Cyanobacteria as Natural Therapeutics and Pharmaceutical Potential: Role in Antitumor Activity and as Nanovectors

Cyanobacteria (blue-green microalgae) are ubiquitous, Gram-negative photoautotrophic prokaryotes. They are considered as one of the most efficient sources of bioactive secondary metabolites. More than 50% of cyanobacteria are cultivated on commercial platforms to extract bioactive compounds, which have bene shown to possess anticancer activity. The chemically diverse natural compounds or their analogues induce cytotoxicity and potentially kill a variety of cancer cells via the induction of apoptosis, or altering the activation of cell signaling, involving especially the protein kinase-C family members, cell cycle arrest, mitochondrial dysfunctions and oxidative damage. These therapeutic properties enable their use in the pharma and healthcare sectors for the betterment of future generations. This review provides a baseline overview of the anti-cancerous cyanobacterial bioactive compounds, along with recently introduced nanomaterials that could be used for the development of new anticancer drugs to build a healthy future for mankind.


Introduction
Cyanobacteria (blue-green microalgae) are ubiquitous Gram-negative photoautotrophic prokaryotes, for which evidence they existed 3.3-3.5 billion years ago was found [1]. They exist in the environment as unicellular, filamentous or colonial forms surrounded by a mucilaginous sheath [2]. Owing to their ubiquitous nature and the vast diversity of their metabolites, they have several applications in the biomedical field. During the last few decades, the emergence of new cancers and their developing resistance to available drugs has led to chemotherapeutic failure. Thus, the identification of new biologically active compounds is immediately required for the development of new drugs. In this regard, the medicinal properties of cyanobacteria have been explored. To date, several secondary metabolites have been isolated from the members of oscillatoriales (49%), followed by nostocales (26%), chroococcales (16%), pleurocapsales (6%) and stigonematales (4%) [2,3]. The capacity of cyanobacteria to integrate non-ribosomal peptide synthetases with polyketide synthases enables them to produce biologically active and chemically diverse compounds, such as cyclic alkaloids, peptides, depsipeptides, cyclic depsipeptides, fatty acid, lipopeptides, swinholides and saccharides [4]. Back in 1500 BC, Nostoc sp. was first used to treat gout, fistula and different types of cancers [5]. However, more focused research utilizing modern technologies was started in this field in the 1990s. Several cyanobacteria species are cultivated in commercial platforms to extract bioactive compounds, which have been shown to possess anticancer activity and potentially kill a variety of cancer cells via the induction of apoptosis or altering the activation of cell signaling [4,6] (Figure 1).  (Table 1). Furthermore, in the rat non-tumoral myoblast A-10 cells, it caused a complete loss of the filamentous (F)-actin at the 30 nM and 60 nM concentrations [8].

Apratoxin
Apratoxin, a cyclic depsipeptide isolated from Micronesia and Lyngbya sp., is a potent cytotoxic marine natural product that showed anti-cancerous activity against various cancer cell lines (Table 1). Apratoxin has antiproliferative effects via the induction of G1 cell cycle arrest and apoptotic cascade [9]. Furthermore, it inhibits the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway by downregulating the interleukin (IL)-6 molecule signal transducer (gp130) and reversibly inhibiting the secretory pathway by preventing cotranslational translocation early in the secretory pathway [10]. Apratoxin A (Figure 3), isolated from Lyngbya sp., induces cytotoxicity on adenocarcinoma cells, and showed cytotoxicity in the nanomolar range when tested against HT29 (colorectal adenocarcinoma), HeLa (cervix adenocarcinoma) and U2OS (osteosarcoma) cancer cell lines. The IC 50 value ranges from 0.36 to 0.52 nM for in vitro studies while, for in vivo studies it was only marginally active against a colon tumor, and was ineffective against a mammary tumor [11]. Furthermore, apratoxin also affects the apoptosis of cancer cells by downregulating receptors and the associated growth ligand. A hybrid synthetic molecule that combines both apratoxins A and E has been synthesized with improved in vivo antitumor properties. It downregulated receptor tyrosine kinases and vascular endothelial growth factor A (VEGF-A) in a colorectal tumor xenograft model [12]. Oxazoline, a synthetic analogue of apratoxin A, inhibits the function of heat shock protein 90 (Hsp90) and promotes chaperone-mediated autophagy [13]. The in vivo pathological studies against the pancreatic tumor revealed that the Sec 61 complex is the molecular target of apratoxin A [14]. Recently, apratoxin S10 (Apra S10, an apratoxin A analogue) has been developed, which induced promising antitumor effects in the pancreatic cancer model by exhibiting the downregulation of multiple receptor tyrosine kinases and inhibiting several growth factors and cytokine secretion [15]. Moreover, apratoxin D revealed strong cytotoxicity, with an IC 50 value of 2.6 nM against NCI-H460 lung cancer cells [16].

Aurilide
Aurilide, a cyclic depsipeptide isolated from Dolabella auricularia, showed cytotoxicity ranging from picomolar (pM) to nanomolar (nM) concentrations against several cancer cell lines (Table 1) [17]. It encourages mitochondrial-induced apoptosis by selectively binding to prohibitin 1 (PHB1) in the mitochondria and activating the proteolytic processing of optic atrophy 1 (OPA1) [18]. Aurilides B and C (aurilide analogues, Figure 4) showed in vitro cytotoxicity against an NCI-H460 human lung tumor and the neuro-2a mouse neuroblastoma cell lines, with LC 50 values ranging from 0.01 to 0.13 µM [17]. Aurilide B exhibits potent cytotoxicity against human renal, leukemia and prostate cancer cell lines from the NCI-60 panel, a panel of 60 different human tumor cell lines of different tissue origins, with an average growth inhibition (GI 50 ) value of less than 10 nM [17].

Bisebromoamide
Bisebromoamide ( Figure 5), a linear peptide isolated from an Okinawan strain of Lyngbya sp., contains a unique N-methyl-3-bromotyrosine, a modified 4-methylproline, a 2-(1-oxopropyl) pyrrolidine and an N-pivalamide unit. Based on cell morphological profiling analysis, bisebromoamide was identified as an actin filament stabilizer [19]. It revealed cytotoxicity against HeLa S3 cells with an IC 50 value of 0.04 µg/mL, and exhibited 50% growth inhibition against a panel of 39 human cancer cell lines with a GI 50 value of 40 nM (Table 1). It exhibited strong protein kinase inhibition by phosphorylating extracellular signal-regulated protein kinase (ERK) in normal rat kidney (NRK) cells via platelet-derived growth factor (PDGF) stimulation at a 0.1-10 µM concentration [20]. However, studies have shown that bisebromoamide and its synthetic analogs, whose stereochemistry reveals the presence of a methylthiazoline moiety and a methyl group at the 4-methylproline unit, do not significantly influence cytotoxicity against HeLa S3 cancer cells [21]. Bisebromoamide inhibited the phosphorylation of ERK and AKT (protein kinase) enzymes when tested against human renal carcinoma cell lines 769-P and 786-O. The IC 50 values for 769-P and 786-O cells were 1.63 µM and 2.11 µM, respectively [22]. In another study, it was shown to induce apoptosis through ERK and mTOR inhibitions in renal cancer cells [23].

Biselyngbyaside
Biselyngbyaside A ( Figure 6), a glycomacrolide isolated from Lyngbya sp., exhibits cytotoxicity against HeLa S3 cells with an IC 50 value of 0.1 µg/mL [24]. Moreover, Biselyngbyolide B, C, E and F have an antiproliferative effect in HeLa and HL-60 cells, and biselyngbyolide C was found to trigger endoplasmic reticulum (ER) stress and apoptosis in HeLa cells [25] (Table 1).

Calothrixin
Calothrixins are quinone-based bioactive molecules ( Figure 8) isolated from Calothrix that possess potent antiproliferative activity against several cancer cell lines [27]. Calothrixins A, an indolophenanthridine isolated from Calothrix, induced apoptosis and cell cycle arrest in the G2/M phase of several human cancer cell lines (Table 1). It has shown antitumor activity against human HeLa cancer cells at nanomolar concentrations [28]. Besides this, calothrixin B displayed antiproliferative activity against the HCT-116 colon cancer cell line with an IC 50 value of 0.32 µM [29]. Recently, a series of calothrixin B analogs have been synthesized that inhibit cancer cell growth by extensive DNA damage followed by apoptotic cell death. They have shown cytotoxicity against the NCI-H460 cell line with a GI 50 of 1 nM [30]. Another analog of calothrixin B, isothiacalothrixin B, exhibits cytotoxicity against human colon cancer cells in vitro by inducing irreversible DNA damage, and causes apoptosis. It was found to be a promising anti-cancer agent that caused the strong arrest of the cells in the S and G2/ M phases of the cell cycle in the HCT116 cell line [31].

Carmaphycins
Carmaphycins A and B (Figure 9), isolated from Symploca sp., are structurally related to proteasome inhibitor epoxomicin, and show strong antiproteasomal activity. They exhibited strong cytotoxicity against lung adenocarcinoma (NCI-H460) and colon (HCT-116) cancer cell lines, as well as exquisite antiproliferative effects in the NCI-60 cell lines [32] ( Table 1). Recently, carmaphycins conjugates with peptides and antibodies have been investigated in the design of novel antibody-drug conjugates (ADCs) for targeted delivery in cancer therapy [33].

Caylobolide
Caylobolide A and B are macrolactones ( Figure 10) isolated from Lyngbya majuscula and Phormidium sp. that induce cytotoxicity against several cancer cell lines (Table 1). Caylobolide A exhibits cytotoxicity against the HCT-116 colon tumor with an IC 50 of 9.9 µM [34], while the IC 50 values of Caylobolide B against the HT29 colorectal adenocarcinoma and HeLa cervical carcinoma are 4.5 and 12.2 µM, respectively [35].

Coibamide A
Coibamide A ( Figure 11) is a novel N-methyl-stabilized antiproliferative depsipeptide extracted from Leptolyngbya sp. that induces concentration-and time-dependent cytotoxicity with a half-maximal effective concentration (EC 50 ) < 100 nM in human SF-295 and U87-MG glioblastoma cells and mouse embryonic fibroblasts (MEFs) ( Table 1). It induces mTOR-independent autophagy and cell death in human glioblastoma cells [36]. In addition, it displayed potent cytotoxicity towards the NCI-H460 lung cancer cells and mouse neuro-2a cells when used in nanomolar concentrations (LC 50 < 23 nM). Previously, it was evaluated against an in vitro panel of 60 selective human cancer cell lines, including colon, breast and ovarian malignant tumors cells. The results indicated that coibamide A was found to be more effective against the MDA-MB-231 breast cancer cell line than against other cell lines [37]. Recently, an analog of coibamide A was synthesized that significantly inhibited tumor growth in vivo [38].

Cryptophycins
Cryptophycin, a depsipeptide isolated from Nostoc sp. var. ATCC 53789 and GSV 224, is a potent anticancer agent. It inhibits microtubule assembly, and exhibited antitumorigenic activity against several solid tumors implanted in mice, including multidrugresistant cancer cells ( Table 1). The IC 50 value was found to be less than 50 pM for multidrug-resistant cancer cell lines [39,40]. Several natural and synthetic analogs of cryptophycins ( Figure 12) were reported and progressed to preclinical and clinical trials. Cryptophycin-8, a semi-synthetic analog, showed more efficient in vivo antiproliferative activity, leading it to act as an antitumor agent. Cryptophycin 52 (LY355703), isolated from Nostoc spp., induced cell cycle arrest in the G2/M phase and apoptosis via multiple pathways, such as both caspase-1-and caspase-3-dependent activation and Bcl-2 and Bcl-xL phosphorylation in several human prostate cancer cell lines [41]. A clinical phase II study of cryptophycin-52 revealed anticancer activity against advanced human non-small cell lung carcinoma (NSCLC) and platinum-resistant advanced ovarian cancer in patients [42], but failed due to neurotoxic side effects and limited in vivo efficacy [43]. Cryptophycins are promising drug candidates as their activity is not negatively affected by P-glycoprotein (a drug efflux system commonly found in multidrug-resistant tumor cell lines). Recently, cryptophycin conjugates with antibodies and peptides were developed for targeted drug delivery in cancer therapy [44]. The antiproliferative activities of RGD-cryptophycin and iso-DGR-cryptophycin conjugates were evaluated against human melanoma cells M21 and M21-L. They exhibit anticancer activity at nanomolar concentrations with different expression levels of integrin α v β 3 . In another study, the cryptophycin conjugate (RGDcryptophycin) showed antiproliferative effects against M21 and M21-L human melanoma cell lines at nanomolar concentrations [45].

Curacin A
Curacin A (Figure 13), an anti-tubulin agent isolated from Lyngbya majuscula, was screened against the NCI-60 panel of human tumor cell lines. It inhibits the binding of tubulin polymerization into the colchicine binding pocket, and arrests the cells in the G2/M phase of the cell cycle (Table 1). Due to its therapeutic potential as an antimitotic agent, curacin A was evaluated for clinical trials [46]. It exhibits cytotoxicity against renal, colon and breast cancer cell lines [47].

Desmethoxymajusculamide C
Desmethoxymajusculamide C ( Figure 14), a cyclic depsipeptide isolated from L. majuscula, exhibits strong antitumor potential against HCT-116 human colon carcinoma cells with an IC 50 of 20 nM (Table 1). It helps in the destruction of the cell microfibrils networks by depolymerizing the actin cytoskeleton [48].

Dolastatins
Dolastatins are peptides initially isolated from the sea hare Dolabella auricularia and later from marine cyanobacterial strains that induce cytotoxicity against cancer (Table 1). Dolastatins 10 and 15 (Figure 15a,b) are linear peptides isolated from a cyanobacteria, Symploca sp. VP642 [49]. They show cytotoxicity against several cancer cell lines due to their specific molecular interference with the dynamics of microtubule assembly, and induce an arrest in the G2/M phase of the cell cycle, leading to apoptosis. Several synthetic and semi-synthetic analogs of dolastatins 10 and 15 have been produced and are in clinical trials as anticancer drugs. Symplostatin 3 (Figure 15c), a dolastatin analog isolated from Symploca sp. VP452, disrupts microtubules, and showed IC 50 values ranging from 3.9 to 10.3 nM against KB and LoVo cell lines, respectively [50]. Dolastatin 10 and its synthetic analog auristatin PE underwent early clinical trial phases, but have been withdrawn owing to a lack of efficacy and reports of peripheral neuropathy in patients. Nevertheless, a highly effective antibody-drug conjugate (ADC) named brentuximab vedotin, developed by Seattle Genetics, passed the clinical trial and was granted FDA approval in August 2011 for the treatment of Hodgkin's lymphoma and anaplastic large cell lymphoma. Brentuximab vedotin is a combination of monomethyl auristatin E, linked with an antibody targeting CD30 on cancer cell surfaces [51]. Additionally, three more dolastatin 10 analogs were formulated as antibody-drug conjugate ADCs, namely, glembatumumab vedotin, SGN75 and ASG5ME, which are in the clinical trials [52]. The cyanobacterial peptide named glembatumumab vedotin has been approved for Phase II trials. It exhibited potent activity against breast and melanoma cancers with the maximum tolerant doses of 1.0-1.88 mg/kg [53]. Furthermore, a new analog, auristatin TP (Figure 15d), has been synthesized, which is a tyramide phosphate modification of dolastatin 10. It possesses improved anticancer properties owing to its increased bioavailability, with ED 50 values ranging from <1.2 to 54.6 nM when tested against P388, NCI-H460 and MCF-7 cancer cell lines [54]. Besides this, dolastatin-15 derivative-based ADCs have also been developed that effectively kill human epidermal growth factor receptor (HER)2-positive cancer cells [55]. Recently, using trastuzumab (Herceptin ® ) as the antibody, Dol10-containing ADCs have been synthesized and investigated for in vitro cytotoxicity assays against HER2-positive (SK-BR-3) human tumor cells. Additionally, ADCs derived from Herceptin ® and PEG8-Dol10 effectively delayed tumor growth at a dose of 10 mg kg −1 , and prolonged the survival time in mice bearing human ovarian SKOV-3 xenografts [56].

Grassypeptolides
Grassypeptolides A-C ( Figure 16) are cyclic depsipeptides (containing bis-thiazoline) produced from Lyngbya confervoides, which reveal cytotoxicity against several cancer cells. The IC 50 values for grassypeptolides A were evaluated against HT29 and HeLa cancer cell lines, and were found to be 1.22 and 1.01 µM, respectively (Table 1). Similarly, for grassypeptolides B and C, they were 4.97 and 2.93 µM and 76.7 and 44.6 nM, respectively. Grassypeptolide A and C both induce G1 cell cycle arrest at lower concentrations, but in HeLa cells they induce the G2/M cell-cycle arrest at higher concentrations [57]. Furthermore, grassypeptolide D and E reveal cytotoxicity against HeLa cells at IC 50 values of 335 and 192 nM, respectively, and against mouse neuro-2a blastoma cells at IC 50 values of 599 and 407 nM, respectively [58].   (Table 1).

Hectochlorin
Hectochlorin ( Figure 18) was isolated from L. majuscula, and promotes actin polymerization with an EC 50 value of 20 µM in human non-tumoral kidney cells PtK2. It also displays cytotoxicity against the NCI-60 panel of cancer cell lines (Table 1). Among these, 23 cancer cell lines, such as colon melanoma, renal cells and ovarian cells, showed the strongest cytotoxicity. The IC 50 values range from 20 to 300 nM for CA46 human Burkitt lymphoma cell and PtK2 cells, respectively. However, the dose-response curve of hectochlorin was flat, suggesting that the compound is more antiproliferative than cytotoxic [60].

Hierridin B
Hierridin B (Figure 19), a polyketide isolated from the Cyanobium sp. LEGE 06113, was tested in a panel of eight human cancer cell lines, and showed selective cytotoxicity only against the colon adenocarcinoma cell line HT-29, with an IC 50 value of 0.1 mM [60] (Table 1).

Hoiamide D
Hoiamide D, isolated from Symploca sp, consists of a unique triheterocylic system structurally comprising two consecutive thiazolines and a thiazole, as well as an unusual isoleucine moiety ( Figure 20). The carboxylate anion of hoiamide D was reported to inhibit p53/HDM2 (human homolog of MDM2) interaction with an EC 50 of 4.5 mM (Table 1). Besides this, it also possesses significant sodium channel-activating properties. [61].

Largazole
Largazole (Figure 24), a cyclic depsipeptide isolated from Symploca sp., is a highly potent class I histone deacetylase (HDAC) inhibitor, and is currently being used for the development of anticancer drugs. It has been screened to evaluate its cytotoxicity against many epithelial and fibroblastic cancer cell lines [69] (Table 1). Generally, largazole acts as a prodrug, and upon hydrolysis of the thioester, a largazole thiol releases that function as a reactive molecule. The resultant molecule then complexes with Zn 2+ and inhibits class I histone deacetylase. Beside its HDAC inhibitory property, largazole exhibits a wider range of in vitro and in vivo biological activities, for example antitumor, antiosteogenic and antifibrotic activities [70][71][72]. When largazole was used together with dexamethasone (a synthetic glucocorticoid steroid), it induced E-cadherin localization at the plasma membrane in triple-negative breast cancers and suppressed cancer invasion [73]. In another study, largazole sensitized Epstein-Barr virus-positive (EBV + ) lymphoma cells to ganciclovir (an antiherpes viral drug) at nanomolar concentrations [74]. Recently, largazole and its analogs were found to inhibit a ubiquitin-activating enzyme (E1) during ubiquitination [75].

Lyngbyabellins
Lyngbyabellin A and E (Figure 26a,b), isolated from L. majuscula, are hectochlorinrelated lipopeptides. They possess potent actin polymerization activity. Lyngbyabellin A exhibited moderate cytotoxicity against human cervical (KB) and colon (LoVo) cancer cell lines with IC 50 values of 0.03 µg/mL and 0.5 µg/mL, respectively (Table 1) [78]. In vivo trials suggested that lyngbyabellin A is toxic to mice. It disrupts the cellular microfilament network in A-10 cells at 0.01-5.0 µg/mL concentrations [78]. When compared to the above, Lyngbyabellin B was found to be slightly more cytotoxic in in vitro trials [79]. Furthermore, Lyngbyabellin E showed cytotoxicity against human lung tumors (NCI-H460) and neuro-2a mouse neuroblastoma cell lines with LC 50 values between 0.2 and 4.8 µM [80].

Nocuolin A
Nocuolin A (Figure 29a), a natural oxadiazine isolated from some of the species belonging to Nostoc, Nodularia and Anabaena genera, induces cell death and exhibits antiproliferative activity against various human cancer cells, specifically against p53-mutated cell lines with IC 50 values ranging from 0.7 to 4.5 µM [88] (Table 1).

Scytonemin
Scytonemin (Figure 29c), an aromatic indole alkaloid extracted from Stigonema sp., showed antiproliferative activity against several human fibroblast and endothelial cell lines. It inhibits the human polo-like kinase activity that plays a crucial role in the regulation of the cell cycle at the G2/M transition [90] (Table 1).

Tasiamides
Tasiamides are linear peptides isolated from Symploca sp. and tasiamide B (Figure 29e) that showed strong cytotoxicity against human cervical adenocarcinoma (KB) cells, with an IC 50 value of 0.8 µM [92]. In another study, eighteen analogs of tasiamide were screened against KB cells and human non-small cell lung tumor A549 cells, with IC 50 values between 1.29 and 12.88 µM, respectively [93] (Table 1).

Veraguamides
Veraguamides A-C and H-L cyclodepsipeptides ( Figure 30) were isolated from Oscillatoria margaritifera, and veraguamides A-G were also isolated from Symploca hydnoides. All showed a cytotoxic effect (Table 1). Veraguamide A was found to be highly toxic against the human lung cancer cell line NCI-H460 (LD 50 = 141 nM) [94]. Moreover, veraguamides A-G exhibited moderate to weak cytotoxicity against both the HT 29 and HeLa cell lines [95].

Cyanobacteria as Nanoformulations in Cancer Therapies
Globally, the use of nanomaterials in the biomedical field has attracted the increasing interest of researchers because of their unique ability to interact with cells and tissues at the molecular level with a high degree of specificity and improved efficacy. The small size (ranging from 1 to 100 nm), design flexibility and large surface-to-volume ratio make these materials of significant use. With the appreciation of the enhanced properties of metals at nanosizes, extensive research on different nanometals has been carried out in recent years for exploring their applications in cancer research. Emphasizing the biological synthesis, cyanobacteria are considered as one of the best biological systems for nanoparticle (NP) synthesis, both intra-cellularly as well as extra-cellularly [111]. However, there are only a few reports available on the biological synthesis of noble metal NPs utilizing cyanobacteria as the host system. Silver (Ag) is the most commonly reported NP synthesized [111][112][113]. Besides this, gold (Au), palladium (Pd) and platinum (Pt) NPs of well-controlled size are also formed from cyanobacteria such as Anabaena, Calothrix, Leptolyngbya, etc. [113]. Moreover, several cyanobacterial strains have been used to synthesize selenium NPs (11.8 to 60 nm) [114]. Furthermore, Spirulina subsalsa has been utilized for the extracellular synthesis of Ag and Au NPs [115].
Very few studies on the above have suggested that these NPs could be used for the treatment of cancer (Table 2). Recently, silver NPs (AgNPs) synthesized from the aqueous extract of Oscillatoria limnetica have exhibited cytotoxic effects against human breast (MCF-7) and colon (HCT-116) cancer cell lines with IC 50 values of 6.15 µg/mL and 5.37 µg/mL, respectively [116]. In another study, silver nanoparticles (20-50 nm size) synthesized from L. majuscula were screened against three leukemic cell lines, i.e., K562, MOLT-3, and REH, and showed dose-and time-dependent anticancer activity. The REH cells showed the maximum sensitivity to AgNPs, with an IC 50 value of 620 ± 3.73 µg/mL [117]. Moreover, AgNPs (51-100 nm size) synthesized from the cell extract of the cyanobacterium Nostoc sp. strain HKAR-2 showed a dose-dependent cytotoxic activity against MCF-7 cancer cell lines, with an IC 50 of 27.5 µg/mL [118]. The phycocyanin extracted from Nostoc linckia synthesizes silver nanoparticles (9.39 to 25.89 nm). These AgNPs exhibit in vitro antitumor activity against the human breast MCF-7 cancer cell line (IC 50 = 27.79 ± 2.3 µg/mL), and were shown in vivo to inhibit tumor growth in Ehrlich ascites carcinoma-bearing mice [111]. ZnO NPs synthesized by using the cell extract of the Nostoc sp. EA03 exhibit less cytotoxicity when used in lower concentrations against the human adenocarcinoma alveolar basal epithelial cells (A549 cells) and human lung fibroblast (MRC-5 cells) cancer cell lines [119].
Cells and inhibited tumor growth. Consequently, they showed notable effects as anticancer agents and could be used as nanomedicines in cancer research. However, some cyanobacterial nanoformulations could also be used to deliver therapeutic drugs to cancer sites and monitor the tumor tissues, but limited data are available. As mentioned above, numerous studies show that, even at pico to nano molar concentrations, several cynobacterial secondary metabolites, such as apratoxin, calothrixin, curacin A, dolastatins, etc., are effective, and have shown potent inhibition of several cancer cell lines in vitro, however in vivo studies are limited. As with camptothecin, whose clinical application is limited due to its low solubility, although it has shown potent anticancer activity in vitro [120,121], most compounds isolated from cynobacteria have a low solubility in water, which makes their formulation troublesome or even incomprehensible [122,123]. Nanoformulation of cyanobacterial compounds represents an interesting strategy to overcome the hydrophobicity or low aqueous solubility of most natural bioactive compounds. Although further studies are needed to prove the concept, it can be hypothesized that if one of these compounds is combined with a nanomaterial, as stated above, such as gold, silver, zinc, etc., it could prove more effective. Furthermore, the use of nanomedicines has unique advantages over the systemic administration of free natural bioactive molecules. These advantages include the improved protection of the biological activities of the agents in a serum-rich environment, longer periods of circulation in the blood, improved permeability and effectiveness of tumor targeting, reduction of adverse effects and the possibility of considering reactivity to stimuli for personalized therapies, etc. [122][123][124]. Once encapsulated in nanoparticles, the hydrophobic bioactive compound becomes completely dispersible in water and can therefore be injected intravenously; the drug-loaded nanoparticles will then be able to release the active ingredient. However, only a very few studies have used this strategy with cyanobacterial compounds, which include nanovectorized extracts from Arthrospira platensis with anti-fungal biofilm activity [125] and the anti-inflammatory protein phycocyanin from Aphanizomenon flosaquae (a freshwater cyanobacteria usually known as AFA Klamath), the delivery of which to the deep skin layers was improved when using propylene glycol-containing nanovesicles [126]. However, no attempt to nanovectorise a cyanobacterial compound with an anticancer effect has been reported to date and this would merit future research. Previously, green carbon nanotags (G-tags) developed from cyanobacteria were shown to be an advanced and efficient imaging platform for anticancer therapy due to their high solubility, excellent photostability and low cytotoxicity. These nanotags, when conjugated with doxorubicin, not only induced cell death in cancer cells (HepG2 and MCF-7), but also, the fluorescence of G-tags enables the monitoring of doxorubicin uptake by cancer cells and their intracellular location [127]. Similar studies could also be carried forward by conjugating cynobacterial metabolites with nanotags, or the nanovectorization of canonical anticancer drugs using cynobacteria to enhance the anticancer potential of the drug. Cynobacterial metabolites from Spirulina showed protective effects against the cardiotoxicity induced by doxorubicin, and thus improve the therapeutic index of doxorubicin [128]. Similarly, the biochemical protective effects of Spirulina platensis against oxidative stress caused by doxorubicin were also evaluated [129]. These could find future applicability, with a better shelf-life and stability as natural capping occurs [130].

Conclusions
In the last few decades, the use of cyanobacteria as a novel source of therapeutics has been realized. Several chemically diverse compounds have been screened against different cancer cell lines, but still, very few cyanobacterial compounds have entered clinical trials, as much is not known at the molecular level. Though their economic cultivation expanded their utilization in drug discovery, still there is a need to explore more deeply into cyanobacterial compounds in order to clarify the specific targets and the mechanisms involved in cancer. Thus, more scientific attention and interdisciplinary research would have to be devoted to finding novel compounds by exploring cyanobacterial strains from extreme unexplored habitats. Furthermore, we do not explore in this review paper the structure-function relationship that might be established and would link a structural motif to a particular target. To this end, the reader may refer to the comprehensive review published previously by Salvador-Reyes and Luesch [131], which details the mechanisms of action of cyanobacterial metabolites alongside the methodology used for these discoveries. Besides this, Xu et al. focused on calothrixins synthesis and their biological effects [27]. For example, quinone compounds such as calothrixin A and B are known to be redoxactive, and the generation of ROS through redox cycling of the quinone was thought to be responsible of the induced apoptosis and DNA damage [27]. Structure-function studies showed that this mechanism of action was independent of the presence of calothrixin's ring E, but strongly dependent on the tetracyclic ring quinone structure [27] Further analyses highlighted the crucial role of the nitrogen in ring D, the importance of ring A-D, and finally evidenced the higher potential of calothrixin B [27].
One of the main subjects of interest in the field of natural biomedicines and their nanoformulations is their toxicity and/or that of contaminating toxic substances. Indeed, cyanobacteria could also be the sources of toxin production [86]. Although generally free of contaminants, cyanobacterial products may contain substances harmful to the liver, such as microcystins, toxic metals and other types of harmful bacteria, which are dangerous for human consumption [132]. Contaminated cyanobacteria can cause liver damage, stomach pain, nausea, vomiting, weakness, thirst, rapid heartbeat, shock and even death. The presence of microcystins is well known for its hepatotoxicity [132], and several other compounds cause severe anaphylaxis and neuromuscular toxicity [133]. This biosafety issue may partly explain the very low number of cyanobacterial compounds reaching clinical trials, mainly Brentuximab vedotin for Hodgkin lymphoma and Glembatumumab vedotin for various cancers (Table S1) [6]. Although this is also true for numerous other compounds that exhibit strong anticancer activity but did not reach clinical trial, this might be due to their toxic nature, which hindered the biological activities in vivo or their natural contamination with toxicants while processing the drug.
In addition, the biosynthesis of NP by cyanobacteria is a relatively new idea to which promising results are attributed and, in the future, it will be possible to build datasets for marine pharmaceuticals by developing nanoformulated drugs based on cyanobacteria. However, to bring it to the platform of drug development there is a need to optimize biosynthesis pathways involving the futuristic approach of green synthesis by controlling the size and shape of particles and achieving monodispersity in the solution phase. As cyanobacteria act as natural therapeutic and have pharmaceutical potential, it could be expected that they could be used as an antitumor killer for a better world.
Supplementary Materials: The following are available online. Table S1: Clinical trials involving cyanobacterial-derived compounds.