Terfenadine: A Promising Agent in the Treatment of Colorectal Cancer

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Introduction to Terfenadine and Colorectal Cancer Treatment

Colorectal cancer (CRC) is a type of cancer that affects the colon and rectum, and it is one of the most common cancers worldwide. Every year, many people are diagnosed with CRC, and it is a significant cause of cancer-related deaths. Despite advances in medical treatments, CRC, especially when it spreads to other parts of the body (metastasizes), remains challenging to treat effectively.

One promising area of research in CRC treatment involves exploring new uses for existing drugs. Terfenadine is a drug that was initially developed to treat allergies, specifically allergic rhinitis and hives. However, it was taken off the market due to some serious side effects related to heart problems. Interestingly, recent studies have shown that terfenadine might have potential as a cancer treatment, particularly for CRC.

Researchers are investigating how terfenadine affects the growth and survival of CRC cells. This drug seems to work by interfering with specific pathways and signals that cancer cells use to grow and avoid death. One of the key processes it influences is called apoptosis, which is a type of programmed cell death that helps get rid of damaged or unwanted cells. Cancer cells often find ways to bypass apoptosis, which allows them to survive and proliferate uncontrollably.

Terfenadine’s potential as a cancer treatment is linked to its ability to trigger apoptosis in cancer cells. By doing so, it helps to reduce the number of cancer cells and slow down the progression of the disease. Additionally, terfenadine targets specific receptors and proteins involved in cancer cell growth and survival, making it a promising candidate for further research and development as a CRC treatment.

In summary, while CRC remains a challenging disease to treat, the repurposing of drugs like terfenadine offers new hope. This drug, originally used to treat allergies, is now being studied for its potential to combat cancer, providing a new avenue for improving CRC treatment and outcomes.

Scientific study and analysis of research

Colorectal cancer (CRC) ranks third globally in terms of both incidence and prevalence, with an incidence rate of 10.2% worldwide. The survival rate of metastatic CRC is alarmingly low, at less than 20%. However, recent five-year clinical trials have shown a marked improvement in the overall survival rate and the pathophysiological characteristics of the tumor (Biller and Schrag, 2021). Conventional treatments for CRC vary depending on the disease’s condition, with various combination therapies, including immunotherapy and chemotherapy, being developed to overcome CRC-associated multidrug resistance (Dariya et al., 2020). Despite the availability of various immunotherapies and targeted therapies, metastatic CRC remains a significant challenge in therapeutic management (Messersmith, 2019). The development of new chemotherapeutic agents with enhanced therapeutic activity could be a breakthrough in the successful treatment of CRC.

Apoptosis, a programmed cell death modality, involves a caspase cascade through intrinsic and extrinsic mechanisms and their downstream targets (Kim and Choi, 2013; Kashyap et al., 2021; Morana et al., 2022). Cancer cells often bypass apoptotic cell demise by distorting the homeostasis between antiapoptotic B-cell lymphoma (Bcl) family proteins and proapoptotic proteins (Hanahan and Weinberg, 2011; Kundu et al., 2014). An imbalance between these proteins is a significant factor in many cancers (Elmore, 2007; Park et al., 2014; Yun et al., 2017). Alterations in the proportions of Bax and Bcl-2 destabilize the mitochondrial membrane potential, leading to the transfer of cytochrome c into the cytoplasm, which triggers the simultaneous cleavage of caspase-9, caspase-7, and caspase-3, and PARP, inducing cell death (Kim, 2014; Chae et al., 2020). The expression of Bcl-2 is modulated by the signal transducer and activator of transcription-3 (STAT3) at the transcriptional level (Kundu et al., 2014). STAT3 activation is triggered through phosphorylation by several upstream regulators such as MEK/ERK (Aggarwal et al., 2009) and JAK2 (Slattery et al., 2013; Kim et al., 2016), followed by dimer creation and transfer into the nucleus for transcriptional activity. While transient STAT3 activation promotes normal cell growth and differentiation, constitutive activation is associated with carcinogenesis, apoptosis evasion, and cellular proliferation. The downregulation of STAT3 activation suppresses cellular proliferation and prompts apoptosis (Aggarwal et al., 2009; Johnston and Grandis, 2011; Cho et al., 2014; Park et al., 2019; Raut et al., 2021).

The histamine H1 receptor (H1R) is a Gαq/11-coupled transmembrane receptor (Parsons and Ganellin, 2006; Lieberman, 2011). The activation of this receptor activates phospholipase C, which mediates the stimulation of protein kinase C (PKC) and the discharge of calcium via the assembly of inositol triphosphate (IP3) and diacylglycerol (DAG), which function as second messengers for downstream signal transduction. PKC plays a significant role in the G protein-associated signaling transducer mechanism of H1R through the phosphorylation of several substrates mediating downstream signaling (Massari et al., 2020; Nguyen and Cho, 2021). It is reported to be regulatory upstream of ERK, a mitogen-activated protein kinase (MAPK) and critical player in carcinogenesis (Matsubara et al., 2005). However, the complex formation of G protein-coupled receptors (GPCRs) with β-arrestins can actuate G protein-independent signaling mechanisms (Luttrell and Luttrell, 2004; Park et al., 2016; Moo et al., 2021). Arrestins, which are scaffold proteins, pair with G protein-coupled receptor kinase (GRK)-mediated phosphorylated GPCRs, causing the desensitization of the receptors and reduction of their response to the ligand. In addition to their desensitization as scaffold functions, β-arrestins, particularly β-arrestins 1 and 2, recruit several signaling proteins, including components of MAP kinase cascades and Src family tyrosine kinases, acting as signal transducers irrespective of G protein activation (Miller and Lefkowitz, 2001; Perry and Lefkowitz, 2002; Chun et al., 2010).

Numerous studies suggest that histamine is a critical player in colorectal carcinogenesis. CRC cells display aberrant H1R expression, implying the tumorigenic function of histamine (Wang et al., 2014; Massari et al., 2020). In addition to the predominant expression of H1R, CRC cells demonstrate expression of other types of histamine receptors, including H2R and H4R. Even the mRNA levels of these histamine receptors are reported to be higher in CRC than in normal mucosa (Cianchi et al., 2005). Conversely, another study showed reduced expression of H1R and H4R in CRC compared to normal mucosa (Boer et al., 2008). Microarray studies reflect high expression of H1R positively correlated with poor survival of CRC cases (Wang et al., 2014). A preclinical study reported that exogenously administered histamine accelerates the proliferation of in vivo tumor xenografts in mice, corroborating the link between histamine and CRC (Tomita and Okabe, 2005). Clinical studies have also indicated a positive crosstalk between histamine receptors and CRC progression (Chanda and Ganguly, 1987; Reynolds et al., 1997). Based on these findings, it is speculated that histamine receptor antagonists could exert prominent anticancer effects on CRC.

Terfenadine is a second-generation antihistamine initially exploited for managing allergic rhinitis and urticaria (McTavish et al., 1990). The clinical use of terfenadine was associated with several side effects, such as torsades de pointes and ventricular fibrillation, which led to its withdrawal from the market (MacConnell and Stanners, 1991; Delgado-Ramírez et al., 2021). However, recent studies have dissected the new interface of terfenadine, particularly its antitumor property, which could support its revival for therapeutic use. The idea of repositioning terfenadine as an anticancer agent has garnered considerable interest in the research community for exploring its anticancer potential (Delgado-Ramírez et al., 2021). Although a few studies have delineated the antitumor property of terfenadine, its role against CRC and its underlying mechanism remain unexplored.

The effect of terfenadine on the growth and proliferation of CRC HCT116 cells in vitro and in vivo was explored by elucidating the mechanism of its anticancer properties. The impact of terfenadine on the stimulation of apoptosis and H1R-dependent signaling cascades in HCT116 cells was also investigated. Despite novel advancements in anticancer drug development, successful treatment of CRC remains a significant challenge. CRC is the third most prevalent cancer and is considered the second dominant origin of cancer-related deaths. CRC accounts for 10.2% globally, which is expected to increase in the near future (Biller and Schrag, 2021; Sung et al., 2021). The potential implications of this projection have garnered worldwide attention for the development of effective therapies for CRC, emphasizing the pathophysiological factors contributing to the disease.

Histamine is considered a dominant factor in the etiology and advancement of CRC (Massari et al., 2020). CRC tissues exhibit high levels of histamine compared to healthy tissues (Chanda and Ganguly, 1987; Reynolds et al., 1997). A clinical study revealed that patients with malignant solid tumors had significantly higher blood histamine levels, which normalized after the surgical excision of the cancer tissues (Moriarty et al., 1988). Moreover, the function of the histidine decarboxylase enzyme, involved in histamine synthesis, is reported to be two-fold higher in CRC specimens than in healthy tissues (Garcia-Caballero et al., 1988; Moriarty et al., 1988). Exogenous administration of histamine accelerated the growth of in vivo tumor xenografts in mice, supporting the positive association between histamine and CRC (Tomita and Okabe, 2005). In contrast, numerous studies have suggested an anti-tumorigenic function of histamine in cancer (Parihar et al., 2013; Gao et al., 2017).

These controversial roles of histamine in carcinogenesis are assumed to depend on the variable interplay between histamine and histamine receptors and interactions with other cellular components of the tumor microenvironment (Massari et al., 2020). Thus, the biphasic role of histamine and its differential interactions with histamine receptors prompted the screening of various classes of histamine receptor antagonists on the viability of CRC HCT116 cells. Terfenadine and hydroxyzine, H1R antagonists, were investigated for their concentration- and time-dependent cytotoxic effects on HCT116 cells. Additionally, JNJ7777120, an H4R antagonist, decreased cell growth in a concentration- and time-dependent pattern. However, the intensity of cytotoxicity with JNJ7777120 was lower than that with terfenadine and hydroxyzine. In contrast, neither H2R and H3R antagonists nor histamine elicited antiproliferative effects on HCT116 cells.

Although histamine acts as a physiological agonist for all four types of histamine receptors, the signaling mechanisms elicited by histamine upon interaction with individual histamine receptors are different. The role of individual histamine receptors in CRC is contradictory and context-dependent. While the activation of H1R is suggested to drive CRC progression, the stimulation of other receptors may exhibit opposite effects (Massari et al., 2020; Nguyen and Cho, 2021). Zhongcheng et al. reported distinct roles of H1R and H2R in colonic tumorigenesis. According to the study, H1R signaling stimulates and H2R signaling suppresses the proliferation of CRC cells. Interestingly, H2R activation counteracted H1R signaling (Shi et al., 2019).

Due to the non-specific affinity to histamine receptors and differential activity of histamine, it is postulated that the overall effect of histamine on the viability of HCT116 cells could be neutralized, which could be the reason for no significant alteration of viability when the cells were stimulated with histamine. These findings correlate well with the overexpression of H1R in CRC, representing a positive connection between H1R and the progression of CRC (Nguyen and Cho, 2021). Additionally, the cytotoxic effect of terfenadine in Caki-1, SK-MEL-28, and U87MG cells was investigated, finding that terfenadine suppressed the viability of these cancer cells too, suggesting broad-spectrum anticancer effects of terfenadine. However, extensive research is required to validate its efficacy in other cancer types. The evidence of the variable effects of histamine receptor antagonists on HCT116 cells drove further investigation into the anticancer properties of terfenadine and its underlying molecular mechanisms.

The clinical use of terfenadine for the treatment of allergies was associated with cardiotoxicity, which led to its withdrawal from the market. This raises concerns regarding the clinical translation of the drug as anticancer therapy; however, at that time, the medicine was available in conventional dosage forms. Due to the presence of H1R in the heart, it was more likely to cause such adverse effects due to the direct antagonistic effect of terfenadine (MacConnell and Stanners, 1991). However, various advancements in drug delivery technologies, such as nano-based delivery approaches and active targeting, opened the door to achieving selective drug delivery to cancer cells, thus minimizing the adverse effects and sparing healthy cells from the toxic effects of the drug (Fan et al., 2023).

Histamine-mediated activation of H1R is associated with the upregulation of H1R expression via an autoregulatory loop involving PKC isoforms (Mizuguchi et al., 2011; Hattori et al., 2013; Mizuguchi et al., 2021). Therefore, the effects of terfenadine on the expression of various histamine receptors, i.e., H1R, H2R, H3R, and H4R, were investigated. Interestingly, terfenadine selectively downregulated the expression of H1R without significantly impacting the levels of other types of histamine receptors, confirming its specificity toward H1R (Wang et al., 2014). Consistent with this, it was found that the treatment of U-373 cells with H1R antagonist suppressed the expression of H1R level via PKCα isoform (Mizuguchi et al., 2021). Moreover, another study also demonstrated downregulation of H1R expression upon inactivation of H1R signaling using quercetin through the suppression of protein kinase C-δ/extracellular signal-regulated kinase/poly (ADP-ribose) polymerase-1 signaling pathway in HeLa cells (Hattori et al., 2013).

Additionally, studies report upregulation of the expression of H1R as a result of histamine-mediated stimulation (Mizuguchi et al., 2011; Mizuguchi et al., 2021). Based on these findings, it seems plausible to propose that inhibition of H1R signaling using its specific antagonists is likely to cause downregulation of H1R via PKC-dependent signaling. However, extensive studies are required to unveil the mechanism of H1R downregulation by terfenadine in HCT116 cells. Depending on the cytotoxic effect of terfenadine in HCT116 cells, it was then attempted to elucidate whether terfenadine-induced cell death proceeded through the apoptotic mechanism.

Terfenadine administration triggered concentration-dependent apoptosis in HCT116 cells, demonstrating a good correlation with its cytotoxic effects. These outcomes are congruent with the apoptotic effects of terfenadine unveiled by several previous studies (Liu et al., 2003; Nicolau-Galmés et al., 2011). Moreover, pretreatment with histamine or 2-(2-pyridyl)ethylamine, agonists of H1R, partially restored cell viability. This suggests that terfenadine-induced cell death occurs partially through H1R inhibition. Previous studies from our laboratory demonstrated that aberrant reactive oxygen species (ROS) production can enhance apoptosis in CRC cells (Chae et al., 2014; Park et al., 2014; Kim et al., 2016). Herein, ROS production in HCT116 cells was observed upon terfenadine treatment. Thus, it is speculated that terfenadine-induced ROS production may be a partial contributor to the cytotoxic effects of terfenadine, which requires further research. Several other studies have also supported the notion that terfenadine triggers H1R-mediated and independent pathways as cell death mechanisms (Jangi et al., 2008; Wang et al., 2014).

Caspase-9 actuation serves as a major determinant of intrinsic apoptosis (Goldar et al., 2015). This study depicted that terfenadine provoked the stimulation of the caspase cascade, leading to the disruption of PARP functionality, suggesting that an intrinsic mechanism is involved in terfenadine-triggered apoptosis in HCT116 cells. Alterations in the Bax/Bcl-2 ratio prompt the discharge of cytochrome c from the mitochondria, which in turn triggers the caspase cascade (Elmore, 2007; Park et al., 2016; An et al., 2022). In this study, terfenadine upregulated the constitutive levels of the proapoptotic protein Bax, whereas the level of Bcl-2 was reduced. Thus, this change in the homeostasis of proapoptotic and antiapoptotic proteins may be associated with terfenadine-induced cell death. p53 directs the transcriptional activation of Bcl-2 family proteins, including Bax (Miyashita et al., 1994). Terfenadine upregulated p53 levels, which may be responsible for the shift in the Bax/Bcl2 ratio and activation of the caspase cascade. Moreover, terfenadine-induced downregulation of Mdm2, which is upstream of p53, advocates the crucial function of p53 in cell death.

STAT3 exhibits an oncogenic nature and is constitutively activated in different cancers, including CRC (Dobi et al., 2013; Khatoon et al., 2023). It participates in the transcriptional modulation of various genes linked to cell cycle progression and survival. Phosphorylation leads to the dimerization and nuclear dislocation of STAT3, which is closely linked to its transcriptional activity (Dobi et al., 2013; Raut et al., 2021). Aberrant stimulation of STAT3 facilitates cancer cell growth, whereas the hindrance of STAT3 signaling triggers apoptosis and cell cycle suppression in colon cancer cells (Corvinus et al., 2005; Lin et al., 2005; Chae et al., 2014; Kim et al., 2016). This led to an examination of the effect of terfenadine on STAT3 phosphorylation in HCT116 cells. Interestingly, terfenadine suppressed the phosphorylation of STAT3 at tyrosine705 and serine727 residues along with the downregulation of the STAT3 reporter gene assay. Moreover, terfenadine diminished the expression levels of STAT3-regulated genes, including cyclins and survivin, which is congruent with the results of earlier studies reporting terfenadine-induced suppression of cyclins in other categories of CRC cells (Liu et al., 2003). These results support the oncogenic nature of STAT3 and its involvement in HCT116 cell proliferation.

STAT3 activation is regulated by several upstream kinases, including JAK2 (Slattery et al., 2013; Kim et al., 2017) and MEK/ERK (Aggarwal et al., 2009). This study showed that terfenadine decreased the activation of JAK2 and MEK/ERK. Surprisingly, the total level of JAK2 was also suppressed; therefore, whether the attenuation of phosphorylation of JAK2 by terfenadine was caused by a decrease in the total JAK2 levels is difficult to rule out. JAK2 mutation in leukemia cells may result in the proteasomal degradation of JAK2, consequently leading to a decline in JAK2 levels (Marubayashi et al., 2010). However, CRC cells do not carry the JAK2 mutation (Herreros-Villanueva et al., 2010). Heat shock protein 90 (HSP90), a chaperone protein, maintains the stability of several client proteins, including JAK2 (Park et al., 2015). ROS production can facilitate HSP90 cleavage and inactivation, consequently leading to JAK2 degradation (Chae et al., 2014; Park et al., 2015).

Indeed, terfenadine increased ROS production in a concentration- and time-dependent pattern. Therefore, the terfenadine-induced ROS production may be involved in JAK2 degradation. However, further research is required to unveil the mechanism of total JAK2 reduction by terfenadine in HCT116 cells. Moreover, to investigate the interplay of JAK2 and MEK/ERK in STAT3 activation in HCT116 cells, specific pharmacological inhibitors were used. Remarkably, AG490 and U0126 inhibited STAT3 phosphorylation and its transcriptional functions. Moreover, the reduction in the STAT3 reporter gene activity by the JAK2 and MEK inhibitors provides supporting evidence that these kinases serve as upstream of STAT3 in HCT116 cells. Additionally, the JAK2 inhibitor did not affect MEK phosphorylation and vice versa, highlighting the involvement of two independent signaling mechanisms affected by terfenadine in HCT116 cells.

H1R can activate the MAPK pathway by recruiting β-arrestins or downstream signaling cascade of G proteins, including PKC, which can activate MEK and ERK (Matsubara et al., 2005; Jain et al., 2016). Because the action of terfenadine is partially H1R-dependent, the effect of terfenadine on the phosphorylation of PKC substrates was analyzed. In this study, terfenadine decreased the phosphorylation of PKC substrates. Furthermore, Ro31-8220, a pan-PKC inhibitor, downregulated the phosphorylation of PKC substrates and MEK/ERK in HCT116 cells, indicating that terfenadine decreases STAT3 activity through the PKC/MEK/ERK axis. These results align with earlier findings of the suppression of MEK/ERK signaling downstream of PKC using other H1R antagonists in human epidermal keratinocytes (Matsubara et al., 2005; Aziz et al., 2010). However, the involvement of specific PKC isoforms in this signaling was not investigated, which warrants further research. Moreover, terfenadine decreased the recruitment and complex formation of β-arrestin 2 with MEK. These findings indicate that terfenadine simultaneously downregulates H1R signaling through G protein-dependent and G protein-independent mechanisms in HCT116 cells.

The scope of this research was extended through in vivo studies using an HCT116 xenograft model to validate the cytotoxic effects of terfenadine. Tumor growth was markedly repressed by terfenadine administration to mice. The outcomes of the in vivo study correlate with the in vitro cytotoxic effects of terfenadine, which are in good agreement with the results of other studies where terfenadine administration retarded the growth of MDA-MB-231 cells (Fernández-Nogueira et al., 2018) and hepatocellular carcinoma (Zhao et al., 2020).

This study illustrates that terfenadine provokes apoptosis and retards the growth of HCT116 cells. Terfenadine activates the caspase cascade through the intrinsic apoptotic pathway. Moreover, terfenadine inhibited the activation of PKC substrates and MEK/β-arrestin 2 complex formation, as well as the phosphorylation of JAK2, which ultimately downregulated STAT3 activation and its transcriptional activity, resulting in reduced expression of cyclins and survivin. Based on the results of this study, a proposed mechanism of action for the anticancer effect of terfenadine was outlined. The antitumor effects of terfenadine in the HCT116 tumor xenograft model strongly correlate with the in vitro tumor suppression effects. Overall, the outcomes of this study elucidate that terfenadine is a potential anticancer agent for managing CRC.


reference link : https://www.frontiersin.org/journals/pharmacology/articles/10.3389/fphar.2024.1418266/full

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