Therapeutic value of steroidal alkaloids in cancer: Current trends and future perspectives.

Discovery and development of new potentially selective anticancer agents are necessary to prevent a global cancer health crisis. Currently, alternative medicinal agents derived from plants have been extensively investigated to develop anticancer drugs with fewer adverse effects. Among them, steroidal alkaloids are conventional secondary metabolites that comprise an important class of natural products found in plants, marine organisms and invertebrates, and constitute a judicious choice as potential anti-cancer leads. Traditional medicine and modern science have shown that representatives from this compound group possess potential antimicrobial, analgesic, anticancer and anti-inflammatory effects. Therefore, systematic and recapitulated information about the bioactivity of these compounds, with special emphasis on the molecular or cellular mechanisms, is of high interest. In this review, we methodically discuss the in vitro and in vivo potential of the anticancer activity of natural steroidal alkaloids and their synthetic and semi-synthetic derivatives. This review focuses on cumulative and comprehensive molecular mechanisms, which will help researchers understand the molecular pathways involving steroid alkaloids to generate a selective and safe new lead compound with improved therapeutic applications for cancer prevention and therapy. In vitro and in vivo studies provide evidence about the promising therapeutic potential of steroidal alkaloids in various cancer cell lines, but advanced pharmacokinetic and clinical experiments are required to develop more selective and safe drugs for cancer treatment.

mitochondrial membrane potential

acetylcholinesterase

anaphase‐promoting complex 4

anticancer steroidal alkaloid

autophagy‐related protein 5

butyrylcholinesterase

basic fibroblast growth factor

capillary nuclear magnetic resonance spectroscopy

cyclin D1

cyclin‐dependent kinase inhibitor 2B

C/EBP homologous protein/growth arrest‐ and DNA damage‐inducible gene 153

central nervous system

castration‐resistant prostate cancer

half maximal effective concentration

effective dose, for 50% of individuals getting the drug

eukaryotic initiation factor‐2

E‐prostanoid receptor 4

endoplasmic reticulum

extracellular signal regulating kinase

extracellular signal‐regulated kinase 1 and 2

focal adhesion kinase

Food and Drug Administration

growth arrest and DNA damage‐inducible protein GADD45 α

gastrointestinal tract

human epidermal growth factor receptor

high‐performance liquid chromatography

high‐performance liquid chromatography‐electrospray ionization mass spectrometry

inhibitor of κBα

liquid chromatography–mass spectrometry

liquid chromatography–nuclear magnetic resonance spectroscopy‐mass spectrometry

liquid chromatography‐solid‐phase extraction‐nuclear magnetic resonance spectroscopy

lipopolysaccharide

microtubule‐associated protein 2

Mcl‐1 short form

mass spectrometry

Mucin 1

nuclear factor kappa B

nuclear magnetic resonance spectroscopy

non‐small cell lung cancer

p38mitogen‐activated protein kinase

Poli (ADP‐Ribose) Polymerase‐1

protein kinase C‐α

reversion‐inducing cysteine‐rich protein with Kazal motifs

steroidal alkaloids

self‐contained underwater breathing apparatus

S‐phase kinase‐assoc. protein 2

second mitochondrion‐derived activator of caspases/direct inhibitor of apoptosis‐binding protein with a low isoelectric point

smoothened receptor

signal transducer and activator of transcription 3

transactivation‐responsive

tissue inhibitor of metalloproteinase‐1

tyrosine kinase inhibitors

tumor necrosis factor α

topoisomerase Iiα

thioredoxin reductase 1

urokinase‐type plasminogen activator

vascular endothelial growth factor

Introduction

Small molecules containing a steroidal structure possess a wide range of pharmacological activities (e.g., antioxidant, neuroprotective and anti‐hypercholesterolemic), irrespective of their highly conserved chemical structures and hormonal action (e.g., glucocorticoids, mineralocorticoids and gonadal steroids).1, 2 Various natural products have traditionally been used to treat or cure different diseases.3, 4, 5, 6, 7, 8, 9 Recently, extensive studies have used natural and synthetically derived compounds to develop novel preventive or therapeutic agents for clinical applications for cancer treatment.10, 11, 12, 13, 14, 15, 16 Over 60% of the available anticancer drugs are derived from natural sources, and this underlies the importance of these studies.17 Understanding the structure–activity relationships and traditional applications of natural product‐derived compounds can facilitate the design and synthesis of a vast array of novel therapeutics.

Prospective natural sources for drug discovery can be terrestrial or marine; those of terrestrial origin (plants, microorganisms, vertebrates and invertebrates) are easily accessible and offer a promising foundation for potentially new chemical entities (NCE). According to the World Health Organization (WHO), approximately 80% of the global population (representing approximately four billion people) living in developing countries depend on plant‐based traditional medicines for their primary health care.18, 19 Ethnomedicinal properties of traditional medicines, along with their diverse medical applications in various diseases, have been well‐characterized in several publications.20, 21, 22, 23, 24, 25, 26 Goat bitter apple or soda apple (Solanum aculeastrum), thorn apple (Solanum incanum L.), tomato (Lycopersicon esculentum M.), false daisy (Eclipta alba L.) and Christmas box (Sarcococca saligna) are well‐known plants possessing anticancer activity. Solamargine, tomatidine, solasonine, α‐solanine and α‐chaconine are natural products isolated from terrestrial plants and exhibit potent anticancer activity (Supporting Information S1, Tables 1–4). In addition to anticancer activity, steroidal alkaloids (SAs) also possess analgesic,27, 28 anti‐inflammatory,27, 29, 30, 31 antimicrobial,32, 33, 34, 35, 36, 37, 38, 39, 40 antithrombotic,41 antiandrogenic42 and antiarrhythmic43, 44, 45 properties, which deem them a subject of high research interest.

Conversely, marine organisms comprise ~50% of the total biodiversity of the Earth, and the marine ecosystem is an exceptional and vast reservoir of bioactive natural products with unique chemical features.46 Although few marine‐derived drug candidates are clinically available, significant numbers of anticancer SAs (ASAs) have been discovered from marine sources.47

Over 10,000 bioactive compounds have been discovered from the marine ecosystem, and hundreds of novel compounds with innumerable pharmacological properties are identified annually.48 Notably, sponges and worms are significant sources of ASAs. Although few ASAs have been obtained from the marine environment to date, among them, cephalostatin 1 is one of the most potent anticancer small molecules ever examined by the U.S. National Cancer Institute (NCI). Cephalostatin I is active at nanomolar concentrations and is noticeably more effective in vitro than paclitaxel in dispelling the mitochondrial membrane potential to induce apoptosis.49, 50, 51

Marine sources show great promise for the discovery of anticancer drugs, and the involvement of interdisciplinary fields, including pharmaceutical chemistry, pharmacology, analytical and organic chemistry, ecology, biology and biochemistry, has established marine anticancer drug discovery as a new field. Approximately seven marine‐derived pharmaceutical elements have been approved by the FDA (Food and Drug Administration) for clinical use as drugs for various diseases. Brentuximab vedotin was approved for the treatment of Hodgkin's lymphoma and anaplastic large T‐cell malignant lymphoma, cytarabine for leukemia, trabectedin for ovarian cancer and soft tissue sarcoma, eribulin mesylate for metastatic breast cancer, Ziconotide for pain, omega‐3‐acid ethyl esters for hypertriglyceridemia, vidarabine for recurrent epithelial keratitis caused by HSV, superficial keratitis and acute kerato‐conjunctivitis; many other compounds are undergoing clinical trials.47, 52, 53, 54, 55, 56 Clinical trials involving cephalostatins 1 and 7 have been hindered because of the complications in harvesting the materials (0.1 g and <60 mg of cephalostatins 1 and 7, respectively, from 450 kg of the marine tube worm (Cephalodiscus gilchristi) from 60 to 80 m deep in sea waters near East Africa.57 Synthesis of cephalostatin will render the bis‐steroidal alkaloid more available and allow clinical trials. To expedite the design and development of cephalostatin, the identification and validation of the biological targets of cephalostatin should be coupled with quantitative structure–activity relationship (QSAR) studies.

To date, various articles have been published regarding the isolation, synthesis and anticancer effects of SAs; however, to the best of our knowledge, a review focusing on the mechanisms of action of SAs and their synthetic/semi‐synthetic derivatives in various cancer cell lines, including their sources and inhibitory concentrations, has not yet been published. Secondary metabolites from natural products exhibit more “drug‐likeness” (a qualitative concept for how “druglike” a compound is with respect to various parameters such as molecular weight, hydrogen bond and bioavailability, used in the field of drug design) properties than wholly synthetic molecules.58 Therefore, we summarized the available information regarding these ASAs to assist future efforts toward anticarcinogenic drug discovery.

In this review, we systematically summarize the in vitro (Supporting Information S1, Tables 1–3) and in vivo (Supporting Information S1, Table 4) anticancer effects of natural, synthetic and semi‐synthetic SAs, with emphasis on their mechanisms of action. Hereafter, we also highlight the molecular mechanisms involved in their anticancer effects (Supporting Information S1, Tables 1–4; Fig. 1), along with various in silico approaches involved in the development of anticancer leads from SAs.

Figure 1

Anticancer mechanism of steroidal alkaloids. Reproduced with permission from the publisher of Ref. 54. Copyright © 2015, John Wiley and Sons.

Steroidal Alkaloids

Pharmacological properties

Steroidal alkaloids are secondary metabolites, which can be defined as organic molecules involved in the defense mechanisms of plants, with no role in growth, development or reproduction. In addition to SAs, terpenes, phenolic compounds, flavonoids and coumarins are known as secondary metabolites.59, 60, 61 They constitute an essential class of alkaloids generally isolated in the glycoalkaloid form, which characteristically occurs in higher plants from the Solanaceae, Liliaceae, Apocynaceae and Buxaceae families, and in amphibians and marine invertebrates (Fig. 2).62, 63, 64 In addition to their different bio‐potentialities, including analgesic,28 antimicrobial,39 and anti‐inflammatory29 activities, their anticancer potentials are being explored.65, 66, 67 Numerous synthetic routes have also been developed for synthesizing SA analogs to achieve desired biological effects. Although there are multiple reports regarding the widespread pharmacological activities of SAs, few systematic reviews regarding the anticancer potentials and mechanisms of action of SAs are currently available.

Figure 2

Vital sources of steroidal alkaloids. Reproduced with permission from the publisher of Ref. 54. Copyright © 2015, John Wiley and Sons.

Structural insights and classification

SAs are characterized by a cyclopentanephenanthrene framework (steroidal scaffold), with a nitrogen atom fused as a fundamental fragment of the molecule, or in the ring or side chain.62 Generally, SAs possess a 21‐(pregnane), a 24‐(cyclopregnane), a 27‐carbon heterocyclic ring or combination of these. The C27 heterocyclic ring containing SAs are of two types: cholestane and C‐nor‐D‐homosteroidal alkaloids, mostly occurring in the Solanaceae and Liliaceae families. Table 1 lists the different types of natural SAs.68

Table 1

Examples of major SAs

Major types of SAs	Origin	Instances	Brief Chemistry of the representatives
Salamandra	Amphibians	Cycloneosamandione	In this type of steroidal alkaloid, the ring‐A is altered, commonly into aza‐homosteroids, except for Cycloneosamandione.
Jerveratrum	Solanaceae, mainly found in food crops like potatoes, tomatoes	Jervine	Here the side‐chain of a C27 steroid has been transformed into a piperidine ring.
Spirosolane		Soladulcidine
Solanidine		Rubijervine	In this type, an extra ring is formed by C‐16 to N bond formation.
Cerveratrum	Monocot families such as the Orchidaceae	Cevane	In this type of steroidal alkaloid, an abeo shift occurs from a procevine‐type precursor, where the D ring has been expanded at the expense of the C ring. A widespread stereochemical difference has been observed within the basic cevane skeleton.
Conanine	Apocynaceae	Kurcholessine	Here almost all of the bases comprise an amino or an oxygen function at C‐3 position. Some differences are observed in their skeletons such as methylation at the C‐4 position.
Buxus		Cortistatin	This type of steroidal alkaloid is one of the largest groups, where the maximum structures are pentacyclic 4, 4, 14‐trimethyl‐9, 19‐cyclopregnanes. Some of this group posses a tetracyclic system where the 9, 10 bond has been fused to give a seven‐membered ring B. In some representatives, one or both carbon atoms attached to C‐4 have been lost. Presence of a nitrogen function at C‐3 and/or C‐20 has been observed, which may be partially methylated, fully methylated or unmethylated. Slight structural differences remain to be isolated from various Buxus spp. In this group, the suffix letters used in the nomenclature designate the degree of methylation of the nitrogen atom(s).
Pregnane	Apocynaceae, Buxaceae and Didymelaceae		In this type, major representatives contain an amino group or related functional groups at C‐3 or C‐20, and regularly at other positions .
Cephalostatins/ ritterazines	marine worm Cephalodiscus gilchristi (Cephalostatin 1) and tunicate Ritterella tokioka (Ritterazine A)	Cephalostatin 1, Ritterazine A	The cephalostatin 1 structure is categorized by an asymmetric blending of the central pyrazine ring with two extremely oxygenated steroidal spiroketal subunits. In ritterazine A, the left half is similar to that of cephalostatin 7, excluding an additional C‐7′ hydroxyl group. In the right half, the steroidal nucleus is totally rearranged.
Assorted steroidal alkaloids, including non‐nitrogenous steroids connected by an ester or acetal bond to a nitrogen‐containing unit	Marine sponges, toads,	Plakinamines, bufotoxin	In this group, the ester or acetal bond act as a linker between non‐nitrogenous steroids and the nitrogen containing unit.

Abbreviation: SA, steroidal alkaloid.

Mode of action of the anticancer activity exerted by SAs

Induction of apoptosis

Induction of apoptosis is one of the essential anticancer mechanisms of SAs. Apoptosis, or programmed cell death, is a highly synchronized and conserved cellular phenomenon maintained by a highly organized network of intrinsic cellular suicide machinery. When the homeostasis between cell proliferation and death is disturbed, apoptosis‐inducing pathways are altered, which results in oncogenesis.69 Receptor‐controlled extrinsic pathways or mitochondrion‐controlled intrinsic pathways can trigger apoptosis.70 Additionally, other cellular organelles, including the endoplasmic reticulum (ER), cytoskeleton, lysosomes and nucleus, might participate in apoptotic communication by detecting impairments or by incorporating pro‐apoptotic signals.71 Various SAs activate apoptosis in specific cancer cell lines, including briofilin [increases Bax protein expression, suppresses Bcl‐2, caspase‐3 initiation and segmentation of poly (ADP‐ribose) polymerase‐1 (PARP‐1) in HeLa cells], the 3‐O‐(β‐d‐glucopyranosyl) etioline‐induced receptor‐mediated extrinsic apoptotic pathway, solamargine (upregulates TNFR‐1), cephalostatin 1 (uses Smac/DIABLO for inducing apoptosis) (Supporting Information S1, Tables 1–4).50, 51, 57

Cell cycle arrest

Disruption of cell cycle progression plays a role in the anti‐oncogenic effect of SA (Supporting Information S1, Tables 1 and 2). Mitogenic signals permit cells to enter controlled pathways, allowing traversing of the cell cycle.72 CDKs are master regulators in the cell cycle.73, 74 Dysregulation or modification of CDKs results in neoplasia.72 Hyperactivation of CDKs results from mutations in CDK‐regulated genes; CDK‐inhibitor genes are linked to several cancer types. Thus, the inhibitors or modulators offer great promise for use in novel anticarcinogenic therapies.75, 76 SAs such as solamargine, ritterazine b and solanidine derivatives arrest the G0/G1 and G2/M checkpoints in cancer cells (Supporting Information S1, Tables 1 and 2).30, 50, 77, 78, 79, 80, 81

Anti‐proliferative and anti‐metastatic effects

The molecular mechanism underlying the antiproliferative effects of SAs might involve the inhibition of various cell signaling pathways and proteins (such as MMP‐2/9 and AKT) that allow the growth of cancer cells.82, 83, 84, 85, 86, 87, 88 Inhibition of MMP‐2 and MMP‐9 by solanine is associated with the suppression of A2058 human melanoma cell migration and invasion, which contributes to the anti‐metastatic activity of SAs.89 Akt (protein kinase B) phosphorylation, which regulates various proteins involved in metastasis and cancer cell proliferation, is inhibited by α‐tomatine.90, 91, 92, 93 The anti‐proliferative activity of α‐tomatine also involves an increase in p21WAF1/CIP1 levels and checkpoint kinase‐2 activation (Supporting Information S1, Tables 1, 3 and 4). Additionally, SAs such as solasodine and cephalostatin 1 can induce DNA fragmentation and inhibit protein synthesis (Supporting Information S1, Tables 1, 2 and 4).50, 51, 57, 80, 94, 95, 96, 97, 98, 99, 100

Anticancer effects of major SAs

Tomatidine

Goat bitter apple or soda apple (S. aculeastrum) and tomato (S. lycopersicon L.) are rich sources of tomatidine and its glycosides such as α‐, β‐, γ‐ and δ‐tomatine.101 Tomatidine exerts cytotoxic activity against HBL‐100 cells.102 MCF‐7, HT‐29 and HeLa cells were blocked at the G0/G1 phase after treatment with tomatidine.103 Tomatidine also obstructs the invasion and migration of A549 cells by upregulating TIMP‐1 and RECK and downregulating MMP‐2/9 expression.66 By inhibiting the function of ABC transporters, tomatidine efficiently sensitizes carcinoma cells.104

Tomatidine glycosides are more cytotoxic against MDA‐MB‐231, KATO III and PC‐3 cells, compared to tomatidine,105 proving that the carbohydrate moiety plays a pivotal role in its cytotoxicity. α‐Tomatine exerts significant cytotoxicity against MCF‐7, HL60, NCI‐H460, AGS, K562 and HT‐29 cells.106, 107, 108 Lee et al., 2011 reported that α‐tomatine treatment for 1 h kills PC‐3 cells, compared to the normal prostate and liver cells.92 The half‐maximal effective concentration (EC50) of α‐tomatine for PC‐3 cancer cells was 1.67 ± 0.3 μM, whereas those for the normal liver cells WRL‐68 and the normal prostate cells RWPE‐1 was higher (>5.0 and 3.85 ± 0.1 μM, respectively) for 24‐h‐long treatment. The anti‐metastatic activity of α‐tomatine has been well characterized through the inhibition of the signaling of the PI3K/Akt pathway, inhibition of FAK phosphorylation and modulation of IκBα protein expression.108 α‐Tomatine can activate cellular apoptosis via caspase‐3/−8/−9, Mcl‐1 and Bak activation.92

Solamargine and solasonine

Solamargine and solasonine are two important spirosalane SAs. Solamargine exhibits selective and significant cytotoxic effects against HepG2, HCT116, MCF‐7, HeLa, K562 and A549 cells, with an IC50 value of 2.5, 3.8, 2.1, 6.0, 5.2 and 8.0 μM, respectively, which are 2–3‐fold lower than that for HL7702 human normal hepatocytes and H9C2 rat normal cardiomyoblasts (IC50, 13.5 and >20 μM, respectively).77 Shiu et al., 2007 reported that solamargine exhibited more cytotoxicity on breast cancer cells, compared to cyclophosphamide, methotrexate, epirubicin, cisplatin and 5‐fluorouracil.78 Furthermore, intravenous injection of solamargine (2.4 mg/kg) inhibited the progression of hepatocellular H22 cancer cells and Ehrlich ascites tumors in mice by 57.37% and 67.55%, respectively.79

Solasodine

Similarly, solasodine and its derivatives exert effective anticancer action on various human cancer cells, including colon, hepatocellular, lung, cervical, gastric, glioblastoma and breast cancer.80, 94, 95, 96, 97 Solasodine hydrochloride monotherapy (intraperitoneal injection; 30 and 50 mg·kg−1·day−1 of total 14 days) can reduce multidrug‐resistant sarcoma S180 tumor growth by 67.4% and 80.1%, respectively, by boosting LAK (lymphokine‐activated killer cell) and NK (Natural Killer) cell activity, stimulating lymphocyte proliferation and improving IL‐2 production.98 In mice, solasodine hydrochloride (15 mg/ml) combined with cyclophosphamide (20 mg/ml) can significantly reduce multidrug‐resistant sarcoma S180 tumor growth, by inducing apoptosis, followed by the downregulation of topoisomerase II and P‐gp expression.99

Apoptosis induction exerts anticancer effects.109 Similarly, the treatment of human osteosarcoma 1,547 cells with solasodine can induce apoptosis.100 Solamargine can also induce apoptosis by modulating tumor necrosis factor receptor (TNFR) expression,80 upregulating caspase‐3/−9 and downregulating Bcl‐2 and Bcl‐xL expression78, 80, 81 in lung cancer cells.

α‐Solanine and α‐chaconine

α‐Solanine and α‐chaconine are important cholestane SAs. They possess almost similar structures, except side chains, where α‐solanine comprises glucose, galactose and rhamnose molecules, and α‐chaconine is constituted of glucose and two rhamnose molecules.110 Tumor growth and metastasis was inhibited by α‐solanine via the downregulation of miR21 expression.111 By inhibiting the phosphorylation of ERK1/2 and activating caspase‐3, α‐chaconine can also induce apoptosis in the HT‐29 cancer cell line.112 PI3K/Akt and MAPK pathways regulate the expression of MMP‐2/−9, and α‐solanine and α‐chaconine inhibit cancer cell invasion and migration by inhibiting Akt and ERK phosphorylation113 and MMP‐2/‐9 activity.89 The synergistic cytotoxic activities of α‐solanine and α‐chaconine are well established in HepG2 and AGS cells.114

Pregnane alkaloids

Various pregnane alkaloids display potent cytotoxic activity against several kinds of cancer cells. The role of sarcovagine D in PANC‐1, SMMC‐7721, HL60 and A549 cells is well established, with an IC50 value of 0.96–16.69 μM.115 The migration of EGF‐induced human breast cancer MB‐MDA‐231 cells was inhibited by the terminamines A–E and H isolated from Japanese pachysandra (Pachysandra terminalis).65 Sarcovagine D and sarcorucinine A1 also exert potent anticancer activity against K562, PANC‐1 and SK‐BR‐3 cells, with an IC50 value of 2.25–5.00 μM. Wrightia javanica contains another pregnane type SA, wrightiamine A, which acts as a cytotoxic agent against vincristine‐resistant P388 cells.116 SMMC‐7721, MCF‐7, HL60, SW480 and A549 cells become susceptible after treatment with veralkamine 3‐(b‐d‐glucopyranoside), 6,7‐epoxyverdine and 3‐O‐acetylveralkamine, isolated from Veratrum taliense.117 Sarsaligenines A and sarsaligenines B also exert cell growth inhibitory activity in human leukemia HL60 cells with an IC50 value of 2.87 and 3.61 μM, respectively.115

Cyclopamine

Many studies have explored the cytotoxic potential of cyclopamine and its derivative in various cancer cell line (in vitro and in vivo) models. The cyclopamine C‐nor‐D‐homosteroidal alkaloid has been characterized as an antagonist of the Hh signaling pathway and is teratogenic in animals.118 In SMMC‐7721, PLC/PRF/5 and Huh7 cells, cyclopamine is responsible for the induction of apoptosis via inhibition of the Shh signaling pathway and downregulation of Bcl‐2 expression.119 The molecular mechanism of apoptosis induction by cyclopamine includes modulation of the AKT and ERK pathways.120

In prostate cancer, veratramine and jervine (structurally related to cyclopamine) exert anti‐proliferative effects.121 Veratramine inhibits cell proliferation and exerts cytotoxicity against PANC‐1, NCI‐H249, SW1990 and A549 cells, with an IC50 value of 14.5, 8.5, 26.1 and 8.9 μM respectively.67 KAAD‐cyclopamine selectively inhibits the Hh signaling pathway by oncogenic mutations,122 and exo‐cyclopamine and its analogs also block the Hh signaling corridor in a Gli‐dependent reporter assay.123 Cyclopamine and KAAD‐cyclopamine act as in vitro and in vivo anti‐proliferative agents by targeting the smoothened (SMO) receptor.124 Cyclopamine selectively targets and reduces Gli1 expression, resulting in the arrest of cell growth and induction of apoptosis in various leukemia and lymphoma cells.125 Moreover, cyclopamine displays noticeable anticancer effects in breast,126 lung,127 GIT/gastric, pancreatic, esophageal and biliary tract,128, 129 and prostate cancer;130 leukemia;125 and oral squamous cell carcinoma.131

Cyclopregnane alkaloids

Cortistatin A‐D,117 E‐H132 and J‐L133 exert antiproliferative activity against HUVECs, KB3‐1, Neuro2A, K562 and NHDF cells; HUVECs; and HUVECs, respectively, with an IC50 value of 2.3–14 μM. Angiogenesis is the process of formation of new blood vessels from former blood vessels. Angiogenesis regulates tumor development and metastasis. Angiogenesis antagonists can be considered potential candidates as antitumor agents.133 Cortistatin, a cyclopregnane alkaloid isolated from the Indonesian marine sponge Corticium simplex exerts cytostatic anti‐proliferative action against HUVECs via inhibition of angiogenesis.133 Among all cortistatins, cortistatin A exhibits discriminating anti‐proliferative action against HUVECs, by inhibiting the migration and tubular development of HUVECs promoted by VEGF or bFGF.117

Other Multifarious Pharmacological Activities of SAs

Apart from anticancer activity, different SAs possess various other pharmacological actions. For example, solasodine, α‐tomatine and neoverataline A and B possess antifungal activity; α‐chaconine, solanidine, α‐solanine and tomatidine exert anti‐inflammatory activity; saligcinnamide, N(a)‐methyl epipachysamine‐D and tomatidine show antibacterial activity; hookerianamide H and hookerianamide, Na‐methylepipachysamine D and sarcovagine C exhibited antiplasmodial activity; solasodine showed anti‐nociceptive and anti‐estrogenic activity; (+)‐(20S)‐3‐(benzoylamino)‐20‐(dimethylamino)‐5α‐pregn‐2‐en‐4β‐ol and [(+)‐(20S)‐20‐(dimethylamino)‐3α‐(methylsenecioylamino)‐5α‐pregn‐12β‐ol possess antiestrogenic activity; and cyclovirobuxine showed cytoprotective effects.43, 44, 45, 134, 135

Toxicity of SAs

The steroidal jerveratrum alkaloid cyclopamine (Fig. 3) and veratrum alkaloid jervine (Fig. 3) are potent teratogens that cause synophthalmia in sheep, rabbits, mice and hamsters.116, 136, 137, 138 Lipinski et al., 2010 reported that cyclopamine and its semi‐synthetic analog AZ75 can cause palate defects, lateral cleft lip and semilobar and alobar holoprosencephaly in mice, respectively.139 Exposure to α‐chaconine might cause significant hepatotoxicity in normal human Chang liver cells.140 In frog embryos, exposure to α‐chaconine and α‐solanine can induce neurological noxiousness (viz. spina bifida and other deformities).141 According to Chaube et al., 1976,141 Smith et al., 1996,142 and Langkilde et al., 2008,143 the inhibition of cholinesterase enzyme activity in the human central nervous system (CNS) and cell membrane disruption in the gastrointestinal tract (GIT) may be attributed to the toxic effects of α‐chaconine and α‐solanine.144 In hamsters, the teratogenic effects of solasodine may be attributed to the induction of spina bifida, exencephaly and cranial blebbing.145 Structure–activity relationship analyses by Friedman et al., 1991,141 showed that the teratogenic effects of jervanes, solanidanes and spirosolanes were caused by C‐5 and C‐6 unsaturation in the steroidal skeleton and the olefinic linkage at C‐5 and C‐6 renders them more toxic. In pregnant and non‐pregnant mice, three steroidal glycoalkaloids (solasodine, solanidine and tomatidine) induced hepatomegaly after being fed a diet containing these aglycones for 2 weeks.146 Solanum potato glycoalkaloids, namely, α‐chaconine and α‐solanine, the aglycones solanidine and solasodine and the veratrum alkaloid jervine exert toxic effects in rainbow trout (Oncorhynchus mykiss) and the Japanese rice fish medaka (Oryzias latipes).147 The SAs from potato peels (solanidine, demissidine, α‐chaconine and α‐solanine) are highly toxic to humans at concentrations of >1 mg/g (dry weight).148, 149

Figure 3

Chemical structure of typical steroidal alkaloids. [Color figure can be viewed at wileyonlinelibrary.com]

In silico approaches for exploiting anticancer leads from SAs

Computer‐aided drug design approaches are a promising area for the development of anticancer drug‐like compounds using SA scaffolds. To avoid the huge costs and labor intensiveness incurred with random screening, different types of in silico approaches, including virtual screening, molecular modeling, molecular docking, QSAR, pharmacophore modeling, high‐throughput screening, and cloud computing, are efficient and effective for anticancer drug discovery campaigns using SAs.150, 151, 152 This technology has changed the scenario of rational design in the anticancer drug discovery process. Various web‐based programs for QSAR [JRC QSAR Model Database, DemQSAR, OCHEM (Online Chemical Modeling Environment), and MC‐3DQSAR] are available for the generation of potential leads.153, 154, 155, 156 For hit identification to lead optimization, structure and ligand‐based virtual screening, other web‐accessible servers and software (commercial/free versions; Supporting Information S2, Tables 1–3) are available. These computational methodologies provide a powerful toolbox for target identification, hit and lead generation and lead optimization. Synergism between dry and wet labs can facilitate the discovery of cost‐effective and reliable personalized medicines to improve anti‐cancer drug discovery. Figure 4 is a flowchart illustrating the generation of smart anti‐cancer drugs from SAs of natural and synthetic origins.

Figure 4

In silico approaches for smart anticancer drug development from steroidal alkaloids. [Color figure can be viewed at wileyonlinelibrary.com]

Recent trends and future directions

Natural or synthetic secondary metabolites possess potent pharmacological activities. Among them, some have cleared the clinical trial parameters and are clinically available.157 For instance, the FDA has approved vincristine, which is isolated from the leaves of the Madagascar periwinkle Catharanthus roseus (L.) G. Don (formerly Vinca rosea L.), for cancer treatment.158 Dactinomycin, produced by Streptomyces parvulus, is administered as an antineoplastic antibiotic for the treatment of Kaposi's sarcoma, infantile fibrosarcoma and testicular cancer.159, 160, 161 In 2005, paclitaxel (isolated from the bark of the Pacific yew (Taxus brevifolia Nutt.) was approved by the FDA for use in the United States for the treatment of pancreatic, breast and non‐small cell lung cancers.162 In recent times, pharmaceutical companies have been focusing on drug discovery from natural and synthetic origins through combinatorial chemistry, which includes the generation of libraries containing millions of compounds,163 in silico molecular docking, structure‐ and target‐based virtual screening, molecular modeling, pharmacophore designing and receptor‐based QSAR studies. Many biotechnological companies also concentrate on lead identification and validation of natural products (or synthetic derivatives), and the development of these leads into drugs.163 Numerous drugs of plant origin are currently undergoing clinical trials.164, 165

Previously, drug discovery using natural sources was a time‐consuming process, in particular, because of the recognition and structural elucidation of the bioactive compounds, which could take months or years. The rapidity of bioassay‐guided fractionation, identification and structural elucidation of compounds can be significantly enhanced by using instruments and methods such as HPLC, MS or LC–MS, higher magnetic field‐strength NMR, cap‐NMR, LC‐NMR‐MS, LC‐solid phase extraction (SPE)‐NMR, LC‐SPE‐NMR in combination with HPLC‐ESIMS, and robotics, to systematize high‐throughput bioassays.166, 167, 168, 169

Prompt screening of plant extracts for their biological activity is no longer a rate‐limiting step because of the improvement of computerized high‐throughput performances. Compounds with features such as autofluorescence or UV absorption often create problems in the screening process because of interference with the output, which can be overcome by prefractionation of the extracts.170 Additionally, computational filtering methods have been applied with most high‐throughput screening methods to minimize false‐positive results.171 In the future, dereplication can be implicated in the routine use of NMR‐hyphenated techniques.168 Furthermore, simple synthetic methodologies can be implicated for bioassays, and novel analogs of the parent molecules can be designed and developed using upgraded combinatorial chemistry.

To meet the continuous demand, a constant and sufficient supply of anticancer drugs is essential. Plant cell culture is an alternative method for compounds with low synthetic yields, as altering the environmental conditions and medium can result in larger yield.172, 173 Paclitaxel and Dioscorea are successfully produced using cell culture techniques.174, 175, 176, 177

Summary and concluding remarks

The increasing incidence of cancer is a global concern. To avoid the toxicity associated with chemotherapy and irradiation, SAs are being investigated as alternative therapeutic measures. Most SAs are from different sources, and their mechanisms of action also vary. They can exert anticancer effects at micromolar or nanomolar concentrations (Supporting Information Files S1 and S2). This review discussed the potential of SAs as anticancer agents, with a focus on their natural and synthetic sources, in vitro or in vivo models for study, inhibitory concentrations and mechanisms of action.

The key molecular targets of SAs are summarized in Figure 5. They exert their anticancer effects by inducing apoptosis or autophagy by upregulating or downregulating apoptotic (Bax, Bcl2, Bcl‐xL, Caspase‐3/8/9, PARP, TNFR I/II, Fas or HER2) and autophagic (LC3, AKT or mTOR) proteins. The inhibition of cell cycle progression at either G0/G1 or G2/M phase by interaction with CDKs is another mechanism underlying their anticancer activity. The antimetastatic action of SAs can be attributed to their interactions with, and the changes in the expression of ERK, RECK, TIMP and MMP‐2/9 proteins. SAs such as solasodine block P‐gp and inhibit multidrug‐resistant cancers. They also disrupt the integrity of the cell membrane by changing cell morphology and DNA content, thereby exerting anticancer activity. SAs such as cyclopamine inhibits xenograft tumor growth via the Hh pathway; α‐tomatine reduces xenograft tumor growth by inducing apoptosis via interactions with survivin, p50 and p65. Apart from the potent anticancer and other pharmacological effects, some SAs exhibit toxic effects such as hepatotoxicity in normal human liver cells (Chang Liver cell line) and teratogenicity, palate defects and lateral cleft lips in sheep, rabbits, mice and hamsters. SAs such as α‐chaconine and α‐solanine are highly toxic to humans at concentrations of >1 mg/g (dry weight).

Figure 5

Steroidal alkaloids and major cellular signaling molecules. [Color figure can be viewed at wileyonlinelibrary.com]

Although many plant‐derived and synthetic SAs possess anticancer effects, the underlying molecular mechanisms remain to be established, predominantly with respect to the pharmacokinetics around the active site of the target protein. Such data is essential for clearance of standard clinical trial parameters and confirmation that these drugs are effective and safe. Understanding the crosstalk between SAs and associated signaling molecules will assist improved understanding of their molecular mechanisms of action and the development of anticancer drugs. Various biochemical and biophysical approaches, including co‐crystallization and 3D‐structure determination, can help understand the mechanisms underlying these interactions and develop more selective and less toxic anticancer drugs.

Combining existing chemotherapeutic drugs such as solamargine with cisplatin, MTX or 5‐Fu can elevate the combined anticancer activity, thereby proving that combination therapy can exert synergistic or enhanced anticancer activity. In silico tactics, including molecular docking and QSAR studies, can be introduced to elucidate the molecular mechanisms, which can be validated by in vitro and in vivo experiments to aid significant development toward cancer prevention and treatment.

human ovarian cancer cell line

skin epidermoid carcinoma

human lung adenocarcinoma cell

human Caucasian gastric adenocarcinoma

human gastric cancer cell line

murine melanoma

murine melanoma

mouse melanoma cell line

human stomach adenocarcinoma cell line

benign prostate hyperplasia epithelial cell line

osteotropic prostate cancer cell line

human prostate adenocarcinoma cell line

human lung fibroblasts

human lung cancer cells

human adenocarcinoma

multidrug‐resistant counterpart of human NSCLC H460 cells

squamous cell lung carcinoma

large cell lung cancer

small cell lung cancer

human bronchial epithelial cell line SV40‐transformed

human breast transformed epithelial cell line

colon cancer cell line

cervical carcinoma

larynx cancer cell line

hepatocellular carcinoma

liver cancer cell line

human promyelocytic leukemia cell line

normal hepatocyte

colonic adenocarcinoma

human umbilical vein endothelial cells

human leukemia Jurkat T cells

multidrug‐resistant counterpart of K562 cell line

human myelogenous leukemia cell line

multidrug‐resistant counterpart of KB cell line

squamous cell carcinoma cell line

KB epidermoid carcinoma cells

Lewis lung carcinoma cells

Madison lung tumor

breast adenocarcinoma

breast cancer cell line

human melanoma.

human osteosarcoma

human gastric cancer cell line

gastric cancer cell lines

human glioblastoma

human leukemic T‐lymphocytes cell line

human lung large cell carcinoma

murine neuroblastoma cells.

normal human dermal fibroblast.

mouse embryonic fibroblast cells

human bronchopulmonary nonsmall‐cell lung carcinoma cells

parental and the Adriamycin (doxorubicin)‐resistant subline of mouse leukemia cells

vincristine‐resistant murine leukemia.

mouse lymphoid neoplasm.

human prostate cancer cell line

human retinal pigment epithelial‐1 cell line

human normal prostate cell line

human osteosarcoma

Shionogi carcinoma

gastric carcinoma

breast carcinoma

human hepatocarcinoma cell line

human glioblastoma

human osteosarcoma

human glioblastoma

human primary glioblastoma cell line

Chinese hamster lung fibroblasts.

African green monkey kidney cell line

human lung epithelial fibroblast

human melanoma cells line

human melanoma cells line

human normal liver cell line

human breast cancer cell line

File S1.

Table 1. The In‐vitro anticancer activity of steroidal alkaloids from natural sources in various cancer cell lines.

S1 Table 2. The in‐vitro anticancer activity of synthetic‐origin steroidal alkaloids in several cancer cell lines.

S1 Table 3. The in‐vitro anticancer activity of steroidal alkaloids from industrial sources in various cell lines.

S1 Table 4. The in‐vivo anticancer activity of steroidal alkaloids in various xenograft animal models.

Click here for additional data file.

File S2.

Table 1. Important web‐accessible databases for drug target identification.

S2 Table 2. Important web‐accessible databases for small molecule identification.

S2 Table 3. Important web‐accessible and commercially/freely available databases, and software for molecular docking.

Click here for additional data file.