Phytocompounds and Nanoformulations for Anticancer Therapy: A Review

2.2.1. Phytocompounds

Our previous works demonstrated that the extract of Prunus pinose drupes (typical plant of Molise, IT) was effective on 2D, 3D, and in vivo CRC models. Analysis of the chemical composition of the compound (Trigno M^®, Biogroup, Isernia, Molise, Italy) showed that the main active components were phenolic acids, flavonoids, and anthocyanins, known for their antioxidant and antiproliferative activities [93]. Monotherapy with this natural compound (Trigno M^®) reduced HCT116 cell viability, induced apoptosis on 2D and 3D models, and reduced tumor growth in immunodeficient xenografts mice carrying colon rectal cancer [94]. When Trigno M^® was administered in combination with 5-FU, we confirmed the induction of apoptosis on 2D and 3D models and we demonstrated the inhibition of autophagy-mediated resistance [95].

Rosmarinus officinalis L. extract showed an antiproliferative effect on colon cancer cells, upregulating the glucosaminyltransferase 3 (GCNT3), an essential molecule for CRC progression, and downregulating its potential epigenetic modulator miR-15b [96,97].

Resveratrol (Resv, trans 3,5,4′-trihydroxy-stilbene), a polyphenol of the stilbenes group, is a non-flavonoid compound found in many plant species, such as grapes, red fruits, and peanuts. It is produced in plants in response to viral and fungal infection, UV radiation and mechanical injury [98]. Resv was an effective chemosensitizer agent for CRC [99]; in addition, it chemosensitized 5-FU-resistant HCT116 cells, inducing caspase-3-dependent apoptosis; it strongly suppressed TNF-β-induced activation of tumor-promoting factors (as NF-κB, MMP-9, and CXCR4) and epithelial-to-mesenchymal transition factors (increased vimentin and slug, decreased E-cadherin). Moreover, Resv suppressed the formation of cancer stem cells, decreasing CD133, CD44, and ALDH1 expression. A more recent work demonstrated that its chemosensitizing effect was also due to modulation of the β1-integrin/HIF-1α axis that was highly pronounced in CRC [100]. The antitumoral and chemopreventive effect on CRC was also due to activation of autophagy, as demonstrated by the study in HT-29 and COLO 201 human colon cancer cells. Transmission electron microscopy observations and immunoblotting tests demonstrated that Resv induced autophagy, which then induced apoptosis mediated by ROS production [101].

Quercetin (QUE), contained in many fruits and vegetables, suppressed the proliferation of CRC cells and induced apoptosis, especially in cells with activated mutation in the KRAS gene [102]. This mutation, found in 40% of CRC, was associated with resistance to conventional and targeted chemotherapy [103]. QUE antitumor effect was ascribed to AKT inhibition and JNK signaling activation [102].

In vitro studies demonstrated the double role of SFN against CRC: on the one hand, it reduced SW480, DLD1, and HCT116 cell growth by modulating the Wnt/β-catenin pathway with an antitumoral effect [104]; on the other hand, as a phytoantioxidant compound, it protected CRC cells from oxidative stress, activating the Nrf2-mediated cytoprotective mechanism [105].

The molecular pathways involved in CUR anticancer and chemopreventive properties against CRC have been widely clarified, including Wnt/β-catenin, JAK, STAT, MAPK, and NF-kB pathways [106,107]. When used in combination with traditional chemotherapeutics, as oxaliplatin and 5-FU, CUR was able to overcome chemoresistance both in in vitro and in vivo CRC models. For example, oxaliplatin plus CUR reversed the resistance of the CRC cell lines modulating the chemokines/NF-kB signaling pathway [108]. Alternatively, CUR reversed the 5-FU resistance of HCT116 cells inhibiting epithelial–mesenchymal transition progress, acting on the WNT signaling pathway [109].

CBs represent another example of natural compounds with promising effects in CRC [110]. A recent study analyzed the endocannabinoid system, and cannabinoid receptor 2 (CB2 in mice and CNR2 in humans) in particular [111]. The results demonstrated that the activation of endogenous CB2 with cannabinoids modulated the immune response and inhibited colon tumorigenesis. Consequently, this study identified CB2 as a potential target for CRC personalized therapy. An in vitro and in vivo study demonstrated that CBD acted on CRC proliferation, invasion, and metastases. CBD inhibited EMT process acting on the Wnt/β-catenin signaling pathway; in particular, it downregulated APC and CK1 expression and upregulated Axin-1 [112]. Another study on the glycosidic derivative of Δ⁹-tetrahydrocannabinol (THC-9-OG), employing both in vitro and in vivo models, also confirmed the action on EMT when cannabinoids were administered in combination with 5-FU. Moreover, the combination of THC-9-OG plus 5-FU induced ROS production, causing ATM activation and vimentin downregulation, activating autophagic cell death [113]. CBD was also combined with photodynamic therapy for CRC treatment. The combination increased oxygen reactive species, inducing apoptotic cell death. Moreover, it stimulated an immune response that interfered with specific mechanisms regulating CRC tumorigenesis, drug resistance, and metastasis [114].

It is known that betulinic acid (BA) behaves as a chemosensitizer, in combination with 5-FU, irinotecan, and oxaliplatin, on CRC drug resistant cells, inducing mitochondrial-dependent apoptosis [115]. Subsequent studies have shown that BA also displayed a chemopreventive effect on a CRC animal model. COX-2 level and PCNA expression decreased in animals treated with BA plus 1,2dimethylhydrazine (DMH) (carcinogen compound) compared to those treated with DMH alone. So, the chemopreventive effect was ascribed to antiproliferative and anti-inflammatory actions [116]. A recent study investigated via transcriptome analysis the mechanism by which BA induced an antiproliferative effect on CRC. The upregulation of metallothionein 1G (MT1G) induced cell cycle alteration and inhibited cell proliferation [117].

Table 3

Phytocompounds and their main effects in colorectal cancer.

Phytocompounds	Source	Antitumor Mechanism	Refs.
Trigno M®	Prunus spinosa	Antioxidant and antiproliferative activities	[93]
		↓ cell viability	[94]
		↓ tumor growth
		↑ apoptosis
Rosemary extract	Rosmarinus officinalis L.	↓ cell proliferation	[95,97]
		↑ glucosaminyltransferase 3
		↓ miR-15b
		Chemosensitization	[99]
Resveratrol	Grapes, blueberries, raspberries, mulberries, peanuts	↑ caspase-3-dependent apoptosis	[99,100]
		↓ tumor-promoting factors (as NF-κB, MMP-9, CXCR4)
		↓ epithelial-to-mesenchymal transition factors
		↓ cancer stem cells
		↓ CD133, CD44, and ALDH1 expression
		Modulation of β1-integrin/HIF-1α
		↑ autophagy	[101]
		↑ ROS production
Quercetin	Citrus fruits, apples, onions, parsley, sage, tea, red wine	↓ cell proliferation	[102]
		↑ apoptosis
		↓ AKT signaling
		↑ JNK signaling
Sulforaphane	Brassica oleracea	↓ cell growth by modulating Wnt/beta-catenin pathway	[104]
		↑ Nrf2-mediated cytoprotective mechanism	[105]
Curcumin	Curcuma longa	Regulation of Wnt/β-catenin, JAK, STAT, MAPK, and NF-kB pathways	[106,107]
		Reversion of 5-FU resistance	[109]
		↓ epithelial–mesenchymal transition
Cannabinoids	Cannabis sativa	Antiproliferative activities and chemosensitization ↓ epithelial-to-mesenchymal transition factors ↓ cell growth by modulating Wnt/beta-catenin pathway	[112]
		↑ autophagic cell death	[113]
Betulinic acid	Birch, eucalyptus, plane trees	Chemoprevention and chemosensitization ↑ mitochondrial-dependent apoptosis	[115]
		↓ COX2 level and PCNA expression ↓ metallothionein	[117]

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2.3.1. Phytocompounds

Natural products derived from plants, fungi, and marine organisms have attracted considerable attention for their potential to treat various diseases, including lymphoma (see Table 5) [138]. Among the phytocompounds, polyphenols represent the most studied for their multi-target antitumor activities regulating the angiogenesis, inflammation, and apoptosis of CSCs in vitro [139]. Moreover, polyphenols may enhance immune responses through the modulation of T lymphocytes.

Recent studies on natural polyphenols have shown that they can be an adjuvant to conventional therapy to limit the CSCs drug-resistance phenomenon. In this context, CUR suppresses cell proliferation by inhibiting STAT-3 and NF-kB, inducing apoptosis and cell cycle arrest and inhibiting angiogenesis [140,141]. Furthermore, studies reported that CUR treatment increased caspase-3 and caspase-9 levels in CH12F3 cells, effectively reducing cell-proliferation activity [142]. Similarly, CUR has been found to inhibit cell viability, promote cell apoptosis, and arrest the cell cycle in the G2 phase of human DLBCL cells both in vitro and in vivo by upregulating the expression of PPARγ and deactivating the Akt/mTOR pathway [143]. In vivo anticancer effects of CUR on human Burkitt’s lymphoma (Raji cells) have been reported in a xenograft mouse model, resulting in the downregulation of c-Myc oncogene and the upregulation of apoptotic proteins [144].

Different studies have shown the Resv anti-inflammatory, antioxidant, and cell cycle arresting properties in lymphomas. Resv suppresses tumor cell viability by inhibiting the ROS-dependent PI3K/AKT signaling pathway [145]. Frazzi et al. reported that Resv treatment induced cell cycle arrest and apoptosis in the HL-derived L-428 cell line, increasing the expression of p53 and p53 target genes, including Bax and caspase-3 [146]. Resv induced apoptosis through ROS generation in DLBCL cells, dephosphorylating the survival mediator Akt, the transcription factor FOXO1, GSK3, and the apoptotic mediator Bad [147]. Another study reported Resv anti-cancer effects on the anaplastic large-cell lymphoma (ALCL) cell line SR-786 through the increase in Fas/CD95 expression in a dose-dependent manner [148]. In addition, Resv induced cell cycle arrest and apoptosis in malignant NK cells by suppressing the JAK2/STAT3 pathway [149]. Kong et al. reported that pterostilbene, a natural analogue of resveratrol, induced apoptosis in SUDHL-4 and NU-DUL-1 cell lines by activating the Bax/Bcl2 pathway. Pterostilbene significantly induced cell cycle arrest at the G1 phase by inhibiting cyclin-dependent kinase-2 (Cdk-2) and enhancing the effect of checkpoint dependent kinase-2 (Chk-2). In addition, the intravenous administration of pterostilbene inhibited tumor growth in the nude mouse xenograft model [150].

Granato et al. suggested that QUE inhibited the PI3K/AKT/mTOR and STAT3 pathways in primary effusion lymphoma (PEL), an aggressive B-cell lymphoma, reducing the expression of pro-survival cellular proteins such as c-FLIP, cyclin D1, and c-Myc, and the release of the cytokines IL-6 and IL-10, leading to PEL cell death [151]. In addition, this flavonoid induced hyperactivation of PI3K signaling in ascites cells from mice with Dalton’s lymphoma, activating AKT1 and inactivating p53. Glycolytic metabolism was also downregulated by QUE [152]. Furthermore, QUE increased in vitro apoptotic human T lymphoblast MOLT-4 (acute lymphoblastic leukemia) and human B lymphoblast Raji (Burkitt’s lymphoma). Li et al. reported that QUE, in combination with rituximab, increased apoptosis and inhibited cell growth in DLBCL cell lines, potentiating the anti-tumor effect of rituximab through STAT3 pathway inhibition and Mcl-1 and Bcl-xl expression decreases [153].

Table 5

Phytocompounds and their main effects in lymphoma.

Phytocompounds	Source	Antitumor Mechanism	Refs.
Curcumin	Curcuma longa	↓ cell proliferation	[140,141,143]
		↓ STAT-3 and NF-kB
		↑ apoptosis
		cell cycle arrest
		↑ PPARγ expression
		↓ Akt/mTOR pathway
		↓ angiogenesis
		↑ caspase-3 and caspase-9	[142]
Resveratrol	Grapes, blueberries, raspberries, mulberries, peanuts	Cycle arresting ↓ cell viability ↓ ROS-dependent PI3k/Akt signaling ↑ apoptosis	[145,146,149]
		↑ p53 and p53 target genes ↑ Bax and caspase-3	[146]
		↑ ROS production ↓ Akt, FOXO1, GSK3 and Bad phosforilation	[147]
		↑ Fas/CD95 expression	[148]
		↓ JAK2/STAT3 pathway	[149]
Quercetin	Citrus fruits, apples, onions, parsley, sage, tea, red wine	↓ PI3K/AKT/mTOR and STAT3 pathways	[151]
		↑ cell death
		↓ c-FLIP, cyclin D1 and c-Myc expression
		↓ release of IL-6 and IL-10
		↑ PI3K signaling	[152]
		↑ AKT1 activation
		↓ P53 activation
		↓ glycolytic metabolism
Cannabinoid	Cannabis sativa	↓ cell proliferation	[154]
		↑ apoptosis
		↑ NADH
		↑ ROS production
		↓ GSH
		↑ caspase-3 ↑ apoptosis	[155]

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Furthermore, some studies have also provided evidence that CBs, such as CBD and Δ⁹-tetrahydrocannabinol, reduced the growth and survival of lymphomas by disrupting various cellular signaling pathways involving type 1 (CB1) and type 2 (CB2) cannabinoid receptors [154]. Gustafsson et al. have proved that R(+)-methanandamide (R(+)-MA) induced apoptosis in MCL and CLL cell lines, which express higher levels of CB1 and CB2 cannabinoid receptors and significant inhibition of in vivo tumor growth [155]. Moreover, in vivo studies showed that treatment of EL-4 murine lymphoma-bearing mice with THC reduced cell viability and tumor burden and increased the survival of tumor-bearing mice [156]. Strong et al. have proved that CBD in combination with well-known chemotherapeutics, such as ibrutinib, carfilzomib, and Tumorex, displayed synergistic potential in the treatment of DLBCL and MCL cell lines [157]. However, as for other malignancies, despite their proven efficacy, the potential health benefits of phytocompounds are limited and the optimization of delivery systems to target lymphoma cells could be an approach with potential clinical significance.

2.4.1. Phytocompounds

Resv showed chemosensitizing properties and chemopreventive activity in a wide range of cancers such as leukemia, carcinoma, breast, colon carcinoma, and MM [98,178,179,180]. Gatouillat et al. demonstrated that Resv was able to modulate the tumor suppressor gene p53 in chemoresistant B16 MM cells, inducing G1 cell cycle arrest and cell proliferation block. Moreover, Resv enhanced doxorubicin-induced cytotoxicity along with cyclin D1 downregulation [181]. In another in vitro study, Resv caused a cell growth decrease in A375 cells, inducing reactive oxygen species (ROS) generation, endoplasmic reticulum stress, and cell cycle arrest [182]. Larrosa et al. showed that Resv and the related molecule 4-hydroxystilbene induced growth inhibition, apoptosis, S-phase arrest, and upregulation of cyclins A, E, and B1 in human SK-Mel-28 MM cells [98,183]. A number of in vivo studies clearly demonstrated that Resv can delay tumor growth in several cancer types. Caltagirone et al. studied the effects of Resv on the growth and metastatic potential of B16-BL6 MM cells in vivo. After the simultaneous intraperitoneal administration of Resv and the intramuscular injection of B16 cells into syngenic mice, a dose-dependent tumor growth delay and the absence of systemic toxicity were observed [184].

The anti-angiogenic, pro-apoptotic, antiproliferative, and immunomodulatory properties of CUR were widely demonstrated in several human MM cell lines [185]. At the molecular and cellular level, CUR was able to slow down MM progression by influencing several molecules, such as Bcl2, MAPKS, p21, and some microRNAs, as demonstrated by several in vitro, in vivo, and even clinical studies [186,187,188,189,190,191,192]. Zhao et al. demonstrate that CUR was able to induce a block of cell invasion, cell cycle arrest, and autophagy in A375 and C8161 MM cells [193]. Moreover, preclinical animal experiments and phase I clinical studies shown that CUR induced low toxicity even at high doses (12 g/day), suggesting that CUR could be considered as a new therapeutic candidate for the management of MM [185,193,194,195,196].

QUE, a bioactive flavonoid found in fruits and vegetables such as apples, red grapes, and onions, shows multiple antiproliferative and anticancer properties [197]. It has been widely demonstrated to induce cell viability reduction, apoptosis, and autophagy, and to prevent metastasis by reducing VEGF secretion and MMP levels. Also, QUE is involved in different metabolic pathway modulation by inhibiting key enzymes of glycolysis, glucose uptake, and mitochondrial functionality, contributing to its final effects: metastasis inhibition and apoptosis/autophagy induction in cancer cells [198]. Sturza et al. demonstrated that QUE was able to inhibit ATP production at the mitochondrial level in murine MM cells (B164A5), causing tumor cell death [199]. In a study by Peng et al., QUE inhibited mouse MM growth in vivo by suppressing tumor proliferation and promoting apoptosis. In addition, the inhibition effect on migration and invasion in B16 and A375 cells was correlated with RIG-I promoter to induce IFN-I production [200].

Aloe is a medicinal plant with various pharmacological activities; it can contain molecules with important biological and toxicological functions, including flavonoids, vitamins, alkaloids, simple and complex polysaccharides, minerals, enzymes, and hydrocarbons. Aloe flavonoids exhibit anti-inflammatory, antioxidant, antimicrobial, and anti-aging properties and are used in the treatment and prevention of chronic diseases such as diabetes and cancer [201]. Several studies have reported the antitumor activity exerted by Aloe in MM ascribed to its nearly 75 active compounds characterized by high therapeutic value [202]. Aloe emodin is an anthraquinone extracted from Aloe that possesses most of the therapeutic properties attributed to the Aloe plant. Tabolacci et al. demonstrated that Aloe emodin was able to inhibit the metastasic properties of B16-F10 cells in vitro [203]. In another study, Aloe emodin induced cell proliferation reduction and differentiation in SK-MEL-28 and A375 melanoma cells. In addition, Aloe emodin treatment caused inhibition of cell metastatic ability and reduced the proliferation, stemness, and invasive potential of melanospheres, suggesting its potential employment in chemotherapy strategies also directed against resistant MM [204].

Essential oils (EOs) are low-molecular-weight volatile chemical compounds characterized by a strong odor deriving from the plant secondary metabolism. EOs can be extracted from all parts of plants, such as leaves, stems, flowers, seeds, roots, and bark. They are mainly constituted by monoterpenes, sesquiterpenes, and their oxygenated derivatives and are usually obtained via hydrodistillation or steam distillation [205]. Several studies demonstrated that EOs exert antitumor activity against skin cancer, including MM, through in vitro cell proliferation reduction, cell cycle alteration, apoptosis induction, in vitro inhibition of cell invasion and migration, in vivo tumor growth, metastasis, and angiogenesis decrease [206]. Ramadan et al. reported that tea tree oil (TTO), an essential oil extracted from Melaleuca alternifolia, induced apoptosis in A375 MM cells through caspases 3, 7, and 9 activation, upregulation of p53 and Bax proapoptotic proteins, and downregulation of bcl-2 [207]. Furthermore, in another study by Calcabrini et al., TTO and its main active component, terpinen-4-ol, were capable of inducing apoptosis in both sensitive and doxorubicin-resistant M14 melanoma cells. TTO interaction with the plasma membrane and the subsequent reorganization of lipid architecture were identified as a possible mechanism of caspase-dependent apoptosis induction [208,209]. Furthermore, Di Martile et al. demonstrated that TTO, used in combination with dabrafenib/trametenib, synergistically induced cell viability reduction correlated to apoptosis induction (caspase 3 activation, PARP cleavage) along with P-glycoprotein inhibition in MM models [210]. Moreover, they showed that three specific components of TTO (α-terpineol, tepinolene, and terpinen-4-ol) were responsible for MM antitumor action.

Among other terpenes with known anticancer properties, limonene (a monocyclic monoterpene found in citrus fruits) was reported to inhibit cell proliferation and to induce apoptosis in MM cells in both in vitro and in vivo studies [176]. Lupeol (a pentacyclic triterpenoid found in a variety of plants, including Mango, Acacia visco, Camellia japonica), with known anticancer and anti-inflammatory properties, suppressed the growth of several MM cell lines (Mel-928, Mel-1241, Mel-1011) by effectively blocking the Wnt/β-catenin signaling pathway [211]. As widely known, immunotherapy represents an effective treatment strategy for MM allowing for recurrence risk reduction and improvement of patient survival [212]. Unfortunately, not all patients can undergo this therapy, and most of them still present tumor recurrence. Recently, a number of phytocompounds (flavonoids, polysaccharides, terpenes among others) have been demonstrated to improve immunotherapy efficacy thanks to their ability to remodel the tumor microenvironment involved in cancer and their ability to escape from immune system recognition [213]. Among terpene compounds, β-caryophyllene was reported to enhance the antitumor activity of anti-PD-1 immunotherapy in an MM mouse model [214].

The pentacyclic triterpenoid BA, extracted from the bark of plane trees and birches, exerts antitumor activities against MM. Wróblewska-Łuczka et al. showed a significant inhibition of the in vitro growth of several MM cell lines, along with the antiproliferative activity of BA in combination with paclitaxel or docetaxel [215,216].

Ursolic acid (UA) is a pharmacologically active pentacyclic triterpenoid derived from the pomace, cork, flowers, buds, leaves, and bark of several medicinal plants. UA has multiple biological activities, such as antioxidant, anti-inflammatory, and anticancer activities, being involved in many pathways controlling proliferation and apoptosis. Unfortunately, UA exhibits poor bioavailability and absorption; for this reason, the original skeleton of the acid has been modified, and synthetic derivatives showing increased therapeutic effects have been developed [217,218]. Mahmoudi et al. demonstrated that treatment of human MM cells with UA resulted in the induction apoptosis through caspase activation [219].

THC and CBD have been largely studied for their potential therapeutic effects also in MM. Several studies have shown that these cannabinoids can inhibit cell proliferation, induce apoptosis, and suppress tumor angiogenesis [220].

Simmerman et al. demonstrated that CBD was able to reduce tumor growth and improve the quality of life and survival of C57BL/6 mice with MM induced by inoculation of the B16F10 cell line [221]. The anti-cancer effects of CBs in MM raise the possibility of using these compounds as adjuvants in its clinical management. Combining cannabinoids with conventional treatments, such as chemotherapy or immunotherapy, may enhance treatment efficacy and overcome resistance mechanisms [59].

Camptothecin (CPT) is a natural quinoline alkaloid isolated from Camptotheca acuminata, a tree native from Tibet and China. CPT shows anticancer properties deriving from DNA topoisomerase I inhibition, leading to DNA strand breaks and induction of apoptosis. In a study by Rudolf et al. CPT induced cellular stress responses in Bowes melanoma cells, including activation of p53-dependent DNA damage, mitochondrial- and caspase-dependent apoptosis, and p53-independent response to cellular stress [222].

Table 7

Phytocompounds and their main effects in melanoma.

Phytocompounds	Source	Antitumor Mechanism	Refs.
Resveratrol	Grapes, blueberries, raspberries, mulberries, peanuts	↓ cell proliferation	[98,178,181,182,183]
		cell cycle arrest
		↑ apoptosis
		Modulation of p53 gene	[181]
		↑ doxorubicin-induced cytotoxicity
		↓ cyclin D1
		↑ ROS, endoplasmic reticulum stress	[182]
		↑ cyclins A, E, and B1	[183]
Curcumin	Curcuma longa	Immunomodulation	[185]
		↑ apoptosis
		↓ proliferation
		Regulation of Bcl2, MAPKS, p21and microRNAs	[186,187,188,189,190,191,192]
		↓ cell invasion	[193]
		↑ cell cycle arrest
		↑ autophagy
Quercetin	Citrus fruits, apples, onions, parsley, sage, tea, red wine	↓ cell viability	[198,199,200]
		↑ apoptosis
		↑ autophagy
		↓ metastasis	[198]
		↓ VEGF secretion and MMP levels
		↓ enzymes of glycolysis
		↓ glucose uptake
		↓ mitochondrial functionality
		↓ ATP production at mitochondrial level	[199]
		↓ migration	[200]
		↑ RIG-I promoter
		↑ IFN-I production
Aloe emodin	Aloe	↓ metastasis	[203,204]
		↓ cell proliferation	[204]
		↑ differentiation
		↓ stemness
		↓ invasive potential
Tea Tree Oil	Melaleuca alternifolia	↑ apoptosis	[207,208,209,210]
		↑ caspases 3, 7 and 9	[207]
		↑ p53 and Bax
		↓ bcl-2
		Reorganization of lipid architecture	[208,209]
		↓ P-glycoprotein expression	[210]
		PARP cleavage,
		↑ caspases 3, 7 and 9 levels
Limonene	Citrus fruits	↑ apoptosis	[176]
		↓ cell proliferation
Lupeol	Mango, Acacia visco, Camellia japonica	↓ inflammation	[211]
		↓ cell proliferation
		↓ Wnt/β-catenin signaling pathway
Betulinic acid	Birch, eucalyptus, plane trees	↓ cell proliferation	[215,216]
Ursolic acid	Apples, bilberries, cranberries, peppermint, lavender, oregano, thyme, prunes	↑ cell apoptosis caspase activation	[219]
Cannabinoids	Cannabis sativa	↓ tumor growth ↑ survival	[221]
Camptothecin	Camptotheca acuminata	↓ DNA topoisomerase I	[222]
		↑ p53-dependent DNA-damage
		↑ mitochondrial and caspase-dependent apoptosis
		↑ p53-independent response to cellular stress

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2.5.1. Phytocompounds

In this context, a better understanding of GBM resistance mechanisms may lead to the development of new therapeutic strategies based on the employment of new potential molecules with therapeutic action against GBM. In this scenario, several phytocompounds, including soy isoflavones, CUR, EGCG, Resv, cannabinoids, and retinoids, have been demonstrated as promising pharmacologic tools against GBM (see Table 9) [242,243,244,245,246].

QUE, a flavonoid found in several plants and foods such as apples, onions, red grapes, cherries, honey, and green leafy vegetables, has been shown to exert antioxidant, antiviral, and anticancer properties by modulating the cell cycle, inducing apoptosis, and inhibiting angiogenesis in different types of cancer, including GBM [247,248]. This natural product induced apoptotic cell death in p53 mutant glioblastoma U373MG cells by decreasing, in a dose-dependent manner, the mitochondrial membrane potential and causing nuclear fragmentation [249]. As is known, low survival of GBM patients is associated with high expression of interleukin-6 (IL-6) [250,251]. QUE has been demonstrated to decrease IL-6 levels in a dose-dependent mode through STAT3 activation in T98G and U87 glioblastoma cells [252]. Moreover, QUE was able to sensitize two glioblastoma cells lines (U251 and U87) to TMZ treatment by inhibiting the expression of heat shock protein 27 [253].

In recent years, the non-flavonoid polyphenol Resv has attracted substantial attention due to its antioxidant and anti-inflammatory properties for treating ischemia and hypoxia due to its neuroprotective effects in penetrating the blood–brain barrier [254,255]. A number of studies revealed its ability to target different signaling pathways involved in cancer development and progression, resulting in apoptosis, autophagy, and senescence induction in GBM cells; moreover, Resv employment, both in single and combination therapies, achieved the re-sensitization of cancer cells to radiotherapy and induced chemosensitizing effects [256]. In combination treatments, Resv has been demonstrated to enhance TMZ toxicity in several GBM cell lines by inhibiting TMZ-induced G₂/M arrest via the induction of the senescence pathway [257]. A study by Cilibrasi et al. has also demonstrated the effects of Resv in seven glioma stem cell (GSC) lines derived from GBM patients. By modulating the Wnt signaling pathway, Resv was able to inhibit the cell proliferation by increasing cell mortality and reducing motility of the cells [258,259,260]. Furthermore, by regulating the activation of NF-κB in U373MG glioma cells, Resv decreased the TNF-α-induced invasion [261]. In addition, Huang et al. have demonstrated Resv’s ability to modulate TMZ resistance through the downregulation of MGMT expression [262] by increasing the induction of apoptosis in TMZ-resistant T98G cells. These results strongly suggested that Resv may be used as an effective adjuvant molecule in GBM combination therapies.

Among bioactive phytocompounds with anti-GBM activities, polydatin, a stilbenoid polyphenol and a glucoside derivative of Resv, found in such plant families Vitaceae, Liliaceae, and Leguminosae, as well as in red wine, nuts, vegetables, and fruits, represents a promising molecule [263]. Compared to Resv, polydatin shows a higher anti-inflammatory [264] and antioxidant activity [265]. Polydatin treatment efficacy was demonstrated in different GBM cell lines by reducing cell proliferation, migration, invasion, and stemness and inducing apoptosis through the inhibition of EGFR-AKT/ERK1/2/STAT3-SOX2/Snail signaling pathway. In addition, PD showed no or very low cytotoxicity to normal human cells [266].

Numerous literature data suggest that CUR is involved in the modulation of most GBM signaling pathways [267]. CUR potential therapeutic and protective activities have been demonstrated against malignant tumors in the central nervous system thanks both to CUR’s ability to penetrate the BBB and to its lipophilic nature [268].

GBM cell’s ability to penetrate normal brain tissue is related to the very high expression of membrane-associated or secreted matrix metalloproteinases (MMPs) involved in extracellular matrix degradation. By inhibiting AP-1 and MAP molecules, CUR was able to suppress the expression of MMP-1, -3, -9, and -14 in GBM cell lines [267,269]. Wang and Chen reported that CUR affected different steps of angiogenesis process by suppressing the expression of transcription factors, NF-κB, and pro-angiogenesis factors (VEGF and βFGF) [270]. Moreover, Dhandapani et al. revealed that CUR reduced cell proliferation, inducing DNA fragmentation and apoptosis through a caspase-dependent pathway [271] and both increasing the BAX/Bcl-2 ratio and stimulating caspase 8 activation. CUR in combination with TMZ seemed to induce an additive cytotoxic effect in GBM cells by causing cell cycle block in the G₂/M phase [272]. In addition, CUR-TMZ combination was able to induce ERK1/2-dependent autophagy [267]. Moreover, it has been reported that CUR enhanced the action of other numerous drugs, such as cisplatin, CPT, PTX, and doxorubicin, in different human GBM cell lines, leading to a synergistic effect [269,273]. Furthermore, CUR may prevent chemoresistance in GBM cells by reducing the expression of different ABC transporters [274]. Finally, as demonstrated by Trotta et al., CUR was able to sensitize GBM cells resistant to TRAIL therapy via apoptosis [275].

As described above, PTX is currently used for the treatment of several cancer types, including glioblastomas [276]. This natural compound was able to trigger apoptosis via the upregulation of the caspase signal pathway and affected U251 and U87MG cell growth and proliferation through MMP-9 and p38/JNK pathway inhibition [277]. Unfortunately, PTX activity against brain tumors was unsatisfactory in phase II experiments due to the presence of the BBB and the activity of drug-transporter proteins in both in vitro and in vivo conditions [278,279].

In numerous preclinical glioblastoma models, cannabinoids (CBD, THC) have demonstrated anticancer activities via the inhibition of cell proliferation associated with cancer cell death, along with evident effects on angiogenesis [280]. It is also necessary to mention that CB1 and CB2 receptors are expressed in GBM human cells. Twelves et al. reported that submaximal doses of THC and CBD in combination with TMZ treatment had high anticancer activity both in TMZ-sensitive and TMZ-resistant GMB tumors. In addition, the authors have reported preliminary results on the efficacy of nabiximols oro-mucosal cannabinoid spray used in combination with TMZ in GBM patients [281].

Betulin and its derivatives are phytocompounds of great interest. In particular, BA (3-beta-hydroxy-lup20(29)-en-28-oic acid), a derived pentacyclic lupine-type triterpenoid, has been demonstrated to possess different biological activities, such as antioxidants, anti-inflammatory, and anti-GBM action, via the downregulation of NF-kB, the suppression of pro-survival transcription factor Sp1, and the enhancement of TMZ cytotoxic effects [282,283].

Table 9

Phytocompounds and their main effects in glioblastoma multiforme.

Phytocompounds	Source	Antitumor Mechanism	Refs.
Quercetin	Citrus fruits, apples, onions, parsley, sage, tea, red wine	Cell cycle modulation	[247,248,249]
		↑ apoptosis
		↓ angiogenesis
		↓ mitochondrial membrane potential	[249]
		nuclear fragmentation
		↓ IL-6 levels	[252]
		↑ STAT3
		↓ heat shock protein 27 expression	[253]
Resveratrol	Grapes, blueberries, raspberries, mulberries, peanuts	↑ apoptosis	[256]
		↑ autophagy
		senescence induction
		chemo-sensitization
		Wnt signaling pathway modulation	[258,259,260]
		↓ cell proliferation
		↓ cell mortality
		↓ cell motility
		↑ NF-κB	[261]
		↓ TNF-α induced invasion
		↓ MGMT expression	[262]
Polydatin	Grapes, blueberries, raspberries, mulberries, peanuts	↓ cell proliferation	[266]
		↓ migration and invasion
		↓ stemness
		↓ EGFR-AKT/ERK1/2/STAT3-SOX2/Snail signaling
Curcumin	Curcuma longa	↓ MMP-1, -3, -9 and -14 expression	[267,269]
		↓ p38, JNK, ERK
		↓ angiogenesis	[270]
		↓ NF-κB
		↓ VEGF, βFGF and MMPs
		↓ cell proliferation	[271]
		↑ DNA fragmentation
		↑ apoptosis
		↑ BAX/Bcl-2	[272]
		↑ caspase 8
		↓ ABC transporters expression	[274]
		chemo-senzitization	[269,273,274]
Paclitaxel	Taxus brevifolia	Chromosome mis-segregation	[276]
		Mitotic arrest
		↑ apoptosis	[277]
		↑ caspase signal pathway
		↓ cell growth and proliferation
		MMP-9 and p38/JNK pathway
Cannabinoids	Cannabis sativa	↓ cell proliferation	[280]
Betulic acid	Birch, eucalyptus, plane trees	↓ NF-κB ↓ Sp1 ↑ cytotoxicity of TMZ	[282,283]

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