The Anticancer Effect of Natural Plant Alkaloid Isoquinolines

Isoquinoline alkaloids-enriched herbal plants have been used as traditional folk medicine for their anti-inflammatory, antimicrobial, and analgesic effects. They induce cell cycle arrest, apoptosis, and autophagy, leading to cell death. While the molecular mechanisms of these effects are not fully understood, it has been suggested that binding to nucleic acids or proteins, enzyme inhibition, and epigenetic modulation by isoquinoline alkaloids may play a role in the effects. This review discusses recent evidence on the molecular mechanisms by which the isoquinoline alkaloids can be a therapeutic target of cancer treatment.


Introduction
Cancer is a leading cause of death worldwide and has a major impact on society. It is a major barrier to increasing life expectancy this century [1]. The World Health Organization (WHO) estimates that cancer was responsible for an estimated 9.6 million deaths in 2018 [2]. Treatment varies depending on the type and stage of cancer. Most people undergo a combination of treatments, such as surgery with chemotherapy and radiation therapy. However, adverse reactions to conventional treatment and drug resistance have led some to use complementary and alternative medicine (CAM) in conjunction with conventional medical treatments [3][4][5][6]. As interest in complementary therapies increases, so has the value of natural remedies [7]. Isoquinoline alkaloids, a group of plant-derived bioactive compounds, have traditionally been used as alternative treatments for their anti-inflammatory, antimicrobial, and analgesic effects [8][9][10][11][12]. Recently, biomedical and pharmacological developments have begun to uncover the anticancer effects and mechanisms of isoquinoline alkaloids. In this review, we discuss the anti-cancer effects and mechanisms of isoquinoline alkaloids.

Isoquinoline Alkaloids Derived from Various Herb Extracts
Alkaloids that possess an isoquinoline moiety are one of the largest groups of natural substances. Isoquinoline is a heterocyclic compound consisting of a benzene and pyridine ring fused at C3/C4 of the pyridine ring [13]. The biosynthetic pathways of isoquinoline alkaloids proceed via tyrosine generating dopamine and p-hydroxyphenylacetaldehyde ( Figure 1). Tyrosine is converted to dopamine by hydroxylation and decarboxylation, and to p-hydroxyphenylacetaldehyde by transamination and decarboxylation [14]. Through cyclization, hydroxylation, and methylation, dopamine and p-hydroxyphenylacetaldehyde are condensed to form specific scaffold molecules such as norcoclaurine, reticuline, autumnaline, deacetylisoipecoside, or norbelladine, central precursors to several thousand isoquinoline alkaloids [15,16].   Isoquinoline alkaloids have been used in folk medicine and have attracted attention in the pharmacological industry and among researchers due to their potential medicinal benefits. Most of the isoquinoline alkaloids discovered to date have been derived from plants, such as Alangiaceae, Annonaceae, Berberidaceae, Fabaceae, Fumariaceae, Lauraceae, Menispermaceae, Papaveraceae, Ranunculaceae, and Rutaceae [17]. Opium poppy (Papaver somniferum) is one of the oldest plant sources of commercial medicinal isoquinolines in the world. Morphine, codeine, papaverine, noscapine, and thebaine were detected in its latex [18], and more than 40 isoquinoline alkaloids have been isolated from opium [19]. Chelidonium majus L., of the Papaveraceae family, contains sanguinarine, chelidonine, chelerythrine, berberine, and coptisine [20]. 8-oxoberberine, berbidine, berbamine, aromoline, obamegine, berberine, and palmatine were obtained from Berberis vulgaris [21].

Biological Functions
Isoquinoline alkaloids have various biochemical properties related to their binding to various differential biological functional ligands [23]. Isoquinoline alkaloids intercalate with polymorphic nucleic acid structures. Berberine and palmatine bind to B-form DNA and coralyne binds to duplex B-form DNA and a single-stranded poly(A) structure [24]. Spectroscopic and thermodynamic studies suggest that sanguinarine and berberine bind to the DNA and RNA double and triple helical structures [25] and sanguinarine binds to tRNAphe [26]. Interactions between sanguinarine and chelerythrine with DNA were both enthalpy-and entropy-favored actions [27].

Anticancer Effects of Isoquinoline Alkaloids
The anti-cancer activity of isoquinoline alkaloids is noteworthy. Isoquinoline alkaloids and/or isoquinoline-enriched plants have been investigated as alternative regimens to complement chemotherapy. They efficiently induce cell death in various cancer cell lines [52][53][54][55]. The evidence based on in vivo and in vitro models indicated isoquinoline alkaloids exert significant anti-cancer effects through cell cycle arrest, apoptosis, and autophagy (Table 1), leading to cell death.

Apoptosis-Mediated Cell Death
Apoptosis, programmed cell death, is a promising target for anticancer therapy. Apoptosis is triggered by the extrinsic and intrinsic pathways. The extrinsic pathway is triggered by external stimuli. Ligand and death receptor (DR) binding interacts with the Fasassociated death domain (FADD) and tumor necrosis factor receptor 1 (TNFR1)-associated death domain (TRADD). A death-inducing signaling complex (DISC) is then formed and caspase-8 is recruited to DISC. This leads to the activation of caspase-8, which cleaves and activates caspase-3/6/7, initiating apoptosis [56].
The intrinsic pathway is triggered by exogenous and endogenous stimuli, including DNA damage and oxidative stress. The Bcl family members, Bax and Bcl-2, act as proor anti-apoptotic regulatory proteins through binding to the mitochondrial membrane. The release of cytochrome C in the cytoplasm recruits Apaf-1 and procaspase-9 to form the apoptosome, which triggers downstream caspase-9/3 cascades [57].

Caspase-Dependent Apoptosis
Caspase activation is a central process for apoptosis. All caspases are produced as catalytically inactive zymogens and are cleaved and activated during apoptosis [58]. Chelerythrine-induced apoptosis was accompanied by a decrease in the mitochondrial membrane potential (MMP), the release of cytochrome c, activation of caspase-3 and poly ADP-ribose polymerase (PARP), and downregulation of Bcl-2 in BGC-823 cells [59]. Sanguinarine inhibited tumor growth in vivo and in vitro in various cancers, including prostate [60], cervical [61], pancreatic [62], and colorectal cancers [63]. AsPC-1 and BxPC-3 growth were suppressed via an increase in Bax, Bid, and Bak and decreases in the antiapoptotic Bcl-2 and Bcl-xL proteins [62]. Sanguinarine also decreased the tumor size in orthotopical colorectal carcinoma bearing BALB/c-nu mice through increased caspase 3, PARP, and mitochondrial reactive oxygen species (ROS) cleavage [63]. The effect of chelerythrine on A549 and H1299 leads to increased protein levels of cleaved PARP and cleaved caspase 3 [64]. Chelidonine inhibited non-small cell lung cancer growth via regulating epidermal growth factor receptor/AMP-activated protein kinase (EGFR/AMPK) signaling pathways in vivo and in vitro [65]. Berberine induced caspase 3, 8, and 9 mediated apoptosis in A549 and H1299 xenograft mice models [66,67] and triple-negative breast cancer cells [68].

Cell Cycle Arrest
The cell cycle is regulated by several cyclin-dependent kinases and controls cell division and proliferation. Induction of cell cycle arrest and inhibition of cell proliferation by regulation of cell cycle checkpoints is a therapeutic target for treating cancer [74]. Berberine leads to G1 cell cycle arrest with the induction of NAG1 and activating transcription factor 3 (ATF3) expression on HCT116 cells [73]. An antitumor effect has been demonstrated in human colorectal adenocarcinoma by inducing G2/M phase arrest in vivo and in vitro studies [75]. Berberine treatment also caused G2 phase arrest in U251 cells and significantly inhibited tumor progression in the glioma mouse model [76]. Chelerythrine treatment induced S phase arrest to inhibit BGC-823 cell proliferation [59]. Moreover, sanguinarine arrested AsPC-1 and BXPC-3 cells in the G0-G1 phase through modulation of the Bcl-2 family [62].

Autophagy-Mediated Cell Death
Autophagy is a response to a range of cellular stressors to maintain cellular homeostasis. Therefore, autophagy is a critical mechanism of cancer treatments. Mechanistic target of rapamycin (mTOR), a molecular regulator of autophagy, is associated with cell proliferation and is regulated by AMPK. Inhibition of mTORC1 and increased AMPK induces autophagy [77], during which autophagosomes are formed to digest cytoplasmic components and LC3I is converted to LC3II [78,79]. Berberine upregulated LC3-II and induced autophagy in glioblastoma through the regulation of the AMPK/mTOR/unc-51 like autophagy activating kinase 1 (ULK1)-pathway [80] and repressed human gastric cancer cell proliferation through inactivation of the MAPK/mTOR/p70S6K/Akt signaling pathway in vivo and in vitro [81]. In chelerythrine-treated A549 and H1299 cells, LC3-II expression was enhanced [64]. Similarly, neferine upregulated LC3-II and downregulated the phosphoinositide 3-kinase (P13K), Akt, and mTOR pathways, inducing autophagy [82].

Molecular Mechanisms of Anticancer Effects
The molecular or cellular mechanisms behind these anti-cancer effects are of great interest. Molecular functions, such as binding to nucleic acids or proteins and enzyme inhibition, have been suggested as potential anti-cancer mechanisms.

Binding to Polynucleic Acids
Interactions of the alkaloids with DNA and RNA may be responsible for anticancer effects. Specific binding to nucleic acids regulates polynucleic acid stability and may be the therapeutic target of isoquinoline alkaloids with anticancer effects. These bindings disrupt the structure of duplex B-form DNA and affect their interaction with DNA replication, repair, or transcription-related proteins. Sanguinarine and chelerythrine preferred double-helical regions for binding [27] and DNA adduct formed from both isoquinoline alkaloids [101].

Binding to Microtubules
Microtubule polymerization plays a pivotal role in chromosomal segregation during mitosis [104]. Specific binding to mitotic microtubules has been considered the therapeutic target of isoquinoline alkaloids with anticancer effects. Sanguinarine caused microtubule depolymerization and conformational changes in tubulin through tubulin binding and inhibited cell proliferation in Hela cells [105]. Noscapine-treated MCF-7, MDA-MB-231, and CEM cells displayed higher tubulin-binding activity and mitotic arrest followed by apoptosis [83,106]. Chelidonine [107] and hydroxy-substituted indolo[2,1a]isoquinolines [108] disrupt microtubular structure and inhibit tubulin polymerization.

Inhibition of Enzyme Activity
Inhibition of enzyme activity is associated with anticancer activities. The abilities of protoberberine and coralyne as topoisomerase I and II inhibitors are well known [29,30]. Berberrubine's inhibition of DNA topoisomerase II induced DNA cleavage through stabilization of the enzyme-DNA complexes [109,110].
Corydine, parfumine, 8-methyl-2,3,10,11-tetraethoxyberbine, and chelidonine from the Papaveraceae family inhibit CYP3A4, indicating a high-affinity interaction with this enzyme and demonstrating an anticancer effect [31,32]. The binding of chelerythrine to Bcl-2 and apoptotic processes were observed in a dose-dependent manner [115,116]. Berberine inhibited cyclooxygenase-2 (COX-2) transcriptional activity with the regulation of I kappa B kinase (IKK) and nuclear factor-kappa B (NF-κB), and induced apoptosis [33,34]. However, inhibition of AChE and BuChE activity is not related to anticancer effects. Studies have shown that AChE is upregulated in response to apoptotic induction [117]. Its inhibition is considered a potential treatment of Alzheimer's disease (AD). AD is characterized by a loss of neurotransmission due to abnormal synaptic acetylcholine levels [118]. AChE and BuChE are enzymes that break down the neurotransmitter acetylcholine and regulate cholinergic levels in the brain [119].

Epigenetic Modulation
Epigenetics is defined as the heritable changes in gene expression without alteration of the DNA sequence itself [120]. Epigenetic dysregulation of gene expression occurs during stages of cell proliferation, invasion, metastasis, and cancer development [121][122][123]. DNA methylation and histone modifications, as main epigenetic mechanisms, induce chromatin remodeling followed by changes in cellular phenotypes [124]. These mechanisms regulate proto-oncogene, tumor suppressor gene, and DNA repair gene expression.
Natural products including the secondary metabolites found in plants are reported to reverse cancer progression through modulation of epigenetic events, such as modulation of the activities of DNA methyltransferases (DNMTs) and histone deacetylases (HDACs) [125,126]. Remarkably, isoquinoline alkaloids act as putative targets in cancer drug development by affecting epigenetic modulation (Table 2).

Conclusions
Current evidence demonstrates that isoquinoline alkaloids have anticancer effects such as induction of cell cycle arrest, apoptosis, and autophagy (Figure 3), suggesting their potential as a cancer therapeutic agent. The effects are, at least in part, attributed to their binding to DNA or proteins, inhibition of enzyme activity, or epigenetic modulation. Further studies are needed to fully discover the underlying mechanisms of isoquinoline alkaloid-mediated cell death against cancer.

Conclusions
Current evidence demonstrates that isoquinoline alkaloids have anticancer effects such as induction of cell cycle arrest, apoptosis, and autophagy (Figure 3), suggesting their potential as a cancer therapeutic agent. The effects are, at least in part, attributed to their binding to DNA or proteins, inhibition of enzyme activity, or epigenetic modulation. Further studies are needed to fully discover the underlying mechanisms of isoquinoline alkaloid-mediated cell death against cancer.

Conflicts of Interest:
The authors declare no conflict of interest. Toll-like receptor 4 TNFR1