3.1. In Vitro Evaluation of Anticancer Efficacy of Isolated Lichen Compounds
Pro-apoptotic abilities of usnic acid (UA), a secondary lichen metabolite isolated from
Usnea diffracta, was evaluated in human colorectal adenocarcinoma (CaCo2), human rhabdomyosarcoma (RD), human cervical carcinoma (Hep2C), human hepatocellular carcinoma (HepG2), mouse fibrosarcoma (Wehi), as well as in nonmalignant African green monkey kidney (Vero) and mouse subcutaneous connective tissue (L929) cell lines. Both natural and commercial UA had similar cytotoxicity on cancer cells but no significant effect in non-malignant L929 and Vero cells. Indeed, UA increased the expression of Bax and decreased the expression of Bcl-2 and p53 genes in cancer but not in non-malignant cells [
29]. Additionally, the concentration of UA at 8 μM, inhibited SCF-mediated migration of human colorectal cancer (HCT116, LS174 c-KIT+) cells. UA induced an inhibition of mTOR activity in HCT116 cells thereby activated suppression of protein kinase C alpha type (PKC-A) and induced an autophagy followed by the degradation and transcriptional inhibition of mast/stem cell growth factor receptor Kit (c-KIT) as well as to an increase in LDH release [
68]. Another study of UA demonstrated its ability to inhibit the proliferation of human gastric carcinoma (BGC823, SGC7901) cells through the G0/G1 and G2/M cell cycle arrest, respectively while BGC823 cells were more sensitive. Both apoptosis and autophagy of BGC823 and SGC7901 cells with increased ratio of Bax/Bcl-2 and increased activity of caspase-3 and PARP cleavage after UA therapy were identified [
69]. Anti-proliferative effects of UA were also evaluated in human breast cancer cell lines in which 67, 15, and 8 miRNAs in MDA-MB-231, BT-474, and MCF-7 cells, respectively after UA therapy were detected. Indeed, enrichment analysis revealed different groups of miRNA with targets as follows: Apoptosis, Hedgehog, TGF-β, and MAPK pathways identified as prominent [
37]. Pro-apoptotic abilities of UA were demonstrated through the G0/G1 and G2/M cell cycle arrest in both hepatocellular carcinoma HepG2 and SNU-449 cells [
27]. Similarly, an application of UA in lung cancer cells (A549) led to the decrease of cells, induced apoptosis and inhibited cell proliferation, which was accompanied by G0/G1 cell cycle arrest, decreased expression of CDK4, CDK6, and cyclin D1 and increased expression of CDK inhibitor (CDKI) p21/cip1 protein. Consequently, the apoptotic effect of UA co-occurred with enhanced PARP cleavage [
28]. The anticancer effects of UA isolated from
Cladonia arbuscula were demonstrated through the formation of autophagosome as a consequence of H+ shuttling in mitochondria and lysosomes in human breast cancer (MCF-7, T47D) as well as in human pancreatic cancer cell lines (Capan-2) with the decrease in ATP, activation of AMP-kinase and the detection of cellular stress markers [
70]. Similarly, UA induced an apoptosis of MCF-7 cells through the generation of ROS and mitochondrial/caspase pathway. On the contrary, N-acetylcysteine blocked ROS generation, reduced apoptosis mediated by c-Jun-N-terminal kinase, caused a loss of mitochondrial membrane potential, released the cytochrome-c and activated caspases [
71]. In another study, UA isolated from several lichens (
Alectoria samentosa,
Flavocetraria nivalis,
Alectoria ochroleuca, and
Usnea florida) showed the significant inhibitory potential against migration and invasion of human lung cancer (A549, H460, H1650, and H1975) cells. It was also associated with the inhibition of A549 cells motility. Anti-invasive efficacy of UA in H1650 and H1975 cells was demonstrated by the decline in β-catenin-mediated TOPFLASH and KITENIN-mediated AP-1 activity. Additionally, synthetic derivatives of UA exerted anti-proliferative activity in human prostate cancer (PC-3), human epithelial carcinoma (HeLa), and MCF-7 cells. All active derivatives arrested cells at G0/G1 phase and decreased the fraction of HeLa cells in S and G2/M phases. Derivatives 2a and 2b arrested also MCF-7 cells at G0/G1 phase and induced the apoptosis of these cells. Derivatives 2a and 2b also activated strong cytoplasmic vacuolization, which was associated with dynein-dependent endocytosis. This mechanism has not yet been demonstrated in UA and it is the first time reported in synthetic derivatives of UA [
72]. The potasium usnate (PU), which was prepared to enhance the solubility of UA, demonstrated cytotoxic activity in each tested colorectal cancer cells (human HCT116, DLD1, SW480, HT29, SW620, Caco2, COLO320, and mouse CT26) with lower IC50 values than UA except of SW480 and CT26 cells. Potasium usnate showed stronger inhibitory effects on the invasion of Caco2 and HCT116 cells compared to UA. Importantly, PU downregulated the epithelial–mesenchymal markers (EMT) including Twist, Snail, and Snug and decreased the expression of metastasis-related genes CAPN1, CDC42, CFL1, IGF1, WASF1, and WASL in Caco2 cells [
34]. An anticancer efficacy of UA was evaluated together with diffractaic acid (DA) and lobaric acid (LA) in human glioblastoma multiforme (U87MG-GBM) and rat cerebral cortex cells (PRCC). Lobaric acid exerted highly toxic effects demonstrated by the drop of cell viability to 35.09% in PRCC and 30.47% in GBM cells, while DA and UA exerted greater total antioxidant capacity (37.74 and 37.34 mmol Trolox equivalent/L, respectively) in PRCC cells when compared with other compounds [
73]. Cancer inhibitory effect of UA and atranorin (ATR) was also evaluated in human melanoma (HTB-140) and human prostate cancer (DU-145, PC-3) cell lines. Interestingly, both UA and ATR inhibited cancer cells proliferation, migration and actin organization while their effects on apoptosis were less significant [
74]. An impact of UA isolated from
Cladonia foliacea Wild and vulpinic acid (VA) isolated from
Letharia vulpina Hue on proliferation and viability was evaluated in HepG2, mouse neuroblastoma NS2OY and human umbilical vein endothelial (HUVEC) cells. Although UA was more cytotoxic against all cell lines, it had higher anti-proliferative effects in HepG2 cells. On the other hand, VA inhibited the proliferation of NS2OY cells more effectively. Interestingly, the cytotoxic effects of both metabolites against HUVEC were only mild. Moreover, both UA as well as VA exhibited anti-angiogenic abilities evaluated by the endothelial tube formation assay [
75]. Vulpinic acid also decreased viability and induced apoptosis of human breast cancer cells (MCF-7, MDA-MB-231, BT-474, SK-BR-3) compared to human non-malignant breast epithelial cells (MCF-12A). An evaluation of apoptosis-related genes showed that the expression of p53 after VA therapy was almost six times higher in SK-BR-3 cells than in MCF-12A cells [
47]. Similarly, an apoptotic activity of VA was evaluated in vitro in CaCo2, HepG2, Hep2C, RD, Wehi as well as in normal Vero and mouse L929 cells. Vulpinic acid inhibited growth of all tested cell lines in a time and dose-dependent manner and a higher efficacy was found in CaCo2 cells. Vulpinic acid also exhibited significant cytotoxic effects on all tested cancer cells. On the other hand, it did not exert any significant cytotoxicity of on normal L929 and Vero cells, but interestingly, all mRNA, Bax protein levels and p53 were more significantly increased in cancer compared to normal cells. In addition, mRNA and Bcl-2 protein levels showed 7 fold decrease in HepG2 and CaCo2 cells and 5–6 fold decrease in Hep2C, RD and Wehi cells [
76].
Similarly, natural compound ATR, isolated from lichens, was tested against mouse breast cancer (4T1) cells. ATR reduced the clonogenic potential of 4T1 cells compared to normal mammal non-malignant epithelial (NMuMG) cells, in which the clonogenic ability remained unaffected. BrdU incorporation assay did not confirm the anti-proliferative effect of ATR in 4T1 cells. On the contrary, ATR induced caspase-3 activity, PARP cleavage and depletion of Bcl-xL in 4T1, but not in NmuMG cells [
45]. Atranorin, isolated from
Stereocaulon caespitosum also inhibited the growth of human hepatocellular carcinoma (SK-Hep1, Huh-7, SNU-182) cell lines when used in concentration higher than 10 μg/mL. Atranorin arrested SK-Hep1 cells at G2/M phase, induced cell death at 24 h time point and suppressed migration and invasiveness of Sk-Hep1 and Huh-7 cells [
77].
However, only high concentrations of ATR and gyrophoric acid (GA) had similar effect on human melanoma A375 cells, physodic acid (PA) induced apoptosis in A375 cells by mechanism probably involving the downregulation of HSP70 [
17]. In this regard, Emsen et al. analyzed the effect of PA together with olivetoric acid (OA) and psoromic acid (PSA) on U87MG and rat PRCC cells and found a positive correlation between the cytotoxicity associated with the three tested metabolites and their concentrations, lactate dehydrogenase (LDH) activity, and oxidative damage of DNA [
43].
Furthermore,
Parmotrema dilatatum,
Parmotrema lichexanthonicum,
Usnea subcavata,
Ramalina sp.,
Dirinaria aspera,
Cladina confusa and their secondary metabolites were tested on melanoma cancer (UACC-62), murine melanoma (B16-10), and human fibroblast (NIH/3T3) cells. Protocetraric acid (PrA), norstictic (NA), and PSA (depsidones) acids together with divaricatic (DiA) and perlatolic (PeA) (depsides) acids showed a strong cytotoxic effect on UACC-62 cells and reached higher selectivity for melanoma cells compared to 3T3 normal cells. In this regard, NA and DiA was also the most effective against B16-F10. Protocetraric acid proved to be the best candidate for in vivo studies of melanoma since it showed the highest selectivity index against UACC-62 cells [
78].
Paluszczak et al. evaluated effects of lichen-derived compounds on Wnt signaling in colorectal cancer (HCT116, DLD-1) and immortalized keratinocyte (HaCaT) cell lines. Caperatic acid (CA) isolated from
Platismatia glauca downregulated β-catenin-regulated expression of Axin2 gene in both colorectal cancer cell lines, but lecanoric acid (LeA), obtained from
Hypocenomyce scalaris decreased the expression of Axin2 in HCT116 cells just moderately. On the contrary, CA and PA (isolated from
Hypogymnia physodes), downregulated the expression of MMP7 and survivin in HaCat cells in a concentration-dependent manner. Furthermore, CA inhibited the migration of both colorectal carcinomas by 20%, while PA did not have any effect on the cell migration. Caperatic acid showed also the strongest cytotoxic effects on cancer cells at the concentration of 100 μM while other metabolites in this concentration demonstrated just moderate efficacy [
26].
Barbatic acid (BA), isolated from acetone extract of
Usnea longissima, exhibited strong cytotoxic activity against HeLa, A549, MCF-7, and DU-145 cells. Barbatic acid arrested A549 cells in G0/G1 phase with 71% of cell accumulation at the concentration of 1 μM and the same concentration induced the apoptosis of A549 cells with increased caspase-3 activity, PARP cleavage, annexin V staining, and chromatin condensation [
38].
In addition, anti-angiogenic and anti-migratory efficacy of lichen-derived small molecule barbatolic acid (BrA) isolated from acetone extract of
Bryoria capillaris was evaluated on T-47D, HUVEC, and cisplatin-resistant BRCA2-mutated human breast TNM stage IV adenocarcinoma cells (HCC1428). Sub-cytotoxic concentrations (25–100 µM) of BrA dose-dependently inhibited both endothelial tube formation as well as migration determined by the scratch wound healing assay. In fact, the effect of BrA on the migration of cancer T-47D and HCC1428 cells was more effective than on normal HUVEC cells [
35].
Hypostictic acid (HA), extracted from
Pseudoparmelia sphaerospora, exerted significant anti-proliferative efficacy against chronic myelogenous leucaemia (K562), murine melanoma (B16-F10), and renal cancer (786-0) cell lines. Salazanic acid (SA) obtained from
Parmotrema cetratum, showed in vitro anti-proliferative effects also in K562 and B16-F10 as well as in colon cancer (HT-29) cell lines [
79]. In this regard, SA and protocetraric acid (PrA) are major phenolic compounds in lichens including
Parmelia caperata,
Parmelia saxatilis and
Parmelia sulcate. Manojlovic et al. evaluated an impact of the above-mentioned compounds against human melanoma FemX and LS174 cell lines [
80].
Retigeric acid B (RA-B), a pentacyclic triterpenic acid isolated from
Lobaria kurokawae induced apoptosis in PC-3 and DU145 cells through the inhibition of IκBα and p65 (subunit of NF-κB) phosphorylations. In addition, microarray analysis revealed alterations in the expression of genes associated with cellular processes including apoptosis, invasion, and proliferation after RA-B application [
81].
Protolichesterinic acid (PLA) isolated from
Centraria islandica exerted anticancer efficiacy in SK-BR-3 and T47D cell lines while anti-proliferative effects were also observed in SK-BR-3 cells. Indeed, this metabolite increased the expression of fatty acid synthase and decreased the expression of HER2 in SK-BR-3 cells. Additionally, downregulation of ERK1/2 and AKT signaling was observed probably due to reduced HER2. There was also demonstrated some synergistic effect of PLA and lapatinib in SK-BR-3 cells [
31]. Similarly, PLA affected HeLa, K562, and human neuroblastoma cell lines (SH-SY5Y). Interestingly, PLA combined with doxorubicin exerted synergistic cytotoxicicity in HeLa cells but not on SH-SY5Y and K562 cells. The mechanism of synergistic effects might be associated with the apoptosis induced by both PLA as well as doxorubicin, which can induce caspases -3, -8, and -9 activities. Bim expression, which mediates cytochrome-c release might be increased in an additive manner by both PLA and doxorubicin. Protolichesterinic acid also seems to behave as a competitive inhibitor of fatty acid synthase [
82]. Bessadóttir et al. also tested effects of PLA (from
Cetraria islandica) in human multiple myeloma (RPMI 8226), (U266) and human pancreatic carcinoma (AsPC-1). Moreover, PLA inhibited the proliferation of RPMI 8226, U266, and AsPC-1 cells with very low IC
50 values. It induced the cell cycle arrest of pancreatic AsPC-1 cells in G1 phase and the apoptosis of RPMI 8226 and U266 cells but not the apoptosis of pancreatic AsPC-1 cells. Indeed, the pro-apoptotic activity of PLA was cancer cell line-dependent while high concentrations of PLA inhibited the production of 5- and 12-HETE but only in pancreatic not in myeloma cells [
83].
Ramalin (RAM) a metabolite isolated from
Ramalina terebrata inhibited at high concentrations (50 and 100 μg/mL) the proliferation of HCT116 cells. Ramalin induced G2/M cell cycle arrest through the upregulation of TP53 and p21 and downregulation of cyclin B1 and CDK1. The highest concentration of RAM (100 μg/mL) induced significant number of apoptotic cells. Furthermore, wound healing, invasion, and migration of HCT116 cells were also dose-dependently suppressed after the treatment with RAM [
84].
Physciosporin (PHY) isolated from
Pseudocyphellaria granulata, was evaluated in colorectal cancer cell lines (CT26, Caco2, HCT116, DLD1, SW620). While toxic concentrations of PHY induced apoptosis of tested cancer cells, non-toxic ones inhibited migration, invasion, and colony formation of colon cancer cells in concentration-dependent manner. Physciosporin down-regulated downstream transcription factors and/or target genes of EMT, KITENIN, β-catenin and reduced actin-based cell motility [
22]. Another PHY, isolated from
Pseudocyphellaria coriacea demonstrated anticancer effects in A549, H1650, and H1975 through inhibition of migration and invasion of human lung cancer cells through the downregulation of N-cadherin, KITENIN-mediated AP-1 activity, Cdc42, and Rac1. Interestingly, the metastasis suppressor gene KAI1 was also upregulated by PHY [
85].
Interestingly metabolites isolated from acetone extract of
Dirinaria consimilis: antarvediside A (antarA), antarvediside B (antarB), sekikaic acid (SeA), ATR, divaricatic acid (DiA), 2′-O-methyl DiA revealed anticancer potential. In this regard, antarB at the concentration of 30 μg/mL inhibited the growth of HeLa and MCF-7 stronger than the doxorubicin at the concentration of 10 μg/mL. Only metabolites antarB and 2′-O-methyl DiA inhibited the proliferation of A549 cells. Importantly, all metabolites had lower toxicity against human mammary epithelial normal NHME cells in comparison to cancer cells [
86].
Table 2 shows an overview of anticancer effects of above-mentioned isolated lichen secondary metabolites.
3.2. Combined Studies of Lichen Extracts and Isolated Lichen Compounds In Vitro
Notably, several authors studied anticancer potential of lichen extracts in combination with isolated lichen compounds. An acetone extract of
Flavocetraria cucullata or its subcomponent UA exerted a selective cytotoxicity on cancer cells by inducing apoptosis at lethal concentrations. Sub-lethal concentrations of this extract and UA inhibited tumorigenesis and motility of cancer cells, suppressed epithelial–mesenchymal transition (EMT) and inhibited Akt phosphorylation. Interestingly, the anticancer activity of the extract was more potent than that of UA [
87].
Another study analyzed anticancer properties of acetone extract of
Toninia candida and
Usnea barbata as well as their major compounds nortictic acid (NA) and UA against FemX and LS174 cells using tetrazolium assay. The results of the study proved the anticancer activity of NA, UA, and lichen extracts in both cell lines. Importantly, UA demonstrated the strongest cytotoxic effect while both NA and UA induced apoptosis in FemX and LS174 cells [
54]. Moreover, anticancer and antioxidant availabilities of supercritical CO
2 extract of old man’s beard (
Usnea barbata) (SCE) compared to extracts obtained through conventional methods (Soxhlet extracts and macerate) were evaluated in mouse melanoma (B16), rat glioma (C6), and HaCaT cells. SCE exerted the highest cytotoxic effect on both B16 and C6 cells. The cytotoxic effect of lichen extracts correlated well with the content of UA (the most abundant compound) and ROS production. The cytotoxic effects of extracts were evaluated for apoptosis and autophagy processes via monitoring of cells for cell cycle phases and formation of acidic cytoplasmic vesicles. Consequently, both, SCE and UA induced apoptosis and/or autophagy in B16 and C6 cells. Indeed only very low toxicities of all tested extracts were demonstrated against normal HaCaT cells [
88].
Furthermore, acetone extracts of lichens including
Evernia prunastri and
Pseudoevernia furfuraceae and their major secondary metabolites, mainly PA, demonstrated anticancer activity in FemX and LS174 cells using MTT test. Physodic acid exerted the best cytotoxic effect against both cell lines. Additionally, authors also observed a reduction of FemX and LS174 cells in the S and G2/M phase. On the other hand, there was an increase in sub-G1 population of cells after the treatment with mentioned above extracts and metabolites [
89]. Similarly, PA (isolated from
Hypogymnia physodes) demonstrated the most significant cytotoxic effect on MCF-7 cells followed by T47D and MDA-MB-231 cells. The viability of MCF-10A cells was not changed even at concentration of PA higher than 100 μM [
24].
Ethanol extract of
Usnea strigosa as well as its compound NA also showed anticancer effects in MDA-MB-231, MDA-MB-468, MCF-7, T-47D, BT-474, and SK-BR-3 cells. NA exerted anti-proliferative effects in all six breast cancer cell lines with the best inhibition demonstrated in MDA-MB-231 and MDA-MB-468 cells. Nortictic acid inhibited the migration of MDA-MB-468 cells and invasion of MDA-MB-231 cells through the basement membrane. On the other hand, they did not show any significant cytotoxic effects on human non-tumorigenic MCF-10A cells. This acid suppressed also c-Met, STAT3, paxillin/Rac-1and FAK phosphorylation in MDA-MB-231 cells [
33]. Moreover, acetone extract of
Melanelia subaurifera and
Melanelia fuliginosa and their compounds LeA and 2′-O-methyl anziaic acid (2′-O-MA) suppressed HeLa, A549, and LS174 cells. Indeed, LeA and 2′-O-MA metabolites had lower cytotoxic effects compared to both extracts, while both extracts as well as metabolites did not exerted any cytotoxicity on normal MRC5 cells [
25].
Ether extract of
Cladonia salzmannii showed cytotoxic effects on human acute promyelocytic leukemia (HL-60), human laryngeal carcinoma (HEP-2), human lung mucoepidermoid carcinoma (NCI-H292), and murine macrophage (RAW-264.7) cells. In this regard, acetone extract revealed cytotoxic effects on MCF-7 and NCI-H292 cells while purified BA exerted cytotoxicity on HEP-2, MCF-7 and RAW-264.7 cells [
90]. Barbatic acid a secondary metabolite of
Cladia aggregate, was applied against HEp-2, human squamous cell lung cancer (NCI-H292), and human nasopharyngeal squamous cell carcinoma (KB). Lichen extract as well as the isolated compound showed the cytotoxicity against all tested cancer cell lines while the most sensitive ones were HE-p2 cells [
91].
Acetone extract of
Everniastrum vexans inhibited the migration of A549 cells (10 μg/mL). Atranorin identified as an active secondary metabolite of the extract showed the cytotoxic effect on A549 cells at concentrations higher than 5 μg/mL. Atranorin inhibited TOPFLASH activity mediated by β-catenin reducing nuclear import of β-catenin and suppressing the expression of c-jun/AP-1 target genes. Moreover, ATR decreased the mRNA expression of KITENIN and increased the expression of KAI1 mRNA. It also decreased the level of GTP-Cdc42, GTP-RhoA and STAT proteins [
20].
In addition, acetone extract of the endolichenic fungus EL002332 isolated from
Endocarpon pusillum exerted selective cytotoxicity on human gastric cancer AGS and mouse colon cancer CT26 cell lines. It also showed synergistic effects on human gastric cell lines (AGS, TMK-1) in combination with docetaxel chemotherapy. Active compound isolated from acetone extract (myC) had even higher anticancer effect on AGS cells than the crude extract. In fact, myC induced apoptosis through the activation of caspase activity and regulating the expression of Bcl-2 family proteins [
92].
Table 3 summarizes combined studies of lichen extracts and isolated lichen compounds evaluating their anticancer effectiveness.
3.3. Determination of Lichens’ Extracts Effect Against Cancer Cells
In addition, beneficial anticancer activity is associated also with lichen extracts. As revealed by Kosanic et al., extracts of
Umbilicaria crustulosa,
Umbilicaria cylindrica, and
Umbilicaria polyphylla showed strong anti-neoplastic effects against FemX and LS174 cell lines evaluated using MTT assay [
93]. Similarly, methanol extract of
Lasallia pustulata and extracts from
Parmelia caperata,
Parmelia sulcate, and
Parmelia saxatilis showed strong cytotoxic activity against FemX and LS174 [
94,
95]. Another study evaluated the anticancer effect of 70% methanolic extract of
Parmotrema reticulatum against MCF-7 and A549 cancer cells and normal fibroblast WI-38 cells. Analyzed data documented the strong cytotoxic activity of this extract on MCF-7 cells, while low cytotoxicity on A549 and WI-38 cells. Moreover, the extract induced S and G2 cell cycle arrest of MCF-7 cells associated with decreased expression of B1, Cdk2, Cdc25C, and increased expression of p53 and p21. An increased ratio of Bax/Bcl-2 and higher caspase activity were detected in MCF-7 cells leading to PARP cleavage and apoptosis [
96]. Another study demonstrated anticancer activity of methanol extract of
Cetraria islandica on FemX and LS174 cells [
97]. In addition, Kosanic et al. studied biological activity of
Parmelia arseneana and
Acarospora fuscata acetone extracts against FemX, LS174, A549, and human chronic myeologenous leucaemia cells K562. Their results revealed strong anticancer effects of the
P. Arseneana extract in all tested cell lines [
98]. All extracts of unique trans-Himalayan lichens demonstrated a cytotoxic effect against HepG2 and human colon cancer (RKO) cells. Moreover, methanol extract of
Lobothallia alphoplaca and
Melanelia disjuncta exhibited high cytotoxicity against both cancer cell lines [
99]. Additionally, treatment of A549, PC-3, Hep3B, and C6 cell lines with methanol extracts of
Parmelia sulcata Taylor and
Usnea filipendula Stirt led to the cytotoxic, genotoxic and apoptosis-inducing effects in all tested cell lines [
100]. Another in vitro experiment of methanol extract of
Hypogymnia physodes (low concentrations) indicated anticancer and/or apoptosis-inducing effects on MCF-7 and MDA-MB-231 cells and the genotoxic (high concentrations) impact on human lymphocytes [
101]. Moreover, methanol extracts of
Cladonia rangiformis and
Cladonia convolute exerted a significant anti-proliferative effect and these extracts induced also significant apoptosis of MCF-7 cells demonstrated by TUNEL assay [
102]. Additionally, acetone extract of
Xanthoria parietina represents a unique mixture of the secondary metabolites with anticancer abilities.
X. parietina extract inhibited the proliferation of MCF-7 and MDA-MB-231 cells with the maximum effect observed at the concentration of 1.5–3 mg/mL. This extract induced the G1 cell cycle inhibition with the upregulation of p27 and p16 and downregulation of cyclin D1 and cyclin A. Interestingly, therapy of MCF-7 and MDA-MB-231 cells with
X. parietina decreased the expression of anti-apoptotic Bcl-2 and increased the expression of pro-apoptotic TRAIL and pBAD proteins [
103]. In addition, a metanol extract of
Parmelia sulcata had a cytotoxic effect on both MCF-7 and MDA-MB-231 cells. The apoptosis induced in MCF-7 and MDA-MB-231 cells by
P. sulcata extract was rather caspase-independent due to the lack of changes in caspase-3 activation or in PARP cleavage [
104]. Recently, researchers evaluated crude extracts of 17 lichen species against Human Burkitt’s lymphoma (Raji).
Xanthoparmelia chlorochroa and
Tuckermannopsis ciliaris extracts induced apoptosis (dose-dependent), accumulation of cells in the GO/G1 stage and increase of p53 protein [
105]. Acetone extracts of
Parmotrema gardneri,
Pannaria sp., and
Canoparmelia aptata applied to human gastric adenocarcinoma (AGS) and A549 cancer cells as well as normal Canine Madin–Darby kidney cells (MDCK) exerted anticancer potential. On the contrary,
C. aptata extract had just weak cytotoxicity on AGS and A549 cells [
106]. Additionally,
Cladonia rangiformis and
Cladonia convolute represent fruticose lichens with antimicrobial and cytotoxic effects. The extracts from both lichens showed strong cytotoxic impact on MCF-7 cells [
107].
Furthermore,
Caloplaca pusilla grown on G-LBM medium decreased cell viability and induced apoptosis of MCF-7, PC-3 and HeLa cells in concentration-dependent manner. Mycelia of
Xanthoria parietina grown on PDA and G-LBM decreased cell the viability of MCF-7 and HeLa cell lines [
108]. Also, methanol extract of
Cladonia pocillum increased the apoptosis in MCF-7 cells in concentration-dependent manner [
109]. Extract of
Pleurosticta acetabulum exerted strong cytotoxic effect on HT-29 cells. This extract also inhibited the proliferation of HT-29 cells through the presence of cytochalasin E and induced apoptosis [
110]. Furthermore, polysaccharide from
Umbilicaria esculenta decreased the viability of human melanoma A875 and A375 cells but did not exhibit any cytotoxic effect on HUVEC cells. Both, Annexin-V positive as well as TUNEL positive A875 cells were induced after this therapy in concentration-dependent manner. In this regard, apoptosis of A875 but not HUVEC cells was induced by polysaccharide of
U. esculenta (as a result of ROS generation) followed by increased expression of caspase-3 and -9 [
111]. An evaluation of biological activities of five lichen species of Cladonia genus revealed the highest cytotoxic effect of
Cladonia foliacea extract on A549 and human colon cancer (LS174) cells. Moreover, the highest cytotoxic effect on HeLa cells was observed after the treatment with the extract of
Cladonia furcata [
112]. In addition, high concentration of
Candelariella vitellina extract reduced the proliferation (Ki-67) and induced apoptosis and necrosis of Caco-2 cells. The extract of
C. vitelline decreased Bcl-2 but increased Bax and CASP3 protein levels, so the ratio of Bax/Bcl-2 increased [
32]. Anticancer potential of methanol extracts of lichens was evaluated in human lung (H1299, A549) and breast (MDA-MB-231, MCF-7) cancer cell lines. The extract of
Usnea intermedia exerted the strongest anti-proliferative effects especially observed in H1299 and MDA-MB-231 cells. The extract of
U. intermedia also induced apoptosis confirmed via the phosphatidylserine translocation, increased caspase 3/7 activity, loss of mitochondrial membrane potential and the formation of pyknotic nuclei [
113]. Isolates from
Nemania serpens and
Nemania aenea var. aureolatum were the most active compounds against human colorectal cancer (HT-29, HCT116) and human prostate cancer (PC-3, DU145) cell lines. These isolates induced apoptosis characterized by activated caspase 3, 8, PARP cleavage and chromatin fragmentation [
114]. In addition, Nugraha et al. evaluated anticancer activities of nine lichens of East Java Indonesia, of which
Physcia cf.
Milegrana showed the most significant cytoxicity, but only on HeLa cells. Other metabolites exhibited very low cytotoxicity either in cancer or normal African green monkey kidney (Vero) cells [
115].
Table 4 shows an overview of anticancer effects of above-mentioned lichen extracts in various cancer cell lines.