Abstract:
Introduction: Colorectal cancer (CRC) and inflammatory bowel disease (IBD) share pathophysiological features that contribute to their progression, mainly through chronic inflammation. In addition, endoplasmic reticulum (ER) stress, which dysregulates calcium homeostasis, was linked to the progression of CRC and IBD by altering the expression of calcium signaling molecules and junctional proteins. Furthermore, CRC and IBD were shown to modify the expression of specific cationic and anionic transporters (OCTN1,2 and OAT2). OCTN1 and OCTN2 are shown to have anti-inflammatory roles through their specific substrates, acetylcholine, and carnitine, respectively, whereas OAT2 plays a crucial role in transporting anti-cancerous drugs, especially 5-Fluorouracil. Preliminary work in the lab has shown that thapsigargin (TG), a potent ER stress inducer, significantly modulates the protein expression of different proteins involved in cellular integrity, such as Zo-1, Ecad, Cx-43 and in proteins involved in calcium homeostasis, essential for the proper functioning of the ER. However, little information is known about the mechanism of TG-induced ER stress, on the mRNA levels of crucial mediators involved in cell integrity, calcium homeostasis (Pmca1&4, TRPV6, NCX1, Orai-1, Stim 1&2, Serca2&3, RYR1&3, ITRP2&3), and drug uptake (OCTN1,2 and OAT2).
Aim: This study investigates the mechanisms of TG-induced ER stress in two CRC cell lines, Caco-2 and HCT-116, by evaluating 1) the cell cycle profile of two cell lines in response to TG and 2) the mRNA levels of essential mediators involved in maintaining calcium signaling homeostasis and junctional proteins involved in maintaining cellular integrity, and drug uptake. Methods: Two colorectal cancer cell lines (Caco-2 and HCT-116) were used throughout the study. MTT assay and trypan blue assay were used, respectively, to determine the effect of TG concentrations on the metabolic activity and viability of the cells. After selecting the suitable concentration of TG (1 μM), HCT-116 and Caco-2 cell lines were cultured and exposed to 1 μM TG for 4, 8, and 24 hours to induce ER stress. Propidium iodide assay was used to detect variations in the cell cycle profiles of Caco-2 and HCT-116 in untreated and TG-treated cells at different time points. Finally, real-time PCR analysis assessed the mRNA expression levels of ER stress markers (Bip and Chop), OCTN1,2, OAT2, calcium signaling proteins (Pmca1&4, TRPV6, NCX1, Orai-1, Stim 1&2, Serca2&3, RYR1&3, ITRP2&3), and junctional proteins (Zo-1, Ecad, Cx-43). Results: This study showed that 1 μM TG, a concentration known to induce ER stress by blocking SERCA, is suitable for studying TG effects in HCT-116 and Caco-2. TG at 1μM induced cell cycle arrest in both cell lines. Furthermore, PCR analysis indicated differential regulation of key calcium signaling pathways, junctional proteins, and OCTN1, OCTN2, and OAT2, suggesting complex cellular responses to ER stress. Specifically, the stress marker Bip showed a sustained increase at all time points. However, Chop increased at 4 and 24 hours after a significant decrease after 8 hours of treatment. Junctional proteins showed substantial increases after 24 hours of treatment. Channels responsible for calcium efflux (NCX-1, PMCA1&4) showed significant increases after 24 hours of treatment. TRPV-6, a channel responsible for calcium entry, also showed a significant increase after 24 hours. Serca 2&3, RYR 1&3, and ITRP 2&3, channels responsible for calcium transport in the ER, showed substantial increases after 24 hours. Orai-1 and STIM 1&2, elements of the store-operated calcium entry (SOCE) system, also showed significant increases after 24 hours. Finally, OCTNs and OAT2 also showed substantial increases after 24 hours. Conclusion: This study reveals a significant link between ER stress and the dysregulation of calcium signaling and transport mechanisms in two CRC cells. The sustained increase in Bip, a marker of ER stress, indicates that prolonged ER stress critically influences cellular responses in CRC. Notable fluctuations in Chop expression, alongside increases in junctional proteins and calcium transport channels after 24 hours, highlight the complex adaptive processes to ER stress. The upregulation of calcium channels (TRPV-6, NCX-1, PMCA1&4) and SOCE components (Orai-1, STIM 1&2) suggests a compensatory mechanism to restore calcium homeostasis, which is vital for cell proliferation, apoptosis, and survival-key factors in CRC progression. Additionally, the increased expression of OCTN 1/2 and OAT2 may shift cellular transport dynamics, affecting drug metabolism and resistance. However, it is worth noting that previous work in the lab showed ER stress did not affect the protein expression of OCTN2 and OAT2. This finding, along with the increase in the mRNA expression of OCTN2 and OAT2, indicates that the increase in the mRNA levels was not reflected in the protein expression. This exciting observation highlights the importance of further investigating the factors that hindered the expression of these transporters. Overall, these findings advance our understanding of the mechanisms connecting ER stress to signaling pathway dysregulation in CRC. Future research into these pathways, especially in the context of (IBD) and its association with CRC, could further provide new avenues for CRC treatment.