(B) meanSE of the amplitude of 10% FBS-induced intracellular Ca2+ release and SOCE in pCRC and mCRC cells

(B) meanSE of the amplitude of 10% FBS-induced intracellular Ca2+ release and SOCE in pCRC and mCRC cells. technique revealed that constitutive Ca2+ entry was significantly enhanced in pCRC cells and was inhibited by the pharmacological and genetic blockade of Stim1, Stim2, Orai1 and Orai3. The larger resting Ca2+ influx in pCRC was associated to their lower ER Ca2+ content as compared to mCRC cells. Pharmacological and genetic blockade of Stim1, Stim2, Orai1 and Orai3 prevented ER-dependent Ca2+ release, ETC-1002 thereby suggesting that constitutive SOCE maintains ER Ca2+ levels. Nevertheless, pharmacological and genetic blockade of Stim1, Stim2, Orai1 and Orai3 did not affect CRC cell proliferation and migration. These data provide the first evidence that Stim and Orai proteins mediate constitutive Ca2+ entry and replenish ER with Ca2+ in primary cultures of CRC cells. However, SOCE is not a promising target to design alternative therapies for CRC. experiments using as target cells CRC derived from primary tumor or from metastasis obtained during surgical resection and cultured test). Open in a separate window Figure 2 Expression of Stim1-2, ETC-1002 Orai1 and Orai3 proteins in patients-derived colorectal cancer cellsBlots representative of four (each from a distinct patient) were shown. Lanes were loaded with 30 g of proteins, probed with affinity purified antibodies and processed as described in Materials and Methods. The same blots were stripped and re-probed with anti-beta-2-microglobulin (B2M) polyclonal antibody, as housekeeping. Major bands of the expected molecular weights were observed: Stim1 (A), Stim2 (B), Orai1 (C) and Orai3 (D). Bands were acquired, densitometric analysis of the bands was performed by Total Lab V 1.11 computer program (Amersham Biosciences Europe, Italy) and the results were normalized to the corresponding B2M. In a separate set of experiments, we evaluated the expression of some members of the Transient Receptor Potential (TRP) Canonical (TRPC) subfamily, which may mediate SOCE in cancer cells [9, 30, 31]. The comparison of Ct values showed that TRPC3 and TRPC5 transcripts were up-regulated, while TRPC4 and TRPC5 mRNAs were down-regulated in mCRC cells (Supplementary Figure 1). Nevertheless, western blot analysis revealed that there was no difference in the expression levels of TRPC proteins between pCRC and mCRC cells. In more detail, immunoblots displayed a major band of about 92 kDa for TRPC1 ETC-1002 (Supplementary Figure 2A), whereas TRPC3/6/7 and TRPC4 exhibited major bands of 96 kDa (Supplementary Figure 2B and Supplementary Figure 2C, respectively). Therefore, TRPC channels are expressed and have the potential to mediate extracellular Ca2+ entry in CRC cells. Constitutive SOCE is significantly larger in pCRC cells as compared to mCRC cells In order to assess whether Stim and Orai proteins mediate SOCE in CRC cells, we exploited the single-cell Ca2+ imaging technique by loading the cells with the Ca2+-sensitive fluorophore, Fura-2/AM, as described for our types of cancer cells [15, 26, 27]. Our preliminary recordings showed that intracellular Ca2+ levels were stable in both pCRC and mCRC cells, which lacked spontaneous Ca2+ activity. There was no difference in resting Ca2+ levels between the two cell types, as the basal Fura-2/AM fluorescence was 0.840.009 a.u. (n=314) and 0.790.016 a.u. (n=150) in pCRC and mCRC cells (Supplementary Figure 3), respectively. Then, in order to assess whether they displayed a constitutive Ca2+ entry, we simply removed Ca2+ from the extracellular solution (0Ca2+). This maneuver caused a rapid and reversible drop in basal Ca2+ levels (Supplementary Figure 3), which was significantly larger Myh11 in pCRC cells and was consistent with a resting Ca2+ permeability in both cell types. To further characterize the nature of this resting Ca2+ influx pathway, ETC-1002 we turned to the Mn2+-quenching technique. Extracellular Mn2+ is able to flow through most of Ca2+-permeable channels, including Orai channels, thereby causing a drop in Fura-2 fluorescence, which is independent on intracellular Ca2+ concentration ([Ca2+]i) and is more evident at 360 nm, i.e. the isosbestic wavelength for Fura-2 [15, 17]. As shown in Figure ?Figure3,3, there was a clear decay in Fura-2 fluorescence upon substitution of extracellular Ca2+ with Mn2+ in both pCRC and mCRC cells, which displayed a rather linear quenching of Fura-2 fluorescence. This finding further corroborates the notion that a constitutive Ca2+ entry pathway is active in ETC-1002 both cell types. As discussed elsewhere [15], the.