The Effect of Thapsigargin-Induced ER Stress on the Expression of Different Proteins(Structural and Functional) and Transporters (OCTN2 and OAT2) in HCT-116 and Caco-2 Cells

Abstract

Introduction: Colorectal cancer (CRC) remains one of the most lethal cancers worldwide, necessitating a comprehensive understanding of its underlying molecular mechanisms to develop more effective therapeutic strategies. Interestingly, among the various risk factors, inflammatory bowel disease (IBD) has been strongly associated with an increased risk of CRC. The chronic inflammation observed in IBD contributes to CRC through several mechanisms, including disrupting junctional proteins that maintain the intestinal epithelial barrier. This disruption compromises the barrier's integrity, facilitating inflammation and promoting tumorigenesis. Junctional proteins, crucial for maintaining epithelial homeostasis, are thus a focal point in understanding how IBD contributes to CRC development. In IBD, chronic inflammation can be exacerbated by endoplasmic reticulum (ER) stress, which can be induced by many factors, among which is calcium depletion. The ER, the calcium store, is the milieu where protein synthesis occurs, and many chaperons responsible for properly folding the proteins are calcium-dependent. It is thus not surprising that ER stress induced by calcium depletion would affect the synthesis of many essential structural and functional proteins that are important for the proper function of the cells. Additionally, the SLC22A family transporters, particularly the organic cation transporters 2 (OCTN2) and organic anion transporters 2 (OAT2), are known for their involvement in cellular transport mechanisms and the pathophysiology of several diseases, including CRC. These transporters play a significant role in the uptake of chemotherapeutic agents, and their differential expression in CRC cell lines versus normal colon cells suggests they may be critical players in cancer progression and treatment resistance. Yet, nothing is known about their expression levels under inflammatory conditions or ER stress and whether their expression profile is compromised in ER stress conditions as observed in IBD, similar to CRC. Aim: Thapsigargin (TG), a potent inhibitor of the Sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA), induces ER stress by disrupting calcium homeostasis. Therefore, under TG-induced ER stress, this study aims to evaluate the expression profile of different junctional proteins responsible for cell integrity alongside the expression of calcium transporters responsible for calcium homeostasis and transporters (OCTN2 and OAT2) involved in many physiological and pathophysiological episodes. The ultimate aim is to uncover potential links between ER stress, calcium regulation, and protein expression regulation in CRC cell lines under ER stress, as observed in IBD/CRC. Methods: HCT-116 and Caco-2 cells were used for all experiments. MTT and Trypan blue assays were used to determine the effect of different concentrations of TG, known to induce calcium depletion, on cell proliferation. Furthermore, western blot analysis was performed to evaluate the expression levels of OCTN2, OAT2, calcium signaling proteins, and junctional proteins in HCT-116 and Caco-2 cells in response to 1 μM TG for 4, 8, and 24h. At each time point, proteins were extracted, quantified using the BCA assay, separated by SDS-PAGE, and detected using specific antibodies. Following western blot analysis, densitometry quantification of the protein bands was conducted using ImageJ to assess relative protein expression levels. The intensity of the bands was normalized against GAPDH to ensure accurate quantification. These normalized values were used to determine the fold change in expression of OCTN2, OAT2, calcium signaling proteins, and junctional proteins under ER stress conditions induced by TG treatment. Results: MTT and trypan blue assays demonstrated that HCT-116 cells are more sensitive to TG-induced-ER stress than Caco-2 cells, as indicated by a more pronounced reduction in cell viability and metabolic activity in response to TG. 1 μM TG, known to induce calcium depletion, was then selected to perform the additional experiments. TG-induced ER stress was confirmed by the significant increase in the ER stress markers BIP and CHOP in both cell lines. Caco-2 cells exhibited a rapid disruption of junctional proteins (ZO-1, CX-43, and ECAD), while HCT-116 cells showed a delayed disruption, highlighting differences in cellular responses between the two CRC cell lines. This is because CX-43 in HCT-116 showed a considerable decrease at 24h-post treatment with TG, while it showed a significant reduction at all time points in Caco-2 cells. However, ECAD showed no change at early time points in HCT-116 with a substantial increase at later time points, while in Caco-2, it showed a significant decrease at early time points with a remarkable increase at 24h. Moreover, the different studied calcium transporters and channels (TRPV6, PMCA1, PMCA4, PMCAT, NCX-1, SERCA2, SERCA3, Orai-1, STIM1, STIM2, ITPR2, ITPR3, RYR1, and RYR3) were significantly disrupted in both cell lines due to calcium depletion induced by TG, reflecting the critical role of calcium homeostasis in ER function. Starting with TRPV6, its expression in HCT-116 at 4h after treatment with TG showed no change; however, it was reduced in Caco-2 cells. On the other hand, PMCA1, PMCA4, and NCX-1 showed a significant decrease in HCT-116 at all time points post-treatment with TG, while no detectable expression was observed in Caco-2 cells at any time point. Nevertheless, ORAI-1 showed no observable expression in HCT-116 at 4h, with a significant reduction at later time points following treatment with TG, while in Caco-2, ORAI-1 expression showed a slight decrease at 4h with no change in its expression at later time points. Besides, SERCA 2 expression in HCT-116 showed a notable increase at 4 and 8h with a critical decrease at 24h; however, in Caco-2 it showed an increase at 8h, followed by a decrease at 24h. Notably, ER stress did not significantly affect the expression of OCTN2 and OAT2 transporters. Conclusion: The obtained findings underscore the complexity of the cellular stress response in CRC, providing insights into the roles of calcium signaling, junctional integrity, and transporters in ER stress. Calcium transporters were significantly modulated in both cell lines, although to a different extent, due to calcium depletion induced by TG, reflecting the critical role of calcium homeostasis in ER function. Moreover, junctional proteins were affected by ER stress, which affects cell-cell contact. These differential expressions observed between HCT-116 and Caco-2 cells may be attributed to genetic factors, particularly the tumor suppressor gene p53 status. HCT-116 cells express wild-type p53, which may contribute to their heightened sensitivity to ER stress, whereas Caco-2 cells carry a mutated version of the gene p53, potentially influencing their adaptive responses to TG-induced stress. Notably, the unchanged expression of OCTN2 and OAT2 in the two cell lines could be due to their roles in ionic transport, suggesting that their translation might be deprioritized under ER stress conditions, possibly as a strategy to avoid exacerbating ER stress due to further disruption in ions. Another possibility is that the unfolded protein response prioritizes synthesizing ER chaperones and components of the ER-associated degradation pathway over these transporters to maintain cellular homeostasis. Likewise, it is possible that factors other than excessive ER stress and calcium disturbance could contribute to their observed differential expression in different CRC cells. Finally, ER stress, as observed in IBD, could further disrupt junctions; hence, it might contribute to IBD-CRC progression. Nevertheless, future studies are warranted to explore the therapeutic implications of targeting these pathways in IBD-CRC progression.