making it a more reliable system. Although the antigen used in this ELISA was not highly purified and the structure has not been completely elucidated, the strong correlation between this ELISA and the bioassay indicate that this assay may be useful in studies that will further our understanding of this substance. Highly pure antigen, as well as improved understanding of the etiology of PHF-related disease, will be required for this assay to be used as a routine clinical tool.In the normotensive group, PHF was not correlated with blood pressure, but in the hypertensive group PHF was correlated with blood pressure. The number of hypertensive samples was much lower than the number of normotensive samples because of the restriction placed on sample selection. In most cases, once people are diagnosed with high blood pressure, therapy is initiated. Blood pressures in the hypertensive group were therefore in the lower range of hypertension. In both groups, there were some values that fell outside the expected correlation. This observation is not unexpected because our previous studies have demonstrated that PHF may occur in some patients with other conditions, such as type 2 diabetes mellitus (14 ). PHF expression appears to precede increases in systemic blood pressure, and because hypertension is a heterogeneous disease, not all hypertensive patients express PHF.Creation of an ELISA for PHF in biological samples may facilitate future research into the biology and pathology of PHF in humans. Future clinical studies with larger sample sizes will be required to clearly define the relationship of PHF to hypertension. Availability of a test with which clinicians may subclassify hypertension may ultimately allow for selection of the most appropriate therapeutic regimen without requiring the empirical approach that is currently used.We would like to acknowledge the technical contributions of Lynda Tang and Lei Zhang. This work was supported by University-Industry Grant UI-13521 from the Medical Research Council of Canada (to C.G.B.).
Using gene-transduced pancreatic cancer cells, we examined whether survivin expression is directly involved in regulation of radiosensitivity. Ordinarily radiosensitive MIAPaCa-2 cells transduced with wild-type survivin gene (MS cells) proliferated more rapidly than cells transduced with control vector. MS cells were significantly less radiosensitive than control vector-transduced cells. Radiation-induced activity of caspase-3, but not caspase-7, was significantly inhibited in MS cells. On the other hand, transduction of a dominant-negative mutant survivin gene into radioresistant PANC-1 cells augmented radiosensitivity. Further, the radiation-induced increase in caspase-3 activity was enhanced, indicating that survivin function was truly inhibited. These results indicate that survivin expression directly down-regulates radiosensitivity.
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