Vincamine, from an antioxidant and a cerebral vasodilator to its anticancer potential

Abstract

Vincamine is a naturally occurring indole alkaloid showing antioxidant activity and has been used clinically for the prevention and treatment of cerebrovascular disorders and insufficiencies. It has been well documented that antioxidants may contribute to cancer treatment, and thus, vincamine has been investigated recently for its potential antitumor activity. Vincamine was found to show cancer cell cytotoxicity and to modulate several important proteins involved in tumor growth, including acetylcholinesterase (AChE), mitogen-activated protein kinase (MAPK), nuclear factor-κB (NF-κB), nuclear factor erythroid 2-related factor 2 (Nrf2), and T-box 3 (TBX3). Several bisindole alkaloids, including vinblastine and vincristine and their synthetic derivatives, vindesine, vinflunine, and vinorelbine, have been used as clinically effective cancer chemotherapeutic agents. In the present review, the discovery and development of vincamine as a useful therapeutic agent and its antioxidant and antitumor activity are summarized, with its antioxidant-related mechanisms of anticancer potential being described. Also, discussed herein are the design of the potential vincamine-based oncolytic agents, which could contribute to the discovery of further new agents for cancer treatment.

Graphical Abstract

1. Introduction

The free-radical oxidants, including reactive oxygen species (ROS), reactive nitrogen species (RNS), and reactive sulfur species (RSS), are produced in cells that are exposed to radiation, environmental pollutants, and metabolites of drugs or toxins.^1,2 These oxidants can induce oxidative damage to lipids, proteins, and DNA, to lead to various diseases, such as cancer, cardiac insufficiency, diabetes, and neurodegeneration. Thus, antioxidants that scavenge free radicals and non-radical oxidants can protect cells from oxidative stress and damage and hence afford benefits for the effective treatment of human diseases.^1–3

It has been well demonstrated that increases in ROS levels can modify cell-signaling proteins to mediate pathological processes. For example, oxidative stress characterized by the excessive accumulation of ROS contributes to the production of mitochondrial abnormalities, a crucial molecular hallmark in inflammatory idiopathic myopathies (IIMs). IIMs are heterogeneous autoimmune disorders involving nuclear factor-kappa B (NF-κB), endoplasmic reticulum (ER) stress pathways, ROS generation, and protein damage.⁴ Also, oxidative stress is important in acute central nervous system (CNS) injury. The brain consumes a high proportion of oxygen to support ATPintensive neuronal activity, and both Alzheimer’s disease (AD), an age-related neurodegenerative disease, and stroke, a cerebrovascular disorder, are manifestations of brain abnormalities. Thus, oxidative stress represents a promising target for the development of effective agents to treat AD and stroke.⁵ In addition, ROS produced mainly in mitochondria are involved in various physiological activities of the ovary and are critical for regulating the ovarian cycle, and thus antioxidant therapy could be promising for the treatment of ovarian diseases.⁶

The intracellular ROS-mediated signaling proteins include NF-κB, mitogen-activated protein kinases (MAPKs), Kelch-like ECH-associated protein 1 (KEAP1)/nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant responsive elements (ARE), and phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt or PKB),⁷ which are all regarded as important anticancer targets.^8,9 ROS drive tumorigenesis and tumor development, which require an aberrant redox (reduction-oxidation) homeostasis to activate onco-signaling and to avoid ROS-induced programmed death. These processes associate closely with non-coding RNAs (ncRNAs), and thus the ROSncRNAs axis has become a promising target for cancer therapy.¹⁰ ROS also interact with different proteins to function as second messengers to regulate multiple metastasis-related signaling pathways, of which redox signaling is a basis for the control of tumor cell metastasis.¹¹ Also, overproduction of ROS induces oxidative stress in mitochondria to result in mitochondrial DNA damage, mitophagy, inflammation, and apoptosis during the pathological progression of stroke, for which Nrf2 is a master regulator.¹² Activation of Nrf2 that occurs in tumor-associated macrophages (TAMs) may facilitate anti-inflammatory and immunosuppressive activities in the tumor immune microenvironment, associated with tumor resistance to chemo-, radio-, and immunotherapies.¹³ Furthermore, overproduction of ROS can initiate lethal chain reactions, and many anticancer agents exhibit their antitumor activity via ROS activation.¹⁴

Thus far, several antioxidants have been investigated for their anticancer potential, and various clinical trials have been reported for testing their efficacy in cancer prevention and treatment.^15–17 However, the results obtained from these investigations have not been totally successful, probably due to the inefficiency of tumor selective ROS delivery. Thus, different formulations have been prepared for these antioxidants, of which nanoparticles have been used to increase their concentration in tumors and their therapeutic indices.^15–17 Also, ROS have been found to show dual roles in cancer, inducing tumor progression and apoptosis and enhancing the therapeutic effects of cancer radiation therapy and chemotherapy, and thus further investigations on the role of antioxidants in cancer treatment are required.^18,19

Natural product antioxidants, especially dietary phytochemicals, have attracted wide interest for their potential contribution to cancer prevention and treatment. These products have shown synergistic effects with various anticancer agents to reduce their side effects and to improve the life quality of cancer patients.²⁰ In general, these antioxidants show both anti-inflammatory and antioxidant activities, which are all important in cancer therapy, and thus have been regarded as promising agents for the alleviation or prevention of cancer.²¹

2. Vincamine, an antioxidant and a cerebral vasodilator

Indole alkaloids are a large group of natural products showing multiple pharmacological properties, including antimicrobial, anti-inflammatory, antinociceptive, antioxidant, and antitumor activities,^22,23 of which the anticancer drugs, vinblastine and vincristine, along with indirubin, a constituent of a Chinese traditional medicine used as a cancer treatment, have been investigated extensively.^24,25 The Catharanthus or Vinca alkaloids are monoterpene indole alkaloids derived from the Madagascan periwinkle plant, Vinca rosea L. [syn. Catharanthus roseus) (L.) G.Don] (Apocynaceae), and other Vinca species, including vinblastine, vincristine, and vincamine. Of these, vinblastine and vincristine were the first plant-derived antimitotic agents used as cancer chemotherapeutic drugs in western medicine.^23,26

Vincamine or (+)-vincamine (1) was isolated initially from the leaves of Vinca minor L.,²⁷ following which it was also identified from the leaves of Vinca rosea (Catharanthus roseus), an important medicinal plant showing multiple bioactivities, including antibacterial, antitumor, antioxidant, antihyperglycemic, antihypertensive, antidiabetic, and wound-healing activities.^28,29 In addition, vincamine has been identified from other Vinca species, such as the roots of Vinca erecta³⁰ and cultivated Vinca major L.,³¹ as well several plants in the family Apocynaceae, including the leaves of Alstonia pneumatophora Backer,³² the aerial parts of Ambelania occidentalis Zarucchi,³³ the leaves of Tabernaemontana corymbosa Roxb. ex Wall.³⁴, and the bark of Tabernaemontana rigida Miers.³⁵

The structure of vincamine was assigned in 1961 by analysis of its spectroscopic data, along with a series of chemical derivatization reactions.³⁶ The 3S,14S,16S absolute configuration of vincamine was determined from the crystal structure of vincamine hydrobromide salt, but the X-ray coordinates for vincamine were not reported correctly in this early study. Thus, the structure of vincamine was obtained from inversion of these coordinates, which is the same as that determined for vincamine hydrobromide.³⁷ Following this, in 2013, the absolute configuration of vincamine was also established by analysis of the Flack parameter obtained from the single-crystal x-ray diffraction data collected using Cu Kα radiation (Figure 1).³⁸

Fig. 1.

Structure of (+)-vincamine (1). The crystal structure plot of vincamine (right) was drawn using ORTEP-3 for Windows-version 2020.1,³⁹ based on reference data.³⁷ The ORTEP plot was drawn with 50% probability displacement ellipsoids (oxygen atoms are red, nitrogen atoms are pink, carbon atoms are blue, and the small white circles represent hydrogen atoms, which are drawn with an artificial radius).

This complete structure determined for vincamine has been confirmed by a follow-up synthesis of vincamine, which was accomplished from tryptamine condensed with dimethyl 3-ethyl-3-formylpimelate followed by hydrolysis. Then, dl-vincamine was furnished from oxidation of the amino ester with p-nitrosodimethylaniline and triphenylmethylsodium followed by controlled acid treatment.⁴⁰

Synthesis of (+)-vincamine has been summarized recently, for which several strategies have been developed, including Bischler-Napieralski, Pictet-Spengler, and pericyclic cyclizations, annulation/rearrangement reactions, and Michael-like alkylation.⁴¹ The tetracyclic system formed through a Bischler-Napieralski reaction changed to the so-called Oppolzer’s aldehyde, on which a Horner-Wadsworth-Emmons reaction resulted in the production of an α,β-unsaturated ester. This ester was hydrogenated followed by cyclization with indole to afford a heptacyclic lactam. Isonitrosation of this lactam yielded an oxime and then an oxo-lactam, which was converted to vincamine by methanolysis (Scheme 1).⁴¹

Scheme 1.

Synthesis of (+)-vincamine (1), based on a Bischler-Napieralski cyclization. Reagents: a, POCl₃; b, NaBH₄ or H₂, Pd/C or Zn/AcOH or LiAIH(tBuO)₃; c, i. LiAIH₄ or DIBALH; ii. DMSO, (COCl)₂, TEA, or other conditions; d, i. triethyl phosphonoacetate, NaH; ii. H₂, Pd/C, then NaHMDS; e, t-BuCONO, NaHMDS; f, HCHO, HCI or CAN, or HCHO, p-TsOH, or AcOH; g, MeOH, Na₂CO₃, or MeONa, MeOH, or tBuOK, MeOH.⁴¹

A Pictet-Spengler cyclization performed on tryptamine and a chiral lactone afforded an epimeric mixture, which was reduced by LiAlH₄ to yield a primary alcohol. This alcohol was mesylated and then alkylated to generate a precursor, which was then changed to (+)-vincamine (Scheme 2).⁴¹

Scheme 2.

Synthesis of (+)-vincamine (1), based on a Pictet-Spengler cyclization.⁴¹

V. minor was found to show antioxidant and antihyperlipidemic effects,⁴² and the potential antioxidant activity of a major component, vincamine (1), has been investigated extensively. Vincamine was found to protect human corneal epithelial cells from lipopolysaccharide (LPS)-induced inflammation and oxidative stress via activation of thioredoxin reductase.⁴³ Both zafirlukast and vincamine showed hepatoprotective properties through relieving tamoxifen-induced oxidative stress and liver injury by inhibition of the p-JNK/p-ERK and NF-κB pathways, while they exhibited a synergistic effect with each other.⁴⁴ Vincamine protected lungs from acute injury resulting from LPS-induced inflammation and oxidative stress by inhibiting the Nrf2/NF-κB signaling cascade.⁴⁵ Both vincamine and pantoprazole mitigated renal ischemia/reperfusion injury (IRI), a complex disease involving restoration of the flow of blood and oxygen, through the inhibition of apoptosis, attenuation of the extracellular signaling pathways, and suppression of MAPK (ERK1/2, JNK, p38)-NF-κB intracellular signaling.⁴⁶

Vincamine also exhibits hypoglycemic, hypolipidemic, and antioxidant activities, indicating that it possesses therapeutic benefits for diabetes, in which oxidative stress plays a significant role.⁴⁷ Thus, vincamine was found to protect against streptozotocin (STZ)-induced INS-1 832/13 rat pancreatic β-cell apoptosis by regulating the G-proteincoupled receptor 40 (GPR40)/cAMP/Ca²⁺/IRS2/PI3K/Akt signaling pathway. It also increased glucose-stimulated insulin secretion (GSIS) by modulating the GPR40/cAMP/Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) pathway. Thus, vincamine ameliorated effectively glucose homeostasis in mice when six-week-old db/db male mice were injected intraperitoneally (i.p.) with a single dose (100 mg/kg) of STZ followed by daily treatment with 15 or 30 mg/kg vincamine hydrochloride (i.p.) for five to six weeks.⁴⁸

The high consumption of oxygen required by the brain, due to enhanced mitochondrial oxidative phosphorylation in neuronal cells, may result in excessive production of free radicals to lead to oxidative stress. This increases the susceptibility of the neural tissues to damage and ultimately causes functional impairment and unrepaired tissue damage. Thus, modulation of the abundance of prooxidants and antioxidants in the brain can be used to improve neuroprotection in response to oxidative stress.⁵ As mentioned above, vincamine (1) shows potent antioxidant activity and thus has been investigated extensively for its effects on cerebrovascular diseases. When tested for its possible role in the development of oxidative stress, inflammation, and brain damage in rat brain induced by aluminum chloride, a low dose (10 mg/kg, i.p.) treatment (daily for 45 days) with vincamine showed antioxidant and neuroptotective effects, while a high dose (20 mg/kg, i.p., daily for 45 days) exhibited prooxidant and proinflammatory activities.⁴⁹ Furthermore, treatment with vincamine (40 mg/kg, orally for 10 successive days) ameliorated the renal injury in rats induced by methotrexate, an anticancer agent showing nephrotoxicity. This activity was mediated via increasing the expression of Nrf2 and HO-1, suppressing oxidative stress, decreasing the expression of NF-κB and caspase-3, and enhancing ATP levels. Thus, vincamine was found to restore locomotor activity and memory functions.⁵⁰ In addition, vincamine improved cerebral function by targeting the phosphodiesterase-1 enzyme, voltage-dependent Na⁺ channels, voltage-operated Ca²⁺ channels, and glutamate receptors. It thus has been introduced clinically for the treatment of cerebral insufficiency in certain countries in Europe.^51,52

Thus, vincamine shows protective effects on the brain, heart, liver, lung, kidney, and pancreas, which all result from its potent antioxidant activity. As a result, it exhibits antidiabetic and cerebral vasodilatative activities and was used clinically for the treatment of cerebral insufficiency (Figure 2).

Fig. 2.

Antioxidant-related bioactivities reported for vincamine.

After extensive structural modifications, three derivatives of vincamine have been developed as cerebral vasodilators used to treat brain disorders, based on their beneficial properties on brain circulation and neuronal homeostasis. These include (+)-brovincamine (11-bromovincamine or sabromine) (2), (–)-eburnamonine (cervoxane, vinburnine, or vincamone) (3), and (+)-vinpocetine (ethyl apovincaminate or Cavinton^®) (4) (Figure 3).⁵²

Fig. 3.

Structures of (+)-brovincamine (2), (–)-eburnamonine (3), and (+)-vinpocetine (4).

(+)-Brovincamine (2) was synthesized from the regioselective bromination of a tetrahydro-β-carboline precursor. Condensation of the product from bromination of the HCl salt of 1-methoxycarbonyl-1-methyl-1,2,3,4-tetrahydro-β-carboline and 5-bromo-2-ethylpentanal with sodium benzoate yielded 16-bromovincadifformine. This product was treated with hydrogen peroxide followed by reduction with Raney Ni, rearrangement, and epimerization in MeONa/MeOH to afford (+)-bromovincamine (2) (Scheme 3).⁵³

Scheme 3.

Synthesis of (+)-bromovincamine (2).⁵³

(–)-Eburnamonine (3) was prepared from cyclopropanecarboxyaldehyde and tryptamine, from which a lactam was generated from a highly stereoselective tetracyclic intermediate produced by a Pictet-Spengler reaction. This lactam was hydrolyzed and then reacted with indole nitrogen to afford an hexacyclic lactam, which was treated with Meerwein’s reagent followed by methanolysis to lead to an amino ether. This amino ether was alkylated followed by a Dieckmann cyclization to form the required pentacyclic scaffold, which was converted into (–)-eburnamonine (3) (Scheme 4).⁴¹

Scheme 4.

Synthesis of (−)-eburnamonine (3).⁴¹

(+)-Vinpocetine or vinpocetine (4) can be synthesized from vincamine through the combination of two Lewis acids (Scheme 5).⁵⁴ Different conformations of the substituent at the C-14 position were shown in its crystal structures reported, of which one differs from that of vincamine (1). This probably results from the absence of the hydrogen bond formed between the carbonyl and hydroxy groups at the C-14 position of 1.^37,55,56

Scheme 5.

Synthesis of vinpocetine (4).⁵⁴

(+)-Brovincamine (2) was launched commercially in Japan in 1986 for its propensity of selectively increasing cranial and coronary blood flow, and a similar effect was also found in the optic nerve head of albino rabbits, suggesting its therapeutic potential for the treatment of ophthalmologic disorders.⁵² Thus, in a clinical trial investigation, it was concluded that oral treatment with brovincamine may retard visual field deterioration in patients with normal-tension glaucoma.⁵⁷ When compared with vincamine (1), the cerebral metabolic and hemodynamic effects of (–)-eburnamonine (3) were enhanced. The oxygen blood supply, the oxygen extraction coefficient, and cerebral oxygen consumption all increased with (–)-eburnamonine treatment, while they decreased when using vincamine. Thus, (–)-eburnamonine (3) was launched in 1977 by SmithKline Beecham as a cerebral vasodilator.^51,52 (+)-Vinpocetine (4) was found to be more active than vincamine in terms of its cognitive-activating ability, in which it blocks sodium channel activity and inhibits neuronal transmembrane Ca²⁺ influx. It was launched in 1978 by Richter (Hungary) and is still used currently for the treatment of various cerebrovascular diseases.^51,52

In addition, (+)-vinpocetine (4) was found to show anti-inflammatory effects, and it also exhibited antagonist activity against injury-induced vascular remodeling, high-fat-diet-induced atherosclerosis, and cardiac remodeling.^58,59 For example, vinpocetine significantly inhibited receptor activator of NF-κB ligand (RANKL)-induced osteoclasts in bone marrow-derived macrophages (BMMs), by targeting the NF-κB, MAPK, and Akt signaling pathways. It attenuated ovariectomy-induced bone loss in an ovariectomized mouse model by increasing ROS and the expression of Nrf2, heme oxygenase 1, and NAD(P)H:quinone acceptor oxidoreductase 1. This indicates the potential use of vinpocetine for the prevention and treatment of osteoporosis.⁶⁰ Also, vinpocetine protected effectively L02a normal human hepatic cells against H₂O₂ and paracetamol (APAP)-induced cytotoxicity via the Nrf2/HO-1 pathway.⁶¹ It improved the kidney function in rats subjected to renal ischemia/reperfusion injury through modulation of NADPH oxidase/Nrf2, IKKβ/NF-κB p65, and cleaved caspase-3 expression.⁶²

Thus far, several relevant clinical trial investigations have been published for vinpocetine on the https://www.clinicaltrials.gov website. An earlier clinical trial in terms of using vinpocetine to treat acute cerebral infarction, an open, multicenter, and randomized control study started in May 2011 and was completed in February 2013 (NCT01400035, sponsor: Shanghai Rxmidas Pharmaceuticals Co. Ltd.) (https://www.clinicaltrials.gov/ct2/show/NCT01400035?cond=vinpocetine&draw=2&rank=2, accessed May 3, 2023). Another clinical trial study for vinpocetine related to inhibiting NF-κB-dependent inflammation in acute ischemic stroke patients commenced in May 2014 and was completed in December 2015 (NCT02878772, sponsor: Tianjin Medical University General Hospital) (https://www.clinicaltrials.gov/ct2/show/NCT02878772?cond=vinpocetine&draw=2&rank=1, accessed May 3, 2023). A similar clinical trial study on the cognitive effects of vinpocetine in healthy adults and patients with epilepsy began in February 2012 and is due for completion in December 2024 (NCT02011971, sponsor: Stanford University) (https://www.clinicaltrials.gov/ct2/show/NCT02011971?cond=vinpocetine&draw=2&rank=3, accessed May 3, 2023). However, in a recent clinical investigation, vinpocetine was found not to be associated with positive cognitive effects, and thus the use of higher dosages of this compound was suggested for future investigations.⁶³

3. Potential antitumor activity of vincamine

The well-established anticancer drugs, vinblastine and vincristine, were purified from Vinca species, and these compounds mediate their oncolytic activity by targeting tubulin and regulated cell death (RCD), an important mechanism to limit cancer cell survival and the spread of malignancy.^64–66 As a major active component of V. minor, vincamine exhibited antimelanogenesis activity against B16 mouse melanoma cells.³² It reduced the proliferation of human KB oral epidermoid carcinoma and Hep-2 laryngeal cancer cells by induction of cell apoptosis through down-regulation of the expression of Bcl-2 and mutant p53, up-regulation of the expression of Bax and caspase-3, and by excessive ROS generation and mitochondrial membrane depolarization.⁶⁷ In addition, vincamine was found to inhibit the proliferation of A549 human lung carcinoma cells, with very limited effects observed on BEAS-2B human bronchial epithelial and 3T3-L1 mouse embryonic fibroblast cells (Table 1).⁶⁸

Table 1.

Cytotoxicity of vincamine (1).

cell	IC50
A549a,68	309.7
B16b,32	68.9
Hep-2c,67	273.7
KBd,67	232.5

IC₅₀ values (μM) toward the human

^aA549 human lung carcinoma cells (48 h treatment),

^bB16 mouse melanoma cells (96 h treatment), and

^cHep-2 human laryngeal cancer cells and

^dKB human oral cancer cell lines (48 h treatment).

In A549 human lung cancer cells, vincamine triggered cell death by disrupting mitochondrial membrane potential, activation of caspase-3, and augmentation of intracellular ROS generation.⁶⁸ Interestingly, vincamine displayed a renal protective effect against treatment with the anticancer drug, cisplatin, and this beneficial effect results from the antioxidant, anti-inflammatory, and antiapoptotic activities of vincamine via the Nrf2/heme oxygenase-1 (HO-1) and the Toll like receptor 4 (TLR4)/interferon-γ (IFNγ)/CD44 signaling pathways (Figure 4). Thus, vincamine might be able to be used as an adjunct therapy to mitigate cisplatin-induced nephrotoxicity.⁶⁹

Fig. 4.

The molecular targets and signaling pathways for the antitumor potential of vincamine.

In a recently published structure-based virtual screening procedure, vincamine was identified as a potential inhibitor of T-box 3 (TBX3) (Figure 4), a member of a group of the T-box transcription factors that act as key regulators in embryonic development and organogenesis. TBX3 is overexpressed in various epithelial and mesenchymal malignancies to regulate tumor formation and cell migration and thus has been proposed as a possible drug target. Vincamine binds to TBX3 and inhibits its activity, and thus it possesses some promise for the design of specific agents to treat TBX3-induced cancer types.⁷⁰

In an in silico study followed by enzyme kinetic testing, vincamine was also identified as a promising acetylcholinesterase (AChE) inhibitor (Figure 4).⁷¹ AChE is a serine hydrolase that degrades acetylcholine (ACh) and terminates neurotransmission. Alzheimer’s disease (AD) is characterized by reduced ACh uptake in the brain cortical and hippocampus regions, and AChE can block ACh and signaling at cholinergic neural connections. Thus, some inhibitors of AChE are used to treat early-stage AD,⁷² a progressive and irreversible neurodegenerative disorder that leads to brain cell death, and vincamine thus shows promise for the treatment of AD.^71,72 In addition, AChE plays an important role in cell adhesion, differentiation, and proliferation and hence has been regarded as a potential marker and regulator of apoptosis and a promising tumor suppressor.⁷³ Inhibition of AChE was found to result in pancreatic cancer cell viability and invasion being diminished via suppression of pERK signaling, which also reduced tumor-associated macrophage (TAM) infiltration and serum pro-inflammatory cytokine levels.⁷⁴ Thus, AChE inhibitors have been proposed for potential use in the treatment of cancer, and the discovery of further potent and selective AChE inhibitors will provide new knowledge about AChE-regulatory pathways to support the discovery of novel cancer therapies.⁷⁵ In this regard, vincamine could be a potential lead for the design of the AChE-targeted anticancer agents.

Although vincamine shows potential antitumor activity, its cancer cell cytotoxic potency is very weak (IC₅₀ >50 μM) (Table 1).^32,67,68 However, several compounds displaying more potent cytotoxicity have been prepared from vincamine. Hence, the cytotoxicity of (–)-eburnamonine (3) was enhanced by introducing an exocyclic enone group at the C-15 position [e.g., 15-methylene-eburnamonine (IC₅₀ <20 μM)] or a double bond at the C-17 and C-18 positions [e.g., 20(S)-hydroxy-Δ¹⁷-vincamone (IC₅₀ <1 μM)).^76,77 Of these, 15-methylene-eburnamonine showed cytotoxicity against human leukemia, multiple myeloma, breast, and prostate cancer cells.⁷⁶ It also reduced the proliferation of primary acute and chronic lymphocytic leukemia and acute myelogenous leukemia cells, as well as their stem cells, via oxidative stress pathways.⁷⁸

Vinpocetine (4), which exhibits anti-inflammatory and antiatherosclerosic effects by inhibiting NF-κB activation,^79,80 has attracted some interest for its potential antitumor activity. It arrests the cell cycle at the G0/G1 phase to induce cell apoptosis to inhibit the proliferation of different types of human breast cancer cells via a mitochondrial-dependent pathway.⁸¹ Also, it induced apoptosis in human HCT116 colon cancer cells to decrease cellular proliferation,⁸² and inhibits human SW620 colorectal cancer (CRC) cell-induced circular chemorepellent induced defects (CCIDs) related to the blood endothelial cell (BEC) barrier.⁸³ Tumor spheroids may cause the formation of large cellfree areas in the blood endothelial barrier, namely, CCIDs, through which cancer cells penetrate the vasculature, and this could be an early step for the metastatic cascade to resemble a pathological situation. Vinpocetine inhibited the intravasation of SW620 cell spheroids through the blood endothelial barrier, indicating that this compound may show potential antimetastatic activity.⁸³

In an in vivo investigation, breast tumor growth was inhibited significantly when five-week-old female athymic BALB/c nude mice bearing MDA-MB-231 celldeveloped tumors (50 mm³) were treated (i.p.) with vinpocetine (10 mg/kg, every 3 days) for three weeks, with no significant toxicity being observed in the host mice.⁸¹ A mechanistic study showed that vinpocetine inhibited breast tumor progression through the Akt/STAT3 pathway.⁸¹

In addition, vinpocetine displayed a neuroprotective effect in cerebral ischemia/reperfusion injury by inhibiting TLR4-mediated inflammatory responses and proinflammatory cytokine release through the TLR4/MyD88/NF-κB signaling pathway.⁸⁴ It also prevented effectively N,N’-dimethylhydrazine-induced pre-neoplastic colon damage through inhibition of pro-inflammatory cytokines and oxidative stress.⁸⁵ Furthermore, vinpocetine synergized with sorafenib in its cytotoxicity against human HepG2 and Hep3B hepatocellular carcinoma cells via the PI3K/protein kinase B/GSK-3β signaling axis.⁸⁶ It enhanced the antitumor effects of cisplatin in a A549 non-small cell lung cancer (NSCLC) cell tumor-bearing animal model through inhibition of Nrf2.⁸⁷ In nasopharyngeal carcinoma patients with radiation-related brain injury, the anti-inflammatory and antioxidative effects of dexamethasone therapy was enhanced by vinpocetine, and the patient cognitive performance was improved by the combination treatment with vinpocetine and dexamethasone.⁸⁸

4. Bisindole alkaloid anticancer agents

Bisindole alkaloids are well known for their antitumor activity, of which vinblastine (5) and vincristine (6) are long-established anticancer agents.²⁵ The Vinca alkaloids 5 and 6 are constituents of Vinca rosea (syn. Catharanthus roseus),²⁵ and the complete structure, including the absolute configuration, of vincristine methiodide dihydrate (6a) has been determined, based on the anomalous scattering single-crystal X-ray diffraction data. Following this determination, the structures and the absolute configurations of vinblastine (5) and vincristine (6) have been deduced from their chemical relationships,⁸⁹ and the crystal structure of vinblastine sulfate nonadecahydrate (5a) was determined by analysis of its single-crystal X-ray diffraction data (Figure 5).⁹⁰

Fig. 5.

Structures of vinblastine (5) and vincristine (6). The crystal structure plots of 5a and 6a were drawn using ORTEP-3 for Windows-version 2020.1,³⁹ based on reference data.^89,90 ORTEP plots for these compounds were drawn with 50% probability displacement ellipsoids (oxygen atoms are red, nitrogen atoms are pink, carbon atoms are blue, and the small white circles represent hydrogen atoms, which are drawn with an artificial radius).

Compared with the crystal structure of 5a, the methyl ester group at the C-19 position of 6a is rotated by approximately 180 degrees. This could result from intramolecular hydrogen bonding, which is formed between the C-19 carbonyl group and N-1 (O•••H–N-1) in 5a but between the methoxy group at C-19 and N-1 (O•••H–N-11) in 6a.^89,90 In addition, vinblastine is protonated at both N-9 and N-9’ in 5a, and vincristine is methylated at N-9 in 6a while the substituent at the N-1’ position is different in 5 and 6, which may all contribute to this varied orientation of the C-3 substituent. However, in both molecules (5 and 6), the vindoline residue (lower level) is proximate to but not distal from the N-1 position (Figure 5), as generally portrayed in the literature.

As mentioned earlier, the bisindole alkaloids 5 and 6 mediate their anticancer activity by targeting tubulin and regulated cell death (RCD) to limit cancer cell survival and malignancy spread.^65,66 They bind to microtubulin to prevent microtubule formation and to disrupt mitotic spindle assembly, and thus inhibit cancer cell migration and metastatic potential to lead to the cancer cell programmed cell death and apoptosis.^25,26 Structure-activity relationship (SAR) investigations have shown that the ethyl group substituted at the C-7 position is critical for the binding to microtubulin and for mediation of the antitumor activity of these molecules.⁹¹

Microtubules play an important role in the non-mitotic process in both malignant and non-malignant cells, so both vinblastine and vincristine also show activity against non-malignant cells. Thus, these bisindole alkaloids possess undesired side effects, including myelosuppression, mucositis, fever, anemia, and alopecia, and neurotoxicity. To reduce these side effects and to enhance the therapeutic efficiency of vinblastine and vincristine, several drug delivery systems and combination therapy regimens have been developed. These include liposome-entrapped, chemical- or peptide-modified, and polymeric packaging formulations and combinations of vinblastine or vincristine with radiation therapy or other chemotherapeutic agents, including bleomycin, cisplatin, cyclophosphamide, dacarbazine, and doxorubicin.⁹²

Vinca alkaloids are the first plant-derived antimitotic agents, of which up to five agents, including vinblastine (5), vincristine (6), vindesine (7), vinflunine (8), and vinorelbine (9) (Figures 5 and 6), have been thus far developed and approved by the U.S. FDA and/or the European Medicines Agency (EMA) for the treatment of different types of cancer.^26,93 Of these, vinblastine (5) and vincristine (6) were approved by FDA in 1961 and 1963, respectively, while 7 and 9 were approved by FDA in 1994, and 8 was approved by EMA in 2012.²⁶

Fig. 6.

Structures of vindesine (7), vinflunine (8), and vinorelbine (8).

Vindesine (7), marketed as vindesine sulfate, is a second-generation semi-synthetic Vinca alkaloid that possesses broad-spectrum antitumor activity involving in depolymerization of microtubules. In tissue culture studies, vindesine sulfate shows more potent activity than vincristine in blocking the effect of mitosis.²⁵ To improve the cancer therapeutic effects of bisindole alkaloids, vinflunine (8) and vinorelbine (9) have been synthesized from the precursor alkaloids, catharanthine and vindoline. These two compounds bind to tubulin to mediate their antitumor activity,²⁵ and 8 showed lower toxicity than its precursor and more potent antitumor activity than 9.⁹⁴ As a new microtubule inhibitor, vinflunine (8) is clinically effective and well tolerated in several malignancies. It is the first agent to improve survival in patients with metastatic transitional cell cancer of the urothelial (TCCU) tract and was approved for the treatment of second-line TCCU tract in Europe.⁹⁵ Vinorelbine (9) (Navelbine^®) is a third-generation of Vinca alkaloid that suppresses cancer cell proliferation by attaching to tubulin. It shows less potent toxicity and more potent antitumor activity than other previous bisindole alkaloid anticancer agents and has been approved as a chemotherapeutic drug for the treatment of the metastatic non-small cell lung cancer.^25,96

Indigo naturalis is a dark-blue powder prepared from the leaves or stems of Baphicacanthus cusia (Nees.) Bremek. (Acanthaceae), Polygonum tinctorium (Aiton) Spach (Polygonaceae), and/or Isatis indigotica Fort. (Brassicaceae).^24,97 It is named as “Qingdai” and has been used traditionally in China for the treatment of different disorders.⁹⁷ Recently, an oral arsenic formulation, Realgar (an impure form of As₄S₄)-indigo naturalis formula, was found to be effective for the treatment of acute promyelocytic leukemia and thus has provided a preferred frontline approach to leukemia therapy in China.⁹⁸

Two structurally simple bisindole alkaloids, indigo (10) and indirubin (11) have been identified from indigo naturalis,⁹⁷ of which the structure of indigo (10) has been confirmed by analysis of its single-crystal X-ray diffraction data.⁹⁹ As one of the most well-known blue dyes, indigo has emerged as a potential functional motif due to its photochemical properties, from which indigo carmine (10a) has been synthesized by reaction with sulfuric acid.¹⁰⁰ A retrospective cohort study on patients after standardized transanal total mesorectal excision for suspected rectal cancer showed that the identified positive lymph nodes were not increased by ex vivo intra-arterial indigo carmine staining. In addition, this study suggests that indigo carmine injection is safe and may be an alternative to methylene blue that can induce DNA damage in advanced lymph node staging of rectal cancer.¹⁰¹ Thus, the indigo carmine contrast method has been used in chromoendoscopy to detect early gastric cancer while the linked color imaging, a newly developed image-enhanced endoscopy, was approved to be more effective.¹⁰²

The structure of indirubin (11) has been confirmed from its single-crystal X-ray diffraction data,¹⁰³ which shows a different conformation when compared with indigo (10) (Figure 7). Indirubin (11) exhibited potent cytotoxicity against different types of human cancer cells by targeting aromatic hydrocarbon receptor (AhR), Bcl-2 and Bax, cyclin-dependent kinases (CDK), NF-κB-dependent gene C-MYC, ROS, and the JAK/STAT3, Src/STAT3, PI3K/AKT/mTOR, and MAPK signaling pathways.^24,104,105 It cyclin-dependent kinases (CDKs). The water solubility and in vivo bioavailability of indirubin and its derivatives have been improved by employing several drug delivery systems.^106,107 Recently, a nanoparticle formulation of 6′-bromoindirubin-3′-acetoxime (BiA), namely, PPRX-1701, was found to improve significantly survival in the immunocompetent murine glioblastoma (GBM) model. Further transcriptomic studies showed that BiA improves GBM mouse survival through downregulating indoleamine 2,3-dioxygenase 1 (IDO1) and blocking interferon-γ (IFNγ)-induced IDO1 expression.¹⁰⁸

Fig. 7.

Structures of indigo (10), indigo carmine (10a), and indirubin (11). The crystal structure plots of 10 and 11 were drawn using ORTEP-3 for Windows-version 2020.1,³⁹ based on reference data.^99,103 ORTEP plots for these compounds were drawn with 50% probability displacement ellipsoids (oxygen atoms are red, nitrogen atoms are pink, carbon atoms are blue, and the small white circles represent hydrogen atoms, which are drawn with an artificial radius).

5. Concluding remarks and discussion

Increases of the levels of intracellular ROS can modify cell-signaling proteins to mediate various pathological processes, including NF-κB, MAPKs, Keap1/Nrf2/ARE, and PI3K/Akt.⁷ Nrf2 and NF-κB regulate cellular responses to oxidative stress and inflammation, respectively, and there is a functional cross-talk between these proteins,^109,110 while Nrf2 has been demonstrated as a downstream target of PI3K.¹¹¹ All of these ROS-mediated signaling proteins have been identified as important anticancer targets.^8,9 Also, oxidants can induce oxidative damage to lead to different diseases, including cardiovascular diseases, infections, cancer, diabetes, obesity, and brain disorders.^2,3 For these, inflammation is an immediate body response to tissue injury caused by infection or other noxious stimuli, and certain malignancies arise in tissues severely damaged by chronic inflammation, of which one cause is the induction of ROS generation.¹¹² ROS drive tumorigenesis and tumor development,¹⁰ and also interact with different proteins to regulate multiple metastasis-related signaling pathways.¹¹ However, overproduction of ROS can initiate lethal chain reactions, and many anticancer agents exhibit their antitumor activity via ROS activation.¹⁴ Thus, ROS show dual roles in cancer, and they were demonstrated to enhance cancer therapeutic effects.^18,19

Vincamine (1) shows potent antioxidant activity and thus may protect human corneal epithelial cells from LPS-induced inflammation and oxidative stress.⁴³ It also exhibits hepatoprotective activity and protects the lungs from LPS-induced inflammation and oxidative stress.^44,45 As an effective agent for the treatment of cerebral insufficiencies, vincamine also ameliorated mouse renal injury induced by methotrexate and was found to reduce cisplatin-induced nephrotoxicity.^50,69 Consistent with ROS-mediated signaling, vincamine mediates these activities by targeting Nrf2, HO-1, NF-κB, caspase-3, ATP, the phosphodiesterase-1 enzyme, Na⁺ and Ca²⁺ channels, and the glutamate receptors,^50–52 as well the GPR40/cAMP/Ca²⁺/IRS2/PI3K/Akt and GPR40/cAMP/Ca²⁺/CaMKII,⁴⁸ p-JNK/p-ERK,⁴⁴ MAPK/NF-κB,⁴⁶ Nrf2/NF-κB,⁴⁵ Nrf2/HO-1,⁶⁹ TLR4/IFNγ/CD44,⁶⁹ and TLR4/NF-κB¹¹³ signaling pathways. Also, vincamine exhibits cytotoxicity against several human cancer cell lines by targeting Bax, Bcl-2, caspase-3, p53, ROS, and mitochondrial membrane depolarization.^32,67,68 These mechanistic observations suggest that vincamine might be able to be utilized as a conjunct therapy to decrease the risk of multidrug resistance and toxicity occurring in some types of current cancer chemotherapy. In addition, vincamine was identified as an inhibitor of TBX3 and AChE, and hence may have the potential to inhibit cancer metastasis.^70,71

As well demonstrated previously, the anticancer potential of several natural products has been improved considerably by synthetic modification, of which dimerization has been proved as a promising strategy.¹¹⁴ Several dimeric indole alkaloids have been synthesized from vincamine derivatives or analogues,^115,116 and hence the dimerization of vincamine and subsequent biological testing may produce some interesting anticancer lead compounds, which could be further developed by employing new drug delivery systems.

Natural products are well-known as an important source for the discovery of new anticancer agents,¹¹⁷ and thus a further search for new naturally occurring analogues of vincamine could also lead to the elucidation of new indole alkaloid-type cancer chemotherapeutic agents.

Abbreviations

AChE

acetylcholinesterase

Akt or PKB

protein kinase B

ARE

antioxidant responsive element

CCID

circular chemorepellent induced defect

CNS

central nervous system

CRC

colorectal cancer

ER

endoplasmic reticulum

HO-1

heme oxygenase-1

IC₅₀

the concentration required for 50% inhibition of cell viability

IFNγ

interferon γ

IIM

inflammatory idiopathic myopathies

i.p.

intraperitoneal

Keap1

Kelch-like ECH-associated protein 1

LPS

lipopolysaccharide

MAPK

mitogen-activated protein kinase

mTOR

mammalian target of rapamycin

NF-κB

nuclear factor-κB

Nrf2

nuclear factor erythroid 2-related factor 2

PI3K

phosphoinositide 3-kinase

RCD

regulated cell death

redox

reduction-oxidation

RNS

reactive nitrogen species

ROS

reactive oxygen species

RSS

reactive sulfur species

SAR

structure-activity relationship

STAT3

signal transducers and activators of transcription 3

TAM

tumor-associated macrophage

TLR4

Toll like receptor 4