Alkaloids and Colon Cancer: Molecular Mechanisms and Therapeutic Implications for Cell Cycle Arrest

Cancer is the second most fatal disease worldwide, with colon cancer being the third most prevalent and fatal form of cancer in several Western countries. The risk of acquisition of resistance to chemotherapy remains a significant hurdle in the management of various types of cancer, especially colon cancer. Therefore, it is essential to develop alternative treatment modalities. Naturally occurring alkaloids have been shown to regulate various mechanistic pathways linked to cell proliferation, cell cycle, and metastasis. This review aims to shed light on the potential of alkaloids as anti-colon-cancer chemotherapy agents that can modulate or arrest the cell cycle. Preclinical investigated alkaloids have shown anti-colon cancer activities and inhibition of cancer cell proliferation via cell cycle arrest at different stages, suggesting that alkaloids may have the potential to act as anticancer molecules.


Introduction
Cancer is the second most fatal disease in the world, and according to the World Health Organization (WHO), it accounted for approximately 8.2 million deaths and 14 million new victims in 2012 [1]. In 2017, the projected deaths due to different cancers in the United States were 600,920, with 1,688,780 newly diagnosed cancer patients. The overall cancer prevalence and death rate was 20% higher in males than in females [2]. The coronavirus disease 2019 (COVID-19) pandemic impeded cancer detection and therapy in 2020. In 2021, the United States was projected to have 1,898,160 cancer cases and 608,570 cancer-related deaths [3]. Older people, between 85-90 years-of-age, have higher cancer prevalence than younger subjects [4]. The rapid and unmanageable proliferation of abnormal cells leads to cancer origination [5], with abnormalities during cellular division in the mitochondrial genome triggering genetic mutations and cancer progression [4].
Colon cancer (CC) is one of the most prevalent cancers in the world, with the highest prevalence in Black Alaska's Natives and the lowest prevalence in Asian/Pacific Islanders [6]. Colorectal cancer (CRC) is the most prevalent cancer in Saudi men, and the third most prevalent disease in Saudi women [7]. The American Cancer Society has drafted guidelines for the treatment of colon cancer as well as survivorship care, which include long-term complications from chemotherapy [8][9][10].
While diagnostic and treatment options for colon cancer have been stipulated [11][12][13], their outcomes are not always satisfactory, and the number of deaths remains high. Colon transcription in the G1 phase is strongly associated with the transcription factors of E2F coupled with their dimerization partner proteins. Researchers have found functional regulation factors of E2F in cancer [63,64], suggesting that G1-S transcription is critical in the development and proliferation of cancer [65][66][67].

G2-Phase Arrest
The S phase is followed by the Gap 2 (G2) phase, characterized by a synthesis/repair phase coupled with the preparation of cellular machinery for mitosis in the M (mitosis) phase [68,69]. The chromatids and daughter cells separate during the M phase. Subsequently, the cells enter theG1 or G0. The quality and rate of overall cell division is modulated by these checkpoints [70,71]. In this phase, the cdc2-B1 complex is regulated via the activation of phosphate by the Cdk-activating enzyme and the inhibition of phosphatase enzymes [72]. The phosphatase Cdc25C caused dephosphorylation and hence overexpression of Cdk [73][74][75].
The dormant cells are activated by external stimuli [76]. These growth-promoting factors bind to cell receptors and trigger the cellular machinery [77]. Moreover, genetic intervention has been observed as the focusing target of these growth-modulating factors [78]. The response is designated as earlier and delayed. The former induces phosphorylation and activation of the transcription factor proteins that are already present in the cell. In fact, the genes that are involved in early response encode transcription factors, and ultimately modulate the expression of late response genes. The delayed response genes modulate the proteins, G1 cyclin-dependent kinases (CDKs) [79,80], and therefore CDK inhibitors have recently attracted considerable attention as anticancer agents, especially the CDK4 and CDK6 inhibitors [81,82].

Figure 1.
Schematic representation of molecular mechanisms of action involved in anticancer effect of steroidal alkaloids in colonic cancer. Steroidal alkaloids inhibit different pathways such aspg-p, PI3k, Fas, and TNRfs, which are, in turn, linked with different mechanistic pathways exhibiting anticancer effects. Inhibition of Bax and Bcl2 leads to activation of caspase-3,8, and 9 pathways, thus leading to apoptosis.

Activity of Synthetic Derivatives against Colon Cancer
The summary of the activity of synthetic alkaloid derivatives with potential therapeutic effect in colon cancer is shown in Table 2  In 2007, Italian researchers synthesized nortopsentin analogues of marine alkaloids, such as 3,5-bis(3′-indolyl)pyrazol 65 and 1-chloro-3,5-bis(3′-indolyl)pyrazol 66, earlierisolated from Spongosorites (Table 2) . The analogue 1-chloro-3,5-bis(3′-indolyl) pyrazol, bearing a chloro group, was more active both in terms of GI50 and of percentage of sensitive cell lines. Plakinamines N and O, two additional steroidal alkaloids from Corticium niger, as well as two established plakinamine compounds, were isolated (3,4). Using a combination of MS and NMR spectroscopic data, the structures of these molecules were characterized. The antineoplastic effectiveness of plakinamines N, O, and J was examined in the NCI-60 screen, and they demonstrated improved inhibitory activity against all colorectal cell cultures [105]. Table 2. Synthetic alkaloid derivatives with potential in colon cancer.
Berberine 80 is a highly studied isoquinoline plant alkaloid isolated from many species of the genus berberis [114]. Italian researchers synthesized a number of berberine 80 derivatives, which were characterized as [13-(3- which increased to about 50% with the recovery time, whereas NAX018 demonstrated a cytotoxic effect after the treatment in about 50%, reaching 70% after the recovery time. InSW-613-B3 cells, the incubation with 10 µM NAX012 and NAX014 was not effective, while NAX018 affected cell survived by 10% and 20% (at the completion of incubation and after the recovery response, respectively).In particular, when compared to the lead compound BBR, these derivatives showed high potency, being more effective in cells harboring p53wt, promoting cell cycle arrest and DNA damage, and triggering caspase-dependent apoptosis and autophagy [111]. Ilyas and his coauthors established the anticancer efficacy of doxorubicin (of synthetic origin) combined with berberine (of natural origin)by applying an encapsulation/conjugation approach with poly (lactic-co-glycolic acid) PLGA nanoparticles. By using carbodiimide chemistry, doxorubicin has been effectively conjugated to PLGA, and the PLGA-doxorubicin conjugate (PDC) was employed to encapsulate berberine. ROS analysis demonstrated that the PDBNP depicted a dose-dependent rise in the reactive oxygen species (ROS) patterns in MDA-MB-231 cells, but no improvement in ROS was detected in T47D cells. PDBNP caused a change (depolarization) in mitochondrial membrane permeability as well as a cell cycle arrest in the sub-G1 phase, whilst Annexin V/PI assay, followed by confocal microscopy, revealed that MDA-MB-231 cells died due to necrosis.

Effect of Alkaloids on Cell Cycle Arrest and Other Anticancer Pathways
The anticancer effect of alkaloids and the mechanistic pathways involved in the anticancer effect of alkaloids are schematically represented in Figure 2. Piperine 51, an alkaloid commonly isolated from black pepper [98], mediated its anticancer effect via modulation of G1-phase cell cycle arrest and by downstream regulation of cyclins D1 and D3, accompanied by their activating cofactors, cyclin-dependent kinases 4 and 6. Piperine caused downstream expression of phosphorylation of retinoblastoma protein and upstream modulation of p21/WAF1 and p27/KIP1 expression ( Figure 2) [97]. The G1 expression for the initiation of Rb protein phosphorylation shape D-type cyclin complexes with Cdk4 and Cdk6 [115,116].On the other hand, to expresses cell cycle progression, it caused inhibition of cyclin-dependent kinases when this complex attached to p21/WAF1 and p27/KIP1, an inhibitor of cyclin-dependent kinases [117,118]. Isoquinoline alkaloids also inhibit Cyclin D CDK 4/6 and causes cell cycle arrest. mTOR pathway is also activated, which leads to autophagy.
The African medicinal plant Tabernaemontana elegans yielded obasinyl-iboga alkaloids dregamine52, and tabernaemontanine53 exhibited significant antiproliferative activity by modulating its effect through G1-phase arrest in colon cancer cell lines with associated downstream regulation in the S phase [99]. Arun et al. (2017) studied the effect of pyranocarbazole alkaloids, murrayazoline compound 61, and O-methylmurrayamine A compound62 in different stages of the cell cycle; both caused significant cell arrest at the G2/M checkpoint and promoted sub-diploid population at 5.7 µM and 17.9 µM, respectively [102].
The synthetic phenanthroindolizidine alkaloids (92 and 93) showed strong anticancer action by modulating multiple singling pathways. These compounds at a low concentration (5 µg/mL) elicited cell cycle arrest, while targeting the G2/M phase. The synthetic phenanthroindolizidine alkaloids (92 and 93) showed strong anticancer action by modulating multiple singling pathways. These compounds at a low concentration (5 µg/mL) elicited cell cycle arrest, while targeting the G2/M phase. Moreover, a noticeable reduction in cell distribution was observed at the G0/G1 and S phases. In addition, Min et al. (2012) described the effects of another phenanthroindolizidine alkaloid, antofine 41, in HCT-116 cells, causing cell cycle arrest at the G0/G1 phases and inhibiting the expression of cyclin D1, cyclin E, CDK4, and the transcriptional activity of β-catenin/Tcf. Moreover, this alkaloid also potentiated tumor necrosis factor-a (TNF-α)-induced apoptosis and reduced the expression level of β-catenin and cyclin D1 in SW-480 cells [119].
Mechanistic studies on compounds 6 and 7 showed inhibition of the G0/G1 phase after 24 h exposure [83], with directed cell death absent mitochondrial alterations [120]. The antiproliferative effects of alkaloids 42-44 (Table 1) from Zanthoxylum capense in HCT-116 cell were shown to be mediated by apoptosis (Figure 1) [94]. Yang et al. (2010) showed that the arrest of the G1 phase in colon cancer cells and the induction of apoptosis are associated with the suppression of nuclear factor-kappaB (NF-kB) activation. The alkaloid also inhibited the phosphorylation/degradation of IkBa and the phosphorylation/translocation of p65. Furthermore, dauricine down-regulated the expression of various NF-kB-regulated genes, including genes involved in cell proliferation (COX-2, cyclin D1, and c-Myc), invasion (MMP-9 and ICAM-1), antiapoptosis (Bcl-2, XIAP, IAP1, and survivin), and angiogenesis (VEGF). The diversity of anticancer mechanisms was also noted with isostrychnopentamine (ISP) 2, an indolomonoterpenic alkaloid that causes cell cycle arrest in the G2-M phase, and induces apoptosis by the translocation of phosphatidylserine from the inner layer to the outer layer of the plasma membrane, as well as by chromatin condensation, DNA fragmentation, and activation of the caspases 3 and 9 [91].
An experimental finding has illustrated a strong cytotoxic effect of oxymatrine 63, an alkaloid isolated from Sophora flavescens in human colon cancer cells. The underlying mechanism was interfering with the overexpression of human telomerase reverse transcriptase and causing up-regulation of hTERT, c-myc, p53, and mad1 [121]. The overall effect was concentration dependent (Figure 1) [122].

Effects on Chemoresistance
Cancer chemotherapy is limited by cellular drug resistance [123][124][125][126]. In this regard, different mechanisms have been implied for this resistance, including cell cycle arrest and repair [127], apoptosis [128], cancer stem cells [129], drug properties/nature [130], and metabolism of the drug [131]. Cell cycle arrest plays a crucial role in drug resistance to chemotherapy in various cancers [127]. Several studies have confirmed the involvement of checkpoint kinase (CHK1) in chemoresistance and even resistance in radiotherapy [132][133][134]. In the case of DNA damage, the complex cellular signaling network becomes activated and induces cell cycle arrest, thus facilitating DNA-repairing events or, in cases of extensive damage, such events trigger apoptosis [135][136][137].
Damage of the cellular DNA of any origin triggers the activation/upstream regulation of ATM(ataxia-telangiectasia mutated),and ATR(ATM-and Rad3-related) kinases coupled with the DNA-dependent protein kinase catalytic subunit [135,138]. These kinases lead to the translocation of components to the sites of damaged DNA and induce cell cycle progression by modulating effector kinases and checkpoint kinase like CHK1, and CHK2 [139]. The activation of checkpoints controlled by ATM/ATR-CHK1/CHK2 stalls cell cycle progression in the G1, S, or G2 phase [140]. G1 arrest is modulated by p53 through p21CIP1/WAF1 up-regulation [141], while in case of DNA damage, apoptosis charged over the situation [130]. In the course of events, p53 loses its regulatory action; as a result, the chemotherapy-induced DNA damage is unable to arrest the cancer cells in the G1 phase and thus trigger apoptosis. Indeed, to arrest the cell cycle in such cancer cells, following the genotoxic exposure, the S and G2/M checkpoints are required to induce DNA repair before entry into mitosis (M phase). Different polysulfanes have shown cell cycle arrest in colorectal cancer [142][143][144].

Conclusions and Future Prospects
Based on increased cases of cancer and its status as a leading cause of disease worldwide, new effective treatments are required. Among the most common cancer treatments, chemotherapy is an essential tool in getting cancer under control and reducing or treating advanced or aggressive cancers. As noted in the current review, several alkaloids show anti-colon-cancer properties, being able to be cytotoxic against human colon cancer cells. The most exciting substances seem to be those able to act at the level of the checkpoints present at the G1/S and G2/M transitions, which account for the proper replication and division of DNA. These alkaloids hold great potential as novel therapeutic agents for the effective management of colon cancer.