Natural products (NPs) have played an important role in medicine for much of recorded human history. The earliest recorded use of medicinal plants dates back approximately 5000 years to a list of Sumerian drug recipes written on a clay tablet, but there is evidence that Neanderthals may have used plants for medicinal purposes as far back as 60,000 years ago [63,64]. Even today many people in the world rely on medicinal plants for their healthcare needs. It is estimated that 70-95% of people in most developing countries use traditional medicine for their primary healthcare needs . Traditional Chinese and Ayurvedic medicine have historically served as primary healthcare for many people in developing nations, and both systems have drawn the attention of pharmocognosists from around the world.
Active compounds from various organisms have had great success as pharmaceuticals. This is especially true in the case of cancer chemotherapeutics. Between 1981 and 2006, 63% of anticancer drugs being used came from NPs, were inspired by NPs, or were synthesized from a natural pharmacophore . The most profitable chemotherapy drug in history, taxol (or paclitaxel), is a natural product derived from the bark of the Pacific Yew Tree . Taxol was discovered through a random screening of approximately 15,000 species of plants , but targeted screening of known medicinal plants for anticancer properties has also been historically successful. For example, the vinca alkaloids vincristine and vinblastine have been used clinically in cancer therapies for over 50 years . These compounds were isolated from the rosy periwinkle, Catharanthus roseus, a plant used in both traditional Chinese medicine and Ayurvedic medicine. Bacteria have also been a source of successful anticancer agents. Anthracyclines, such as doxorubicin, are isolated from certain Steptomyces bacteria and have been used to treat breast cancer for decades .
With advances in technologies such as high throughput screening (HTS) and combinatorial chemistry in the 90’s, the cancer related drug discovery efforts of many pharmaceutical companies shifted to targeted therapies . These targeted, receptor specific therapies relied upon small synthetic molecules or antibodies that could act as “magic bullets” to treat specific cancer cells. Combinatorial chemistry has allowed vast libraries of new chemical entities to be generated synthetically which can be tested against disease related targets. Thousands of compounds from combinatorial chemistry libraries can be analyzed every day using HTS . In addition, advances in proteomics and genomics have enabled researchers to attempt to model molecules that can interact with specific biological targets. The initial success of these targeted therapies including Gleevec and Herceptin led many to believe that traditional NP based drug discovery had become obsolete .
However, the limited number of successful drug candidates from targeted therapies, the relatively small number of cancers successfully treated with new therapies, and the higher risk of cancer developing a resistance to treatment created a renewed interest in natural product drug discovery in the late 2000’s . The limited efficacy of targeted therapies is of increased likelihood in CSCs, due to the lack of agreed upon universal CSC markers and the many survival mechanisms which they employ. Numerous NPs and their derivatives have shown early clinical success or have received FDA approval for the treatment of cancer since the recent renewal in their interest [46,72]. Despite the obstacles facing the screening of NPs using HTS, they have shown many advantages over synthetic chemical entities. Natural products are thought to possess “privileged structures” that are specialized to interact with biological targets allowing them to influence multiple cellular pathways simultaneously. This ability is crucial in combatting cancer and CSCs, as the robust survivability of cancer is often the result of many different mechanisms. Additionally, the chemical character and diversity of NPs is more favorable than that of synthetic molecules. When compared to synthetic libraries, NP libraries tend to have more chiral centers, higher steric complexity, fewer heavy atoms, more solvated hydrogen-bond donors and acceptors, and a larger variety of molecular properties . Furthermore, historic use of a medicinal plant from which a NP is isolated can speak to the safety of compound for human consumption and the potential to limit sideeffects.
The continued ability of natural compounds to compete with synthetic chemical entities has shown that NP based drug discovery is still relevant and capable of advancing the treatment of cancer. It is likely that the successful screening of NPs for cancer killing potential can be successfully applied to screening for CSC targeting agents. A few promising NPs have been utilized to target CSCs in vivo and in vitro. These compounds may have the potential to sensitize CSCs to conventional treatments, directly induce cell death in CSCs, force CSCs to differentiate, or prevent CSCs from entering a dormant and more resistant state. A brief review of these compounds can be found below. The reader of this review is directed to other reviews for a more comprehensive list of NPs capable of targeting CSCs [12,73,74].
Pomegranate Confirmed To Be A Cancer Stem Cell Killer
Two recent studies have looked at the effects of a pomegranate extract on cancer stem cells.
The first, titled, “In vivo relevant mixed urolithins and ellagic acid inhibit phenotypic and molecular colon cancer stem cell features: A new potentiality for ellagitannin metabolites against cancer,” and published in the journal Food and Chemical Toxicology proposes that colon cancer stem cells (CSCs) “offer a novel paradigm for colorectal cancer (CRC) treatment.”
The researchers looked at how colon metabolites of polyphenols from pomegranate interact with and affect CSCs. Specifically, the researchers looked at:
“the effects against colon CSCs of two mixtures of ellagitannin (ET) metabolites, ellagic acid (EA) and the gut microbiota-derived urolithins (Uro) at concentrations detected in the human colon tissues following the intake of ET-containing products (pomegranate, walnuts).” [emphasis added]
The researchers confirmed that these mixtures were able to suppress colon CSCs taken from the primary tumor of a patient with colorectal cancer. They showed these mixtures altered the phenotypic and molecular features of colonospheres — the cancer stem cell based micro-colonies of cells that can become invasive and lethal cancers with time — consistent with cancer suppression.
According to the study authors,
“These data support a role for polyphenols metabolites in the control of colon cancer chemoresistance and relapse and encourage the research on the effects of polyphenols against CSCs.”
The second study, titled, “Pomegranate Extract Alters Breast Cancer Stem Cell Properties in Association with Inhibition of Epithelial-to-Mesenchymal Transition,” and published in the journal Nutrition and Cancer, evaluated pomegranate extract (PE) for its abiilty to alter the characteristics of both individual breast cancer stem cells, and clumps of them known as mammospheres. The study found that,
“PE inhibited mammosphere formation in two different cell lines, neoplastic mammary epithelial HMLER and breast cancer Hs578T. In addition, mammosphere-derived cells from PE treatment groups showed reduced mammosphere formation for at least two serial passages.”
Additionally, the researchers found that pomegranate extract was able to promote breast cancer stem cells differentiation (an indication of its chemopreventive properties), as well as preventing epithelial-to-mesenchymal transition (EMT), a key program in generating CSCs and maintaining their characteristics.
The researchers concluded from these findings that,
“The ability of PE to suppress CSCs can be exploited in the prevention of breast cancer.”
Many natural products used as pharmaceuticals can be classified as polyphenols. Polyphenols are structurally defined by the presence of aromatic benzene rings bonded to hydroxyl groups, but they encompass a number of structurally diverse compounds. These subgroups include flavonoids, stilbenes, tannins, lignans, and phenolic acids among others. Polyphenols of various groups have been demonstrated to regulate inflammation, angiogenesis, cell growth, invasiveness, and apoptosis in vitro . As a result, they have been studied extensively in the context of cancer prevention and metastasis. Recently, these investigations have been extended to determine the effect of polyphenols on CSCs. The polyphenols resveratrol and curcumin are notable examples of NPs that have been shown to exhibit cytotoxic effects on CSCs.
Resveratrol is a polyphenolic stilbene derivative most commonly found in the skin of grapes and berries. It has undergone extensive examination for its anti-inflammatory and antioxidant properties in addition to many other useful biological properties. These attributes give resveratrol the attractive potential to act as a cancer chemopreventative. Resveratrol has been shown to induce apoptosis and promote S-phase arrest of select cancer cells. This potential was demonstrated in Hep G2 hepatocyte carcinoma cells in vivo at concentrations ranging from 10 to 50 µM . At concentrations higher than 50 µM, however, resveratrol induced G1/G0 arrest which was confirmed in a separate study using a number of ovarian cancer cell lines [76,77]. Resveratrol has further been shown to induce cell death through a non-apoptotic mechanism at concentrations between 50 and 100 µM in a ovarian cancer cell lines . This variety of mechanisms demonstrates the ability of resveratrol, like other NPs, to influence numerous biological mechanisms simultaneously making it an attractive anticancer agent.
Resveratrol may also be able to eliminate CSC populations from tumors. The compound has been shown in a study by Shankar et al to induce caspase-3/7 activated apoptosis in CD44+/CD24+/ESA+ pancreatic CSCs at 10 to 30 µM concentrations. The study also found that 10 to 20 µM resveratrol was able to inhibit both stem cell maintaining factors, such as Nanog and Oct-4, as well as anti-apoptosis proteins of the Bcl-2 family in the pancreatic CSCs. Additionally, EMT proteins, such as Snail and Slug, as well as the EMT capability of the pancreatic CSCs in non-adherent conditions was inhibited in response to 10 to 20 µM of resveratrol. Further, the expression of the drug efflux pump ABCG2 was inhibited after administration of 10 to 30 µM of resveratrol, potentially sensitizing the cells to conventional chemotherapy treatments. The apparent ability of resveratrol to target CSCs and act as a chemopreventative and anti-inflammatory drug was further demonstrated using a mouse tumor model. The frequency of tumor formation in KrasG12Dmice, spontaneous pancreatic tumor forming mutants, was significantly diminished when treated with resveratrol for 10 months . The ability of resveratrol to induce apoptosis in CSCs as well as reduce their tumorigenic potential in vivo was additionally supported in a CD24-/CD44+/ESA+ model of breast cancer stem cells. In this study, apoptosis was induced in the breast CSCs through a FAS mediated pathway after incubation with 50 or 100 µM resveratrol. The tumorigenic potential of the cancer stem cells was significantly diminished in female nude mice through the administration of either an oral gavage or intraperitoneal injection of 22.4 kg/body weight of resveratrol, giving significant evidence that resveratrol is able to disrupt tumor formation by targeting CSCs .
While resveratrol exhibits extremely promising anticancer effects in preclinical studies in vivo and in vitro, resveratrol has failed to translate this success to clinical trials. This is due, in large part, to extremely low bioavailability, high effective dosages, and the rapid metabolism of resveratrol to glucuronide, sulfate, and hydroxylate conjugates [80,81]. These conjugates, once absorbed into the bloodstream fail to provide the same health benefits as free resveratrol. As a result, there have been efforts to engineer resveratrol formulations or drug delivery systems aimed at increasing the bioavailability of resveratrol. These include formulations to stabilize resveratrol in the body, formulations to increase the aqueous solubility of resveratrol, and encapsulation of resveratrol in various lipids, micelles, or polymer structures with the aim of sustained, concentrated, and/or targeted release [80,81].
Curcumin is another polyphenol which has been thoroughly investigated for its anticancer properties. This compound is a major component of turmeric, a spice widely used in Indian and many Middle-Eastern cuisines. Curcumin has been shown to exhibit an anti-inflammatory effect and promote apoptosis in cancer cells . It has been used in clinical trials demonstrating its safety at high doses and activity against pancreatic neoplasms in human patients despite its low bioavailability . The antitumor properties demonstrated by curcumin have led to investigations of its potential to target CSCs.
Curcumin has been used to inhibit the formation of breast cancer mammospheres in vitro by 50% and 100% using 5 µM and 10 µM concentrations, respectively, demonstrating the ability of curcumin to inhibit CSC’s ability to undergo EMT . An analogue of curcumin, GO-Y030, was demonstrated to induce apoptosis, diminish tumorsphere formation, and inhibit STAT3 phosphorylation in ALDH+/CD133+ colon CSCs when used at 2 to 5 µM concentrations. The ability of this analogue to target tumor initiating cells was further demonstrated using a NOD/SCID mouse model. When given a 50 mg/kg intraperitoneal injection of GO-Y030, the average tumor weight resulting from a xenograft implantation of 1 × 105 CSCs was diminished by 58.10% . Curcumin has also been suggested as a supplement to current chemotherapy treatments. Curcumin in combination with FOLFOX, a commonly prescribed combination of leucovorin calcium, fluorouracil, and oxaliplatin, was able to decrease the viability and diminish EMT of colon CSCs to a higher extent than FOLFOX alone .
While curcumin shows great potential as an anticancer agent and has been used in a number of clinical trials against cancer, it suffers similar shortcoming to resveratrol. Namely, the rapid metabolism and excretion of curcumin, along with its hydrophobicity, results in low bioavailability which has been demonstrated using mouse models [87,88]. Numerous drug delivery studies have been conducted to increase the bioavailability of curcumin including the use of adjuvants to interfere with metabolism, encapsulation in liposomes and nanoparticles, and the use of more stable structural analogues .
Flavonoids are a major class of polyphenolic secondary metabolites found in numerous medicinal plants. They are derived from flavone which contains two phenyl rings and one heterocyclic ring. Flavonoids are commonly found compounds throughout the plant kingdom, and as a result, they are widespread throughout the human diet. Due to their abundance in fruits, vegetables, nuts, spices, and herbs, a flavonoid rich diet has been suggested as a feasible means of cancer chemoprevention . Certain flavonoids including, quercetin and kaempferol, have been implicated as apoptosis inducers, antioxidants, inflammation regulators, and angiogenesis inhibitors. Further, certain flavonoids have been shown to have an effect on heat shock proteins, multiple drug resistance, adhesion, metastasis, and angiogenesis . The high number of CSC related properties which seem to be affected by flavonoids have led to their investigation as CSC targeting agents. A review of one such flavonoid, quercetin, is presented below.
Quercetin is a flavonol secondary metabolite found throughout many species of plants. Quercetin is a known anti-inflammatory agent and anti-oxidant which has been demonstrated to induce programmed cell death in many malignant cancer cell lines. Quercetin has been shown to interfere with a number of cellular pathways associated with the formation and maintenance of human cancers including down regulating P53, inhibiting tyrosine kinase, inhibiting heat shock proteins, and inducing type II estrogen receptor expression . Quercetin has further drawn attention as a potential CSC targeting therapeutic.
Not only has quercetin been shown to inhibit the proliferation of CD133+ colon CSCs at a concentration of 75 µM, but it also increases the sensitivity of these cells to doxorubicin (Adriamycin). In fact, when combined with 50 µM quercetin, doxorubicin doses were more effective at inhibiting CSC proliferation in vitro than doxorubicin doses three times more concentrated but lacking quercetin . This finding demonstrates the potential of quercetin and other natural products to enhance the use of other chemotherapeutics to eliminate CSC populations. The use of lower doses of chemotherapeutic agents in combination with natural products such as quercetin may result in diminished off target toxicity while also inducing apoptosis in CSCs, improving patient outcomes, lowering the risk of cancer recurrence, and preventing metastasis formation.
Other CSC models have been targeted using quercetin including CD44+/CD133+ prostate CSCs. At a concentration of 20 µM, quercetin lowers the viability of prostate tumor spheroids grown in non-adherent flasks as well as diminish the migratory, invasive, and colony forming potential of CD44+/CD133+prostate CSCs . In this same publication, quercetin was shown to synergize with epigallocatechin gallate, a catechin found in tea, synergistically amplifying the above effects on these prostate CSCs. As is the case with many other NP’s, however, quercetin’s poor solubility, poor permeability, and instability result in diminished bioavailability . The relatively high dose of quercetin required to elicit a biological response in combination with these issues warrant further drug delivery efforts to increase the lifetime and concentration of the compound at the site of the neoplasm.
Alkaloids are a class of pharmacologically active organic compounds distinguished by the presence of nitrogen and aromatic rings in the chemical structure. Alkaloids are produced throughout the plant kingdom, but are usually found in higher plants . Many alkaloids have been used throughout history in the medical field from quinine for the treatment of malaria to vinblastine for the treatment of multiple carcinomas. Several alkaloids have been used clinically in the treatment of cancer with great success, demonstrating their importance in the field. A small group of alkaloid compounds have even been shown to differentiate between healthy and cancerous DNA, inhibiting in vitro cancer DNA synthesis while leaving healthy DNA unaffected and resulting in a potential cancer treatment with diminished side-effects . New investigations on alkaloids are still being conducted showing further antineoplastic, anti-metastatic, and MDR inhibiting potential . These results suggest a potential for alkaloids to eliminate CSCs, and indeed, a number of compounds belonging to the alkaloid family have been shown to target CSCs in vitroand in vivo. Three promising anti-CSC alkaloids, dihydrocapsaicin, piperine, and berberine, are presented in the following sections.
Capsaicin is the secondary metabolite and alkaloid responsible for the hotness of many species of pepper. Dihydrocapsaicin (DHC), a saturated derivative of this compound, has exhibited numerous anti-neoplastic properties. DHC has been shown to induce dose-dependent and catalase regulated autophagic cell death in colon and breast cancer cells when used at concentrations between 50 and 400 µM . However, when autophagic cell death was inhibited through treatment with the inhibitor 3-methyladenine, DHC instead induced caspase-3 activated apoptosis in these cell lines. Further, when apoptosis was inhibited by the addition of peptide zVAD, autophagic cell death was enhanced. This ability to promote separate modes of cell death is a useful tool in targeting CSCs due to the many cell death evading pathways active in CSCs. This ability further highlights the potential of NPs to influence multiple cellular mechanisms and produce a robust cytotoxic effect on cancer cells.
A review of CSC related patents revealed that DHC is further hypothesized to exhibit a cytotoxic effect on neural CSCs . In one of the patents collected in the review, US20090076019A1, a neurosphere assay was invented to screen potential drugs for activity against neural stem cells. As the percentage of putative CSCs are increased in cancer neurospheres, compounds capable of inducing cell death in these spheres can be thought of as agents targeting neural CSCs. DHC was identified in this patent as one of several lead compounds which showed an ability to target CD133+ neural CSCs. The high IC50 values of DHC, however, limit its use as an effective chemotherapeutic agent, especially when one considers the low bioavailability common for many NPs. Further research is warranted to determine if DHC or an analogue can target any phenotype of CSCs with higher efficacy than what has been shown.
Piperine is a promising antineoplastic alkaloid found in black and long pepper. The use of piperine has previously been suggested as a cancer chemopreventative, but it has also demonstrated the ability to induce cell cycle arrest, endoplasmic reticulum stress, and apoptosis when exposed to colon cancer in vivoat concentrations between 75 and 150 µM . The treatment of colon cancer cells with piperine has been shown to reduce the ability of the cells to form non-adherent spheres and colonies, suggesting the inhibiting effect of piperine on CSCs. The apoptotic effect of piperine has additionally been confirmed using prostate cancer cells .
The ability of piperine to target stem cells specifically has been investigated in a breast tissue model. After pre-treatment with 5 to 10 µM piperine, the mammosphere formation potential, ALDH expression, and Wnt signaling of unsorted breast tissue was significantly diminished . Interestingly, the differentiated population of these cells was seemingly unaffected by the piperine treatment. The potential of piperine to target CSCs without affecting other cells is a fantastic example of the robust ability of NPs to influence molecular pathways while imparting only benign side effects. Piperine has additionally been suggested for use in combination therapies with compounds, such as resveratrol or curcumin, due to its ability to inhibit metabolic pathways. By slowing the glucuronidation of these compounds, piperine inhibits the metabolism and clearing of NPs and increases their bioavailability . By inducing a cytotoxic effect on CSCs and increasing the efficacy of other compounds, piperine acts as an ideal complementary medication to other NP chemotherapies.
Berberine is a tetracyclic, isoquinoline alkaloid found in the roots and stems of numerous plants. Berberine producing medicinal plants have been used as anti-inflammatories in Ayurvedic medicine for years, and the compound has been shown to induce dose-dependent apoptosis, initiated by reactive oxygen species generation, in a broad spectrum of cancers [102,103]. The apoptosis induced by berberine goes through an internal caspase-9 dependent pathway which results in a loss of mitochondrial membrane integrity. Like many natural products, the bioavailability of berberine is low in the body, limiting the potential of berberine as a drug. This obstacle is being overcome through the use of targeting liposomes as a drug delivery system . This delivery system encapsulated berberine into liposomes which were engineered to deliver the compound directly to the mitochondria of CD44+/CD24- breast cancer stem cells. Using this system, 1-50 µM of berberine was able to produce dose-dependent apoptosis in breast CSCs. The drug was further able to induce the expression of the pro-apoptotic protein Bax and activate caspase-9 and caspase-3 leading to apoptosis in CSCs isolated from MCF-7 mammospheres.
Additionally, berberine has been used to inhibit the expression of ABC transporters responsible for MDR in CSCs . Diminishing MDR, especially in CSC populations, makes berberine an attractive complementary medicine when currently accepted cytotoxic agents are unable to kill cancerous cells. An in vivo mouse model in which MCF-7 breast CSCs were injected into female nude mice followed by an array of berberine treatments and formulations demonstrated this synergistic capability. A mixture of 10 mg/kg of berberine liposomes and 10 mg/kg of paclitaxel liposomes was able to reduce the average tumor size in these mice by 85.5% compared to the control after just 21 days . In this way, berberine could be used to either target CSCs alone or in combination with traditional chemotherapy agents.
Many other natural compounds which do not fit into the classifications of polyphenols, flavanoids, or alkaloids have shown promise in targeting CSCs. Retinoids are an example of these compounds. Vitamin A, also known as retinol, generates a number of biologically active retinoids, including All-Trans Retinoic Acid (ATRA). ATRA has found clinical success in the treatment of acute promyelocytic leukemia under the trade name Tretinoin. The drug is marked by its successful induction of remission coupled with relatively mild side effects . The mechanism of action utilized by ATRA is through induction of cellular differentiation of leukemic and hematopoietic cells, and this differentiation induction has further been observed in other types of stem cells . The differentiation potential of retinoids presents a unique potential for cancer treatment, namely differentiating CSCs into a cell population more sensitive to classic chemotherapeutic regimens. Additionally, ATRA acts as an inhibitor of ALDH activity, potentially reversing a cause of MDR in CSCs . ATRA has thus been used to limit the tumorsphere formation ability and CSC percentage of breast cancer cells in vivo .
The lactone antibiotic brefeldin A is another NP that cannot be classified as a polyphenol, flavonoid, or alkaloid. It has shown anticancer potential in a number of cancer types including leukemia, colon, and prostate through p53 independent mechanisms [108,109]. Brefeldin A is produced by certain fungal organisms and acts as a protein transport inhibitor, preventing proteins from traveling from the endoplasmic reticulum (ER) to the Golgi apparatus. Subsequently, brefeldin A initiates ER stress, potentially leading to its apoptotic effects. Recently, brefeldin A has been shown to preferentially induce cell death in suspension cultures over adherent cultures of the human breast adenocarcinoma line MDA-MB-231. In the same publication, brefeldin A also down-regulated the expression of CD44, reduced the ability of the cells to form colonies in soft agarose, and reversed the EMT . Preferential killing of putative CSCs and inhibition of colony forming potential was similarly reported in the human colorectal cancer line Colo 205 . This preferential killing has the potential to diminish CSC populations while limiting the side effects typically associated with chemotherapy.
The cancer stem cell hypothesis, while still being investigated, presents explanations to many of the issues facing cancer treatment today. The CSC hypothesis explains the mechanisms underlying cancer recurrence, metastasis, and, to a degree, multiple drug resistance. Cancer treatments directed toward the eradication of CSCs could lead to higher survival rates and brighter prognoses for patients who fear cancer regression could occur at any time. Current cancer treatments are insufficient in regard to the eradication of CSC populations, likely due to the multitude of survival mechanisms utilized by CSCs and the lack of definitive, universal, single molecule targets that separate CSCs from healthy stem or somatic cells. Natural products have historically been an excellent source of bioactive compounds capable of targeting multiple pathways, and current investigations are underway to screen NPs for their effect on the CSC population of numerous cancer types. Many different NPs have exhibited a range of CSC inhibitory properties, and it is likely that more have yet to be discovered. As a result, NPs should continue to be screened as potential chemotherapy agents, complimentary treatments for compounds already in clinical use, and cancer prevention molecules with special attention focused on their ability to target CSCs. Further, due to the limited bioavailability and rapid metabolism of many NPs, these drug discovery efforts must be coupled with continued efforts to engineer robust drug formulations and delivery systems.