ARP2, a novel pro-apoptotic protein expressed in epithelial prostate cancer LNCaP cells and epithelial ovary CHO transformed cells

Abstract

Neoplastic epithelial cells generate the most aggressive types of cancers such as those located in the lung, breast, colon, prostate and ovary. During advanced stages of prostate cancer, epithelial cells are associated to the appearance of androgen-independent tumors, an apoptotic-resistant phenotype that ultimately overgrows and promotes metastatic events. We have previously identified and electrophysiologically characterized a novel Ca(2+)-permeable channel activated during apoptosis in the androgen-independent prostate epithelial cancer cell line, LNCaP. In addition, we reported for the first time the cloning and characterization of this channel-like molecule named apoptosis regulated protein 2 (ARP2) associated to a lethal influx of Ca(2+) in Xenopus oocytes. In the present study, LNCaP cells and Chinese hamster ovary cells (CHO cell line) transfected with arp2-cDNA are induced to undergo apoptosis showing an important impact on cell viability and activation of caspases 3 and 7 when compared to serum deprived grown cells and ionomycin treated cells. The subcellular localization of ARP2 in CHO cells undergoing apoptosis was studied using confocal microscopy. While apoptosis progresses, ARP2 initially localized in the peri-nuclear region of cells migrates with time towards the plasma membrane region. Based on the present results and those of our previous studies, the fact that ARP2 constitutes a novel cation channel is supported. Therefore, ARP2 becomes a valuable target to modulate the influx and concentration of calcium in the cytoplasm of epithelial cancer cells showing an apoptotic-resistant phenotype during the onset of an apoptotic event.

Figure 1. ARP2 expression induces apoptosis in LNCaP cells.

(A) Western blots detected expression of β-gal-V5 (112 kDa) and ARP2-V5 (54 kDa) in total cell lysates. (B) Normalized cell viability% of controls was assessed using MTT assays with 2×10⁴ cells cultured in the absence of serum or transfected with arp2 cDNA. Viability of these two treatment groups were assayed at 16, 24, 48, and 72 h timepoints. (C) Normalized cell viability% of controls assayed using a trypan blue exclusion method. Cells were cultured in the absence of serum or transfected with arp2 cDNA and assayed at 16, 24, 48, 72, 96, and 120 h timepoints. Mean values are presented (n = 3, X ± S.E.M), ^# p<0.05, *p<0.01, ^¶ p<0.001 compared to control groups.

Figure 2. ARP2 expression induces apoptosis in CHO cells.

(A) Western blots detected expression of β-gal-V5 (112 kDa) and ARP2-V5 (54 kDa) in total cell lysates. (B) Normalized cell viability% of controls was assessed using MTT assays with 2×10⁴ cells cultured in the absence of serum or transfected with arp2 cDNA. Viability of these two treatment groups were assayed at 16, 24, 48, and 72 h timepoints. (C) Normalized cell viability% of controls was assessed using a trypan blue exclusion method. Cells were cultured in the absence of serum or transfected with arp2 cDNA and assayed at 16, 24, 48, and 72 h timepoints. Mean values are presented (n = 3, X ± S.E.M), ^# p<0.05, *p<0.01, ^¶ p<0.001, compared to control groups.

Figure 3. Activity of caspases 3 and 7.

Caspase activity was determined using a fluorometric assay employing Z-DEVD-AFC as a substrate. (A) CHO cells grown in the absence of serum, transfected with arp2 cDNA or incubated with 10 µM ionomycin assayed at 16, 24, 48 and 72 h timepoints. (B) LNCaP cells grown in the absence of serum, transfected with arp2 cDNA (72 h) or incubated with 10 µM ionomycin assayed at 16, 24, 48 and 72 h timepoints. Mean values are presented (n = 3, X ± S.E.M), ^# p<0.05, *p<0.01, ^¶ p<0.001 compared to control groups.

Figure 4. Confocal microscopy of ARP2 fusion protein expressed in CHO cells.

CHO cells transfected with egfp cDNA were observed 24 h post-transfection using confocal microscopy (A) and DIC microscopy (B). CHO cells transfected with arp2-egfp cDNA were also observed 24 h post-transfection using confocal microscopy (C) and DIC microscopy (D). Scale bar 50 µm. White arrows indicate cells that present ARP2 localized to the peri-nuclear region.

Figure 5. Confocal microscopy of ARP2 fusion protein expressed in CHO cells.

CHO cells transfected with arp2-egfp cDNA examined at 16, 24, 48 and 72 h post-transfection (A, C, E and G respectively). Same using DIC microscopy (B, D, F and H respectively). Scale bar 10 µm.