Streptomyces, Micromonospors, Saliniospors and Actinomadura, respectively, in which Streptomyces, as the predominant contributor generating over 76% of compounds exclusively. Besides it provides a valuable snapshot of the chemical structure-activity relationship of compounds, highlighting the presence or absence of some specific atoms and chemical units in the structure of compounds can greatly influence their biological activities. To the best of our knowledge, this is the first comprehensive review on natural actinobacterial compounds affecting different types of CRC. Our study reveals that the high diversity of actinobacterial strains and their NPs derivatives, described here provides a new perspective and direction for the production of new anti-CRC drugs and paves the way to innovation for drugs discovery in the future. The knowledge obtain from this review can help us to understand the pivotal application of actinobacteria in future drugs development.Colorectal cancer (CRC) is a common, and deadly disease. Despite the improved knowledge on CRC heterogeneity and advances in the medical sciences, there is still an urgent need to cope with the challenges and side effects of common treatments for the disease. Natural products (NPs) have always been of interest for the development of new medicines. Actinobacteria are known to be prolific producers of a wide range of bioactive NPs, and scientific evidence highlights their important protective role against CRC. This review is a holistic picture on actinobacter-derived cytotoxic compounds against CRC that provides a good perspective for drug development and design in near future. This review also describes the chemical structure of 232 NPs presenting anti-CRC activity with the being majority of quinones, lactones, alkaloids, peptides, and glycosides. The study reveals that most of these NPs are derived from marine actinobacteria followed by terrestrial and endophytic actinobacteria, respectively. They are predominantly produced by  Colorectal cancer (CRC) is the third most common cancer globally with an annual rate of 10% of new cancer cases. Following lung cancer which is the deadliest cancer worldwide, CRC is the second deadliest cancer in the world, accounting for 9.4% of cancer deaths . The gloNatural products (NPs) are bioactive substances found in plants as well as fungi, bacteria and marine organisms. These compounds and their derivatives have been increasingly considered in the design of drugs including antibiotics and anti-cancer drugs owing to the variety of structural features and biological activities, as well as low toxicity and side effects. Natural compounds and their derivatives account for about one-third of FDA-approved drugs in the last 20 years . These cStreptomyces, the largest genus of actinobacteria, are still silent under normal culture conditions, further studies on these microorganisms can be promising to explore more effective biological compounds in which indicates the unknown high potential of these bacteria in the production of secondary metabolites , which contains 1 mm of sea surface, also seabed habitats containing marine sediments and a suitable place for microorganisms to coexist with microphones and macrofauna, fish surface, beach, coastal waters and even on the seabed  , having a fused γ-lactam β-lactone ring, and also its derivatives, salinosporamides D-J, have shown significant antitumor effects with inhibitory properties on the 20s proteosome ((1) has been isolated from marine actinobacteria Salinospora tropica CNB-392 (50 = 11 ng/ml) and HCC-2998, as well as a wide range of other tumors such as myeloma, non-hodgkin’s lymphoma, CLL and pancreatic carcinoma have been studied (1) with common chemotherapy agents including 5FU, leucovorin, and oxaliplatin, was also reported to increase their anticancer effects significantly (actinomadura sp. M048 ((2), N-acetylquestiomycin A (3) and chandrananimycin A (4), B (5) showed antitumor activity against several cell lines, including human colon carcinoma cell line, HT-29.Salinosporamide A oteosome . The inhoteosome . Salinos CNB-392 . The ant studied . This co studied . A combificantly . In 2003sp. M048 . The comStreptomyces strain BL-49-58-005 were studied to evaluate their cytotoxicity effects against cancer cell lines. Indole number two  (6) showed activity against various cell lines, including LOVO and LOVO-DOX colon adenocarcinoma cell lines ((7) and B (8) are two compounds with moderate anti-cancer activity against the HCT-116 cell line, isolated from the marine derived Salinispora arenicola CNT-088  showed moderate cytotoxic activity against HCT-116 cell line with an IC50 value of 30 μg/ml. In 2004, a polyketide called aureoverticillactam (10) was isolated from the marine derived S. aureo-verticillatus NPS001583 , 2 (12) and 4 (13), showed cytotoxic effects against the HCT-116 cell line with the IC50 values of 2.4, 0.97, and 1.84 μg/ml, respectively. Cyanosporasides A (14) and B, structurally related to sporolides, were isolated from S. pacifica strain CNS103. The former one had poor toxicity effects against HCT-116 cell line, with an IC50 value of 30 μg/ml  with an IC50 value of 3.15 μg/ml  were reported to have potent antitumor effects against various cell lines, including HT-29. On average, trioxacarcins A (16) had the highest effect among the rest, while gutingimycin (20) had the lowest toxicity , associated with angucyclines and other aromatic polyketides. The compound exhibits cytotoxic effects against human cancer cell lines, HT-29 (colon), SK-MEL-28 (melanoma) and A-549 (lung), as well as P-388 mouse leukemia ((22), was isolated from the actinobacterium Micromonospora sp. L-25-ES25-008 and its anti-cancer effects examined against various cell lines, including HT-29. However, these antitumor effects were reported to be four times more potent in P-388 mouse leukemia  and B (24) showed significant anti-cancer effects against the cell line with IC50 values of 0.20 and 11 µM, respectively.The marine derived leukemia . A macroleukemia . The antell line . Types A(25), isomarinone (26), hydroxydebromomarinone (27), and methoxydebromomarinone (28), are compounds of a polyketide-terpenoid mix nature produced by actinomycete isolate CNH-099. These compounds exhibited moderate cytotoxicity against HCT-116 cell line with an IC50 value of 8 μg/ml  and 46 μM for type B (30). The chemical structure of compounds 1–30 are presented in Marinones, including neomarinone  8 μg/ml . Streptoineosins . These c(31) and B (32), two bioactive natural compounds isolated from the marine actinobacterium Streptomyces strain CNH990, have potent anticancer effects on a variety of cancer cell lines  for Marmycin B.Marmycins A ll lines . In the Streptomyces sp. YM14-060 in 2007 ((33) among those compounds induces cytotoxicity in the cancer cell line via suppressing the accumulation of GRP78 protein (a drug resistance factor) , show antitumor activity against colon (HCT-116), melanoma, prostate, and central nervous system cancer cell lines. The toxicity property of piperazimycin A against solid cancer lines was stronger three times those of piperazimycins B (35) and C (36).Piericidins were isolated from  in 2007 . Another in 2007 . Pierici factor) . Streptozimycins . All of Streptomyces sp. KS3 and S. staurosporeus AM-2282. Staurosporine, produced by Micromonospora sp. L-31-CLCO-002 and its natural analogues 4′-N-methyl-5′-hydroxystaurosporine (37) and 5′-hydroxystaurosporine (38), were examined in terms of antitumor activity against various cancer cell lines namely lung, melanoma and HT-29 colon carcinoma. The latter two analog compounds had the highest activity against the HT-29 cell line (Streptomyces sp. NB-A13 in which 14 of them (39–52) had significant effects against SW-620 colon cancer cell lines ((44) and No. 14 (51) were reported to have the strongest cytotoxic function. In 2018, the structure and biological effects of staurosporine derivatives from Streptomyces coelicolor M1146 were investigated ((53) showed activities against all of these cell lines, including HCT-116 (IC50 = 1.0M) and staurosporine M2 (54), against HCT-116 cell lines (IC50 = 3.9 µM) and Huh 7.5 (IC50 = 2.7 µM).Staurosporines and their derivatives, are isolated from different species such as komodoquinones-producer ell line . In a 20ll lines . Staurosstigated . The com(55) produced by Streptomyces sp. KORDI-3238 showed significant antitumor effects against HCT-15 colon cancer and other cancer cell lines such as MDA-MB-231, leukemia K-562, lung NCI-H23, etc. ((56), produced by Micromonospora (marina) sp. L-13-ACM2-092 isolated from soft coral from the coast of Mozambique, Indian Ocean, has documented against HT-29 cell line. The compound, by stopping the cell cycle in G1 phase and slowing the progression of S phase of the cell cycle to G2/M, has antiproliferative effects on the LOVO and SW620 cell lines of the human colon with IC50 values of 15 and 500 nM, respectively. The inhibitory effect of compound is due to the inhibition of DNA polymerase α as well as its bilateral entry into the DNA strand  showed potent antiproliferative effects against CRC cell lines.In a 2007 study, the streptokordin 23, etc. . The antA strand . In 2014 pathway . In 2015ll panel . DerivatStreptomyces strain M491 ((58), with moderate toxicity effects, the rest of them left no noticeable effects on the t tested cell lines. Macrodiolide tartrolon D (59), isolated from Streptomyces sp. MDG-04-17-069 has shown strong cytotoxic effects against human cancer cell lines including colon (HT-29), lung (A549) and breast (MDA-MB-231) ((60) produced by marine Streptomyces sp. SCA29, was stated to inhibit α-glucosidase and α-amylase enzymes, and show antibacterial activity and cytotoxicity against various cancers, including HT-29 cell line (The eight t-muurolol compounds were isolated from marine actinobacterium ain M491 . Seven o-MB-231) . 4-methoell line . The che(61) has been identified from Micromonospora strain FIM07-0019 collected off the shallow coastal waters of Chile (50 = 16.4 µM).A compound called levantilide C of Chile . The comActinoalloteichus cyanogriseus and its mutant species, can induce cytotoxic effects on cancer cell lines. Cyanogrisides F (62) and G (63) have shown cytotoxicity against HCT-116 and leukemia HL-60 cell lines  had a significant effect on SNU638 gastric cancer and HCT-116 colon cancer cell line. The other two had little toxic effect.Cyanogrisides and their related analogues, produced by ll lines . In 2014 effects . AmycolaMicrobacterium sediminis spp. nov. YLB-01 exhibits significant cytotoxic effects against a panel of cancer cell lines. Microbacterin B (65) was stated to have cytotoxicity on all of tested cell lines, including HCT-8 human intestinal adenocarcinoma cell line (Pseudonocardia sp. HS7 and its synthetic derivatives exerted a proliferative inhibitory effect on various cell lines ((66–70) and their three synthetic acyl derivatives (5a-5c) (71–73) were measured using SRB Assay. They had significant effects on two human CRC lines HCT-15 and SW620.Compounds called microbacterins A and B, produced by marine actinomycete ell line . The isoll lines . The ant(74) and PM100118 (75), isolated from the marine actinobacterium Streptomyces caniferus GUA-06-05-006A, lead to necrosis and significant antitumor effect on CRC HT-29, lung NSCLCA549 and breast MDA-MB-23 cancer cell lines, by altering the permeability and integrity of cell membranes ((76–79) isolated from marine actinomycetes associated with starfish, Streptomyces sp. 112CH148, from a region in Micronesia, have shown the effects of cytotoxicity against ten human cancer cell lines including HCT-15 and HCT-116 the Colon carcinoma cell lines ((80) compound, produced by the marine actinomycete species BM-17, belonging to the genus Nocardia dassonvillei, was investigated (Streptomyces sp. (NPS008187), collected off Alaskan marine sediments, were examined towards cancer cell lines ((81), showed good cytotoxic effects against colon cancer cell line HT-29.Two polyhydroxyl macrolide lactone compounds, named PM100117 embranes . Α-pyronll lines . In 2012stigated . In addill lines . Glaciap(82–84) with azoxy properties, were isolated from Streptomyces sp. strain HKI0708  compound , were demonstrated toxicity against HCT-116 cell line with IC50 values ranging from 1 to 9 μg/ml ((92), produced by Saccharomonospora CNQ-490, as a specific alkaloid, left relatively little toxic effect against HCT-116 cell line (IC50 = 3.6 µM) (Marinopyrroles produced by the marine  9 μg/ml . Another 3.6 µM) .(93) and B (94) were isolated from Streptomyces CNR-698, which showed a good toxicity with an IC50 value of 320 nM ((95) was isolated from another species called Streptomyces variabilis SNA-020, which had a weaker cytotoxicity with an IC50 value of 3.2–4.9 µM  and B (97) and IC50 value of 5.7 μM for type D (98). In 2013, the cytotoxicity of another halometabolite, chlorizidine (99), isolated from the marine Streptomyces strain CNH-287 was investigated against HCT-116 cell line, and reported to have a significant cytotoxicity with IC50 values of ranging from 3.2 to 4.9 µM ((100–105), have been isolated from marine actinomycetes MAR4 CNS-284 and CNY-960  and 6-OMe-7′,7″-dichorochromopyrrolic acid (107) were reported by  and B2-B4 (114–116), isolated from Streptomyces strain CNQ-329 and CNH-070, were stated to have a weak cytotoxic effect against HCT-116 cell line (IC50 = 4.19–16 μM/ml)  as well as other known compounds including B1, B3, B4, A80915A (121), A80915B (122), A80915C (123), A80915D (124), CNQ525.512 (125), and SF2415B3 (126). In the study of the toxicity and inhibitory effect of these compounds against HCT-116 cell line, a range of results with IC50 values less than 1 µM to more than 100 µM was obtained.Napyradiomycins A – F 6 μM/ml) . They alStreptomyces strain ART 5, collected from the Arctic (eastern Siberia), were identified as a producer of fijiolide compounds A and B as well as C-1027-chromophore-V (127), and C-1027-chromophore-III  and compound number 4  (129) showed good cytotoxicity with IC50 values of 12.7 and 6.2 μM, respectively. In a 2002 study, kosinostatin (130) obtained from marine actinomycete Micromonospora sp. TP-A0468 showed a strong antibacterial effect against gram-positive bacteria and moderate effects against gram-negative bacteria and yeasts  and B, isolated from Micromonospora lomaivitiensis, were examined against a wide range of human cancer lines including several colon cancer cell lines such as HCT15, CACO2, SW948, COLO205  (132), daidzein-7-(2-deoxy-α-l-fucopyranoside) (133) and daidzein-4′,7-di-(2-deoxy-α-l-fucopyranoside) (134), showed a good toxicity on A549 and HepG2 cancer cell lines, in addition to their appropriate effects on HCT-116 cell line ((135), was isolated from a marine bacterium related to Streptomyces, and its cytotoxicity was examined against HCT-116 colon cancer cell line and reported to have a significant effect with LD50: 2.0 μg/ml ((136–138), were found in marine actinomycete Actinoalloteichus hymeniacidonis 179DD-027, isolated from marine sediments on Dokdo Island, Republic of Korea  was reported to have antibacterial effects against gram-positive and negative pathogenic bacteria as well as a significant cytotoxicity against different human cancer cell lines  and one of the induced metabolites (143) showed significant antiproliferative activity against human cancer cell lines HT-29 and HL60 . Due to the inhibitory effect of these two compounds on Pim-1 kinase, it has been suggested that the inhibitory effect of these compounds on cancer cells is due to the induction of this function.Many NPs and metabolites were identified from marine actinomycetes honella. . Among tStreptomyces sp. HNA39 were defined. Among them, compound number 2 (144), showed significant toxicity against HCT-116 with IC50 values of 8.3 µM and prostate cancer PC3 with IC50 values of 0.52 µM. In addition, the compound, along with compounds No. 5, 7 and 8, was stated to inhibit moderately ROCK2 protein kinase ((145) is a compound that has been shown to have a strong inhibitory effect on cell division with an IC50 value of 1.16 μg/ml against HCT-116 cell line. Arenimycin is an antibiotic consumed against rifampin- and methicillin-resistant Staphylococcus aureus produced by marine actinomycete Salinispora Arenicola  were documented to possess good cytotoxic effects on colon cancer HCT-116  cell line. Three new compounds called kendomycins B, C, D (148–150) were identified from the marine Actinomycete Verrucosispora sp. SCSIO 07399  (151), cyclo (Pro-Ala) (152), cyclo  (153), and cyclo (Pro-Leu) (154) with significant effects against HCT-116 cell line have been isolated from Streptomyces nigra sp. nov., collected from the rhizosphere soil of the Avicennia marina mangrove in China, which is closely related to S. coerulescens DSM 40146, S. bellus DSM 40185, and S. coeruleorubidus DSM 41172  has been isolated Streptomyces sp. VN1 that was reported to have a good cytotoxic effect (IC50 = 123.7 µM) on various cell lines, including HCT-116 ((156) and 2,3-dihydroxybenzamide (157) were identified from Streptomyces sp. SBT348, isolated from a Marine Sponge . In another study conducted in Turkey  and compound (159) had IC50 values of 7.4 and 27.9 µM, respectively.A unique furan-type compound  HCT-116 . This ste Sponge . These cn Turkey . Two com(160), neo-actinomycin B (161), along with actinomycin D (162), and actinomycin X2 (163), were isolated from Streptomyces sp. IMB094 , isolated from marine Streptomyces sp. SNJ042, was also reported to have a cytotoxic effect on the same CRC cell line with an IC50 value of 7.61 µM , polycyclic compound was isolated from the terrestrial actinobacter Amycolatopsis sp. IRD-009  showed non-selective toxicity against 36 human cancer cell lines, including two colon cancer cell lines HT-29 and HCT-116 with an average IC50 value of 6.8 µM. Karamomycin C (167) with slightly higher toxicity than karamomycin A (IC50 = 1.3 µM), showed good results.In 2019, the pradimicin-IRD  IRD-009 . In addid Caco-2 . Karamomd Caco-2 . Unlike (168), granaticins A (169), B (170) and methyl granaticinate (171) were isolated from the soil Streptomyces sp. 166, collected from the Tibetan highlands of China , produced by soil Streptomyces sp. CC8-201, in addition to demonstrating a strong antibacterial effect against gram-positive bacteria, has shown a significant toxicity against various human cancer cell lines, including HCT-116 with an IC50 value of 0.59 µM (Xiakemycin A  0.59 µM .(173–176), isolated from the soil Streptomyces sp. CPCC 204980, showed strong cytotoxicity effects against two cancer cell lines namely pancreatic BxPC-3 and colon HCT-116, with IC50 values in the range of 0.9–801.0 nM  and hydroxyiso-9-methylstreptimidone (178), produced by the terrestrial Streptomyces sp. HS-NF-780, have shown a moderate cytotoxicity against lung, leukemia and colon cancer cell lines , identified from Streptomyces sp. KIB-H714. Unlike other Z- type actinomycens, this compound has an additional link between actinoyl chromophore and β-peptidolactone  showed a significant cytotoxicity effect against several cancer cell lines including SW480 with an IC50 value of 9.3 µM. The compound also showed an inhibitory effect on the enzyme indoleamine 2,3-dioxygenase, involved in tryptophan metabolism.Three new glycoside compounds, including ulleungoside, 2-methylaminobenzoyl 6-deoxy-α-l-talopyranoside, and naphthomycinoside, were isolated from the terrestrial actinobacter species CB13F030 . Ulleung(181), an anthracycline metabolite, was produced by the terrestrial Streptomyces sp. NEAU-L3, and its cytotoxicity was studied against three cancer cell lines of lung, liver and human colon in which showed a moderate cytotoxicity with an IC50 value of 20.82 μM on HCT-116 colon cancer cell line (Streptomyces sp. KIB-H1318 ((182) showed a weak toxicity effect against HeLa and SW480 cancer cell lines, with IC50 values of 36.8 and 37.8 µM, respectively.The new compound, tetracenoquinocin A ell line . Three nIB-H1318 . The sec(183), isolated from mangrove soil derived actinomycetes Streptomyces sp. (172614) was studied against HCT-116. It was reported the compound showed a significant toxicity with an IC50 value of 0.37 µM ((184) and Azalomycin F 2-ethylpentyl ester (5a) (185) are produced by Streptomyces sp. 211726, isolated from mangrove rhizosphere soil amino)-2″-methyl)-phenylaminocarbonylstaurosporine  0.37 µM . The twoere soil . Both coEndophytic actinobacteria are group of Gram-positive bacteria that live in symbiotic relationships inside plant tissues . This un(186) is produced by the endophyte Streptomyces sp. YIM66403, isolated from the root of Isodon eriocalyx which is a traditional medicinal plant collected in China  is a natural compound produced by the endophytic bacterium Streptomyces sp RLe8 isolated from the Brazilian plant Lychnophora ericoides that has shown significant cytotoxicity on the MDA-MB435, HCT-8, SF-295 and HL-60 cell lines with an IC50 value of 1.10 μg/ml on the HCT-8 colon cancer cell line ((188) produced by another endophytic species isolated from this plant called Streptomyces cattleya RLe 4 also showed significant cytotoxicity on these cells.2,3-dihydro-2,2-dimethyl-4 (1H) -quinazolinone ell line . AnotherStreptomyces sp KIB-H1083, isolated from the traditional medicinal plant Diaphasiastrum veitchii ((189) had a good cytotoxic effect on various cell lines such as HL-60, SMMC-7721, A-549, and MCF-7, although this compound was the weakest one against the SW480 CRC cell line with an IC50 value of 32.59 µM.Two new compounds of glycosylated piericidins and four known compounds were identified from veitchii . Among t(190–191) from the endophytic species Micromonospora lupini Lupac 08 in non-toxic doses, was able to inhibit the invasive carcinoma cells, murine colon 26-L5, with IC50 values of 0.21 and 0.3 µM, respectively  compound, was identified from Streptomyces sp. 1H-GS5, isolated from ant , have significant cytotoxic effects on various cell lines, including BGC-823 human gastric carcinoma, Caco-2 colonic adenocarcinoma, H460 lung carcinoma, and SMMC-7721 hepatocellular carcinoma cell lines, with an IC50 range of 0.54–5.02 µM.A total of 16 natural compounds of bafilomycins and odoriferous sesquiterpenoids were described from  maximus . Three c(196) was produced and identified from Salinispora sp. in genome-mining studies (50 < 0.076 μg/ml) on the HCT-116 cell line. Retimycin A, is a member of the quinomycin-like depsipeptide family. It was identified through the biosynthetic pathway of the rtm gene cluster using HR-MS/MS data, as compared to normal authentic.Retimycin A  studies . This co(197) was isolated from Streptomyces curacoi mutant strain R25 (50 = 10.5 nM) and HCT-116 colon carcinoma (IC50 = 8.6 nM).Using genome mining, a peptide called curacozole rain R25 . The sighalophilic actinomycete strain (no. HK10) found in salt ponds on Shinui Island in the Republic of Korea. The toxicity of these compounds on 6 human cancer cell lines, including colon, was investigated. The most significant toxicity was related to salternamide A (198) compound on HCT-116 colon cancer and SNU638 gastric cancer cell lines (IC50 = 0.96 µM)  . The Sub0.96 µM) .Streptomyces sp. SCSIO 1666/17C4 was obtained by inserting cosmid p17C4 into the marine derived Streptomyces sp. SCSIO 1666 ((199) and known compound l-rhodinose-l-rhodinose-l-rhodinoserhodomycinone (200), with IC50 values of 0.3 and 0.2 µM, respectively. The chemical structure of compounds 192 to 200 are illustrated in In 2018, a mutant species SIO 1666 . Cosmid In addition to producing effective bioactive secondary metabolites, actinobacteria have been reported to be one of the four main phyla of the gut microbiota, revealing another aspect of their relationship. It is evident that one of the most associated risk factors for precancerous and cancerous intestine conditions is a change in the gut microbiome and microbiota dysbiosis. The variation in the gut microbiome may generate CRC by destructing host DNA, creating and maintaining a pro-inflammatory condition, and influencing host immune responses . EvidencFirmicutes, Bacteroidetes, Actinobacteria and Proteobacteria. However, patients who are dealing with ADK, Firmicutes and Actinobacteria are significantly decreased, which can, in addition to the Lachnospiraceae family, be considered as good specific biomarkers for H, LRA and HP individuals. Also, in people with preneoplastic/neoplastic lesions, in general, a decrease in the Firmicutes/Bacteroidetes ratio can be considered as a marker for intestinal dysbiosis. Comparison between healthy people, people with inflammatory bowel disease (IBD) and those suffering from CRC have shown that the frequencies of the three phyla, Firmicutes, Bacteroidetes, and Actinobacteria, involved in metabolic pathways, are significantly different among them  and groups with hyperplastic polyps (HP), low-risk adenomas (LRA), high-risk adenomas (HRA), adenocarcinomas (ADK), and the last group included patients with ADK who received chemotherapy or radiotherapy (ADK-T) . Bacteriong them . Phyla FClostridia, Fusobacteria, Actinobacteria, and Bifidobacterium were introduced as potential biomarkers in CRC patients of different ages. Actinobacteria at the phylum and class level were more abundant in the older group than in the younger volunteers. Interestingly, the abovementioned frequency was lower in CRC patients than in both healthy groups.In a cohort study between healthy young people (below 30 years old), healthy old group (over 60 years old) and CRC patients, several bacterial species were reported to be distributed inequality in these groups . ClostriFirmicutes and Actinobacteria was observed in tumor tissue as compared to adjacent healthy tissues  were reported with the greater frequency in the group without adenoma as compared to the group of patients having adenoma, which is a common precursor for CRC, confirmed the previous results  and ecteinamycin (202) compounds produced by soil-derived Actinomadura strain, in addition to exhibiting strong antibacterial effects against gram-positive bacteria, have shown anti-invasive effect against mouse colon carcinoma cell line 26-L5  showed good cytotoxicity against 5 human cancer cell lines including colon carcinoma HCT-116 with IC50 values of 6.7, 5.8 and 8.0 µM, respectively. In the study of the effect of these compounds on adhesion, invasion and migration of SMMC7721 hepatocarcinoma cells, fusicomycin B showed a significant anti-adhesion effect and also reduced the number of migrating cells and therefore the anti-invasion and migration effect was recorded in these studies.Six new fusicoccane-type diterpenoids were identified from anoleuca . Fusicom(206), C (207), produced by Streptomyces sp. GKU 220 isolated from sea sponge were examined for biological activity , isolated from marine actinomycete Nocardiopsis, strain CNT-189 was reported to exhibit antitumor activity against colorectal cell lines in a variety of ways. The effect of this compound on three human cell lines adenocarcinoma (AGS), HCT-116 and Caco-2 showed cytotoxicity with IC50 values of 18, 21 and 13 µM, respectively . The chemical structure of these compounds is displayed in (209) and 7-deoxyechinosporin (210), produced by the marine derived S. albogriseolus A2002, collected in Jiaozhou Bay, China, were documented to have replication inhibitory effects on different types of cancer , human leukemia (HL60), human lung carcinoma (A549 and SPCA4) and liver carcinoma (BEL-7402) ((211), was isolated from the non-marine Streptomyces sp. JS520. It was conjugated to gold nanoparticles and showed lethal effects through induction of apoptosis on melanoma A375, lung carcinoma A549, breast cancer MCF-7 and colon HCT-116  . The com HCT-116 .actinomycete Z2039-2, was documented to induce apoptosis in the leukemia cell line K562 , a compound with the inhibitory activity of Ras farnesyl transfrase, from the marine Streptomyces sp. M045 inhibits the growth of Ki-ras-activated mouse fibrosarcoma (Manumycin A osarcoma . A 2000 osarcoma . Howeverosarcoma .(214) and B were isolated from Micromonospora sp. Strain TP-A0316. Arisostatin A showed a good activity against gram-positive bacteria as well as cytotoxicity and antitumor activity against myeloid leukemia cell line U937 and human colon cancer cell line HCC2998 (IC50 = 0.22M), breast, lung and brain , produced by Serinicoccus sp., an actinobacter from the family Ornithinimicrobiaceae on various cell lines, including HCT-15 colon cancer cell line , from marine actinomycetes Streptomyces sp. WBF16 were showed significant antitumor effects against several cancer cell lines, including HCT-116 and HepG2, SGC7901, A549, and COC1. In 2016, a study found that the compound chromomycin A2 exerts its antitumor effects on cancer cells by inducing autophagy through members of the TP53 family  was reported as a compound with selective toxicity against solid tumors in hypoxic conditions. Hypoxia is a hallmark of solid tumors and has been linked to the angiogenesis and related factors such as HIF-1 and VEGF in tumor progression and spread. the various cell lines, including HCT-8 and DLD-1, related to human colon cancer, were examined and this compound showed 17.5 times more toxicity in hypoxia as compared to normal conditions. In further studies, no change in HIF-1 transcriptional level was observed and the mechanism of action of this compound in hypoxic conditions remained unknown ((220–222) compounds were isolated from the marine derived Micromonospora chalcea FIM 02–523 ((223), were tested against pancreatic PANC-1 and colon HCT-8 cancer cell lines in both normal oxygen and hypoxia conditions in which they were 18.2–20.3 times more toxic in hypoxia conditions.In 1995, for the first time, the production of rakicidins A and B, from the soil ell line . In subs unknown . In 2018M 02–523 . These cStreptomyces genera.The distribution of NPs having anti-CRC activities, produced by actinobacteria based on genus is summarized in The NPs presented in this paper include a wide range of chemical compounds with unique properties. We have tried to put all of them in 20 different chemical categories. As you can see in Here are provided some examples of effective NPs on CRC cell lines with different structural features in order to express the importance of the smallest changes in the chemical structure of compounds in their cytotoxic and anticancer performance. having aromatic ring (phenyl and indole rings respectively) showed 75 and 13.6 times more cytotoxicity than lucentamycins C and D (which lack of this moieties) against HCT-116, respectively. The obvious difference in the biological function of lucentamycins, suggests the presence of aromatic rings in the structure of compounds is pivotal for their cytotoxic activity . So that have been reported as reasons for the superiority of type A in causing cytotoxicity on the HCT-116 cell line as compared to type B of the compound , and strong cytotoxic activity for staurosporine derivative No. 7 (44) and No. 14 (51), showed the importance of sugar bridge and bisindolocarbazole in those whose exhibited stronger cytotoxicity against cancer cell lines  and terpenoids ((99), the presence of the chlorinated 5H-pyrrolo  isoindol-5-one ring, which had not previously been reported in natural compounds, was a possible factor in the effect of cytotoxicity against HCT-116 cell line  in their structure can usually add and lead to increase the efficacy and properties of a compound including stability and bioactivity. Having various functions, such as the production of toxins and antibodies and other biochemical properties, are due to the presence of halometabolites in their hosts . The hig(86–91), ammosamides A (93) and B (94) and marinocyanins A-F (100–105) are types of halometabolites, produced by actinobacteria that have been reported to possess significant cytotoxicity against CRC cell lines (Compounds such as marinopyrroles A-F ll lines .(219) which is cytotoxic against various cell lines , also called adriamycin, is an Anthracyclines compound produced by nd so on . Althougnd so on .Kitasatospora sp, isolated from Taxus baccata  is a compound first isolated from soil Streptomyces hygroscopicus. Due to its inhibitory effect on the mTOR pathway, this compound and its derivatives can treat a wide range of diseases such as diabetes, tuberous sclerosis, neurodegenerative and others. Among the therapeutic effects of this compound, anti-CRC properties have also been reported (Sirolimus (Rapamycin) reported .Everolimus  is a derivative of abovementioned compound that has received FDA approval for the treatment of kidney and pancreatic cancers. The use of Everolimus, in combination with compounds such as bevacizumab and mFOLFOX-6 as combination therapy to treat metastatic colorectal cancer, has had significant effects which is in the clinical trial phase .streptomyces strain exclusively. Marine actinobacteria are predominant producers of the anti-colorectal cancer compounds (79.02%), following by terrestrial and endophytic strains counting for 11.16 and 2.68%, respectively. The rest of compounds (7.14%) have been produced by strains living in unconventional environments, synthetic strains , as they constitute the largest genus of actinobacteria. Micromonospora and Salinispora, both members of the family Micromonosporaceae, and Actinomadura, with production of 9.91%, 3.02% and 3.02% of anti-CRC compounds, respectively, appear to be other important genera in this field.As expected, most of the NPs reported in this review are produced by 50 values in ng scale). Even though the mechanism of action of most compounds was not addressed and determined by abundant of published papers, it is notable that these compounds are able to exert their antitumor properties and toxicity through various pathways. Therefore, the biological process and mechanism of actions of compounds involved in the anti-cancer effects is needed to be investigated further. In addition, further studies in this field can reveal the secret of the protective effects of actinobacteria associated with gut and colorectal cancer more clearly. The knowledge of how gut actinobacteria along with lifestyle and microenvironment factors can interfere with the development of CRC at early stage is pivotal to design biomarkers for CRC.The compounds show enormous biological activities, from autophagy, angiogenesis, invasion, migration, apoptosis, to a range of cytotoxicity, some in terms of values comparable to conventional drug compounds to very strong levels of toxicity (with ICDespite the advances in compound discovery and structure elucidation, our knowledge on structure activities relationship is still limited. Therefore, mining of structure activity relationship is another field, which requires further exploration. In addition, the molecular docking studies might indicate the role of halogen bonding in complexes and uncover the secret of structure activities relationship.Another point to note is the use of diverse CRC cell lines in previous studies, while in most of them, the reason for selecting each cell line was not specified. It is also clear that the frequency of use of these cell lines is very unequal. Based on the present evidence, the anti-CRC effect of natural actinobacterial compounds, less attention has been paid to the two groups CMS1 and CMS2 and their associated cell lines. In other words, although most of these researches are preliminary studies of the cytotoxic and antitumor effects of natural compounds, it is worth to use cell lines belonging to two or more different CMSs simultaneously, in order to consider the heterogeneity of this cancer and to comprehensively investigate the antitumor effects of a compound.


BAX and CASPASE-3. All these findings support its study as a potential anticancer therapy.To overcome some limitations of platinum-based chemotherapy agents, new active metallodrugs based on other transition metals are being researched. Runat-BI is a ruthenium-based compound which was synthesized and characterized at the University of Valencia. We investigated the in vitro effect of this compound on eight cell lines of different cancer types. Runat-BI reduced tumor growth and migration significantly in three cancer cell lines, showing selectivity for tumoral cells and little effect on non-tumoral ones. Its mechanism of action is still unknown but seems related to DNA synthesis as the cells with higher growth rates are the most affected by this chemotherapy agent. However, additional mechanism(s) likely play a role in its selectivity for several cancer cell lines. Moreover, Runat-BI slightly increases expression of the proapoptotic genes Ruthenium compounds have demonstrated promising activity in different cancer types, overcoming several limitations of platinum-based drugs, yet their global structure–activity is still under debate. We analyzed the activity of Runat-BI, a racemic Ru(III) compound, and of one of its isomers in eight tumor cell lines of breast, colon and gastric cancer as well as in a non-tumoral control. Runat-BI was prepared with 2,2’-biimidazole and dissolved in polyethylene glycol. We performed assays of time- and dose-dependent viability, migration, proliferation, and expression of pro- and antiapoptotic genes. Moreover, we studied the growth rate and cell doubling time to correlate it with the apoptotic effect of Runat-BI. As a racemic mixture, Runat-BI caused a significant reduction in the viability and migration of three cancer cell lines from colon, gastric and breast cancer, all of which displayed fast proliferation rates. This compound also demonstrated selectivity between tumor and non-tumor lines and increased proapoptotic gene expression. However, the isolated isomer did not show any effect. Racemic Runat-BI is a potential drug candidate for treatment of highly aggressive tumors. Further studies should be addressed at evaluating the role of the other isomer, for a more precise understanding of its antitumoral potential and mechanism of action. Cancer represents a major public health problem. With an estimated 19.3 million new cases worldwide in 2020, it remains among the main causes of death (10.0 million deaths) ,5,6. CurThe most widely used drugs in conventional chemotherapy are platinum-based, among which cisplatin is one of the most commonly utilized to treat various solid cancers, either as a single agent or in combination. Cisplatin exerts anticancer activity via multiple mechanisms, mainly by generating DNA damage followed by activation of several signal transduction pathways which finally lead to apoptosis . DespiteRuthenium species have demonstrated promise in different cancer types, opening the door to the design of novel metal-based chemotherapeutic agents . Those aGlobal structure–activity relationships are still under debate for ruthenium metallodrugs as regards in vitro antiproliferative/cytotoxic activity and in vivo tumor-inhibiting properties as well as pharmacokinetics . The mecThe increasing focus on and promising results shown by ruthenium-based antitumor agents led to our study and synthesis of new Ru(III)-based anticancer compounds. Among these, we selected Runat-BI to study antitumor activity, with the aim of analyzing its effect on viability, migration, cell doubling time (CDT) and expression of pro- and antiapoptotic genes in six breast cancer (BC) cell lines , a colon cancer cell line (HCT116), and a gastric cancer cell line (AGS). The MCF10A cell line from a human mammary gland was used as the non-tumoral cell line control.−1) with 25 scans and a spectral resolution of 4 cm−1 (α radiation (λ = 0.71073 Å). Crystal parameters and refinement data, along with selected bond lengths and angles, for Runat-BI are summarized in All starting chemicals and solvents used in the synthesis of Runat-BI were purchased from commercial sources and used without further purification. The elemental analyses  and X-ray microanalysis were performed through Central Support Service for Experimental Research (SCSIE) at the University of Valencia. The results of scanning electron microscopy (SEM-EDX) were obtained through a Hitachi S-4800 field emission scanning electron microscope with 20 kV of accelerating voltage and 9.0 mm of working distance. Electrospray Ionization Mass (ESI-MS) spectrum of Runat-BI was performed on a SCIEX TripleTOF 6600+ mass spectrometer by using a direct infusion electrospray ionization source . The inff 4 cm−1 . X-ray d2. Cells were seeded for 24 h before treatment with Runat-BI or PEG (control) for MTT, wound healing and CDT . Cell lines were cultured in RPMI 1640 or DMEM medium supplemented with 1% L-glutamine and 10% fetal bovine serum . The culture conditions were identical in all cell lines: 37 °C and 5% CO and CDT .50 was calculated using GraphPad Prism, with the R indicating the goodness of fit.Cell proliferation was analyzed by colorimetric MTT  assay. We seeded and cultured 3000 cells in 96-well plates. Next, cells were treated with a specific drug dose and the MTT assay was performed 24, 48 and 72 h after treatment. DMSO was used as a dissolvent to solubilize the compound and convert the formazan into a colored solution. Absorbance of this colored solution was quantified by measuring through a spectrophotometer at 590 nm. Each experiment was performed in triplicate and repeated at least twice. Average values for triplicates were calculated. Absorbance observed at different drug concentrations was compared with the respective non-treated controls and cell viability was calculated. The IC5 cells/well and incubated overnight until they reached 70% confluence. A pipette tip was used to create a wound in the cell layer. Cells were then treated with Runat-BI (21 μM). Each experiment was performed in triplicate and repeated at least twice. Images were obtained at 0, 24, 48 and 72 h at the same position and closure area of migrating cells was measured using Image J software, and the percentage of wound closure was calculated and compared with time zero. The assay is particularly suitable for studying the effects of cell–matrix and cell–cell interactions on cell migration.Cells were seeded in 6-well plates at a density of 4 × 105 cells. Duplicate counts were made in a Neubauer chamber using Automated Cell Counter TC10TM (BioRad) every 24 h for 10 days after staining cells with trypan blue. Each cell line was cultured in duplicate. From the cell count data, the cell growth curve was constructed during the time studied and the CDT was calculated using the online tool http://www.doubling-time.com/compute.php (accessed on 20 March 2020).We investigated the growth rate and CDT of the nine cell lines used in this study. On day 0, cells were plated in 6-well plates with an initial concentration of 1 × 106 total cells. RNA concentration was measured using a NanoDrop ND 2000-UV-vis Spectrophotometer . cDNA synthesis was performed using the High-Capacity cDNA Reverse Transcription kit . We used TaqMan® Gene Expression Assays . Primers used in the assay were BAX and CASPASA-3 (proapoptotic genes); BCL-2 (antiapoptotic gene) and GAPDH as endogenous control, carried out by quantitative real time-PCR (qRT-PCR) in RNA extracted from cell lines. Normalization was performed with GAPDH. The data were managed using the QuantStudio™ Design & Analysis Software v1.4 . Relative expression was calculated using the comparative Ct method and obtaining the fold-change value (ΔΔCt). Total RNA from all cell lines was isolated using High Pure RNA Isolation Kit from Roche following the manufacturer’s protocol. RNA was extracted from cellular pellet from around 1 × 10p-value < 0.05. The IC50 analysis and graph representations were performed using GraphPad Prism.All statistical analyses were performed using R Bioconductor. Results were considered significant when 3·H2O  and 2,2’-biimidazole  were reacted by means of a solvothermal synthesis in HCl  at 90 °C for 20.5 h, followed by a 20.5 h cooling process to room temperature. Dark blue crystals of Runat-BI were thus obtained and were suitable for X-ray diffraction data collection : 445.90 (4.8%), 444.93 (5.4%), 442.89 (16.4%), 441.82 (94.6%), 440.94 (41.9%), 439.97 (100%), 438.90 (62.0%), 437.93 (36.5%), 436.86 (30.6%), 435.98 (12.7%), 434.90 (3.1%), these m/z values support the stability of Runat-BI in solution  PF6  showed the best results and was selected to be studied in three BC cell lines: MDA-MB-231, MCF-7 and MCF-10A. This complex was more efficient in inhibiting proliferation, adhesion, migration and invasion in MDA-MB-231 cells than non-tumor cells (MCF10A). Nevertheless, the Ru(III) complex showed inconsistent results in MCF-7 [The compound did not show great effectiveness against HCC1500, HCC1937 and MCF-7, all belonging to BC. Furthermore, BC cell lines belonging to the TNBC subtype (HCC1806 and MDA-MB-231) and luminal B (BT474) exhibited a greater response to Runat-BI treatment than those belonging to the luminal A subtype, such as MCF-7, which showed less remarkable results in both viability and migration assays. Despite the lack of specificity of Runat-BI for a particular cancer type, a selective effect has been observed between non-tumorigenic cell lines such as MCF10A and tumorigenic cell lines. The MCF10A cell line was not affected by the agent, showing no reduction in viability or migration. Similar results in the selectivity of action in non-tumor cell lines have already been demonstrated with other ruthenium agents ,22,25,31in MCF-7 . Similarin MCF-7 ,25. Our Despite the anticancer properties found in Runat-BI, no significant results were obtained for isolated isomer 1 from this complex, which is therefore not solely responsible for its antitumor action. Work is currently underway on the isolation and purification of the second isomer to verify whether Runat-BI agent requires both isomers to exert its action or if only one is responsible for the activity.There seems to be a relationship between Runat-BI effectiveness and the growth rate of cell lines, since the fastest growing ones generally show the greatest response, probably due to the binding to DNA, which has been considered the main target of action of the agent. However, the relationship is still unclear, as Ru-based drugs seem to exert their anticancer effects through a multitarget mechanism , which mBAX and CASPASE-3 and the antiapoptotic gene BCL-2. Our results showed that Runat-BI upregulated the expression of proapoptotic genes BAX and CASPASE-3  but exerted no effect the antiapoptotic gene expression of BCL-2. This is in agreement with results obtained in previous studies on BC cell lines [We further investigated the expression of proapoptotic genes ll lines ,29. HoweRunat-BI has shown very positive in vitro results, better even than other earlier reported ruthenium systems . NeverthRuthenium-based antitumor agents such as Runat-BI are an interesting alternative to current chemotherapy agents, overcoming many disadvantages of the latter due to their specific activity against different cancers, more favorable toxicity profile and multitargeted mechanism of action. Altogether, our novel complex Runat-BI significantly reduced viability and migration in three cancer cell lines taken from colon, gastric and breast cancer, all of which displayed fast proliferation rates. This supports the effectiveness of the complex in highly proliferative and aggressive cancers that have limited available therapeutic options. Its selective action between tumor and non-tumor lines as well as its potential multitargeted effect on DNA and upregulation of proapoptotic gene expression make it a potential drug candidate for cancer treatment. The effectiveness of the racemic mixture Runat-BI is not due to the isolated isomer 1, and hence further studies should be addressed at evaluating the role of isomer 2 to more precisely elucidate the anticancer potential of Runat-BI and its mechanism of action.Oficina Española de Patentes y Marcas (OEPM) with No. P202130624. https://www.uv.es/oferta-cientifico-tecnologica/es/resultados/salud/salud-lo-largo-todo-ciclo-vital-1286222426775/OCTResultats.html?id=1286236582025 (accessed on 15 November 2022).Runat-BI has the international patent certificate PCT/ES2022/070415, Universitat de València and Fundación INCLIVA (2021): Ruthenium-biimidazole compound (RUNAT-BI) and its therapeutic use. It has been registered in


Klotho gene (kl/kl). These mice have a shortened lifespan, stunted growth, renal disease, hyperphosphatemia, hypercalcemia, vascular calcification, cardiac hypertrophy, hypertension, pulmonary disease, cognitive impairment, multi-organ atrophy and fibrosis. Overexpression of Klotho has opposite effects, extending lifespan. In humans, Klotho levels decline with age, chronic kidney disease, diabetes, Alzheimer’s disease and other conditions. Low Klotho levels correlate with an increase in the death rate from all causes. Klotho acts either as an obligate coreceptor for fibroblast growth factor 23 (FGF23), or as a soluble pleiotropic endocrine hormone (s-Klotho). It is mainly produced in the kidneys, but also in the brain, pancreas and other tissues. On renal tubular-cell membranes, it associates with FGF receptors to bind FGF23. Produced in bones, FGF23 regulates renal excretion of phosphate (phosphaturic effect) and vitamin D metabolism. Lack of Klotho or FGF23 results in hyperphosphatemia and hypervitaminosis D. With age, human renal function often deteriorates, lowering Klotho levels. This appears to promote age-related pathology. Remarkably, Klotho inhibits four pathways that have been linked to aging in various ways: Transforming growth factor β (TGF-β), insulin-like growth factor 1 (IGF-1), Wnt and NF-κB. These can induce cellular senescence, apoptosis, inflammation, immune dysfunction, fibrosis and neoplasia. Furthermore, Klotho increases cell-protective antioxidant enzymes through Nrf2 and FoxO. In accord, preclinical Klotho therapy ameliorated renal, cardiovascular, diabetes-related and neurodegenerative diseases, as well as cancer. s-Klotho protein injection was effective, but requires further investigation. Several drugs enhance circulating Klotho levels, and some cross the blood-brain barrier to potentially act in the brain. In clinical trials, increased Klotho was noted with renin-angiotensin system inhibitors , a statin (fluvastatin), mTOR inhibitors , vitamin D and pentoxifylline. In preclinical work, antidiabetic drugs  also enhanced Klotho. Several traditional medicines and/or nutraceuticals increased Klotho in rodents, including astaxanthin, curcumin, ginseng, ligustilide and resveratrol. Notably, exercise and sport activity increased Klotho. This review addresses molecular, physiological and therapeutic aspects of Klotho.The α-Klotho protein (henceforth denoted Klotho) has antiaging properties, as first observed in mice homozygous for a hypomorphic  Klotho (kl), which is an antiaging gene, and the corresponding protein α-Klotho (henceforth denoted Klotho or KL). The gene was first identified in mice in 1997  (Klotho gene (Kl−/−)   or full l−/−) . Klotho-l−/−) , 2021. Ol−/−) .Klotho insufficiency appears to play a role in human aging and, specifically, in many of the diseases that are associated with aging. Klotho expression declines with age, renal failure, diabetes and neurodegenerative disease. The age-related decline in serum levels appears to be similar in men and women; and reference values have recently been reported . Notably1) FGF23-dependent phosphate, calcium and vitamin D regulation.2) Antioxidant and anti-inflammatory activities.3) Prevention of chronic fibrosis.4) Protective effects against cardiovascular disease.5) Anti-cancer (tumor suppressor) activities.6) Metabolic regulatory functions relevant to diabetes.7) Anti-apoptotic and anti-senescence functions; stem cell preservation.8) Protection against neurodegenerative disease (Alzheimer’s and other).Klotho can exist as a membrane-bound coreceptor for fibroblast growth factor 23 (FGF23) , or a soIn addition to these topics, we will report on several factors that increase Klotho, including commonly prescribed drugs, recombinant proteins, gene therapies, traditional medicines, nutraceuticals, and exercise.2+ reabsorption. Conditional gene knockout of Klotho in the proximal tubule reproduced many of the features of systemic gene knockout, confirming the importance of proximal tubular functions , but is also found in the brain , pancreatic β cells, blood vessels and skin . Recent work also documents expression in peripheral blood circulating cells . It is aunctions .Klotho consists of a single-pass membrane protein of 1,012 aa (130 kDa), in humans, with a very short intracytoplasmic segment (10 aa) . The extThe regulation of Klotho shedding is not fully elucidated, but the role of the α-secretase ADAM10 and ADAM17 is well documented, especially in renal tubular epithelial cells . The expThe crystal structure of Klotho and its binding sites to FGF receptors  and FGF23 have been reported . No specific receptor for s-Klotho has been identified. However, it binds to some receptors that are widely distributed, on many cell types; notably FGFRs and the TGF-β receptor.Several factors regulate Klotho expression, as reviewed . This maKlotho gene expression, including those binding at promoter sites E-box, Ap-2, PAX4, Sp1 and Oct-1 . Of major physiological significance, FGF23/Klotho/FGFR1c suppresses the expression of 1α-hydroxylase in the proximal renal tubule, which inhibits synthesis of active vitamin D, denoted 1,25(OH)2D3 . Among other effects, vitamin D promotes phosphate and calcium absorption in the gut, and this will be reduced. Furthermore, in a Klotho-independent manner, FGF23 reduces secretion of parathyroid hormone (PTH), which additionally influences phosphate calcium balance  to form a high affinity receptor for FGF23  Chen G,. In term balance .A deficiency of either Klotho or FGF23 in mice results in excessive 1α-hydroxylase activity, overproduction of active vitamin D, and associated hyperphosphatemia and hypercalcemia. In Klotho-deficient mice, hypervitaminosis D and hyperphosphatemia both appear to contribute to the accelerated aging phenotype , 2021. HThe most common clinical cause of Klotho deficiency is kidney failure, which can result from many acute or chronic conditions . Rare huThe Klotho homologue denoted β-Klotho (KLB) is an obligatory coreceptor for FGF19 and FGF21, which are both eFGFs . KLB, unin vivo.A key protective function of Klotho is the prevention of fibrosis , which has been attributed in large part to its ability to block TGF-β, although other pathways contribute . This isPractically all cell types express TGF-β receptors, and its importance is considerable. TGF-β signaling and its regulation are complex , and onlThe activation of several non-canonical (non-Smad2/3 dependent) pathways adds more complexity . In someThe studies of Klotho inhibition of TGF-β mentioned above pertain primarily to renal disease, but are likely applicable to fibrosis elsewhere such as pulmonary fibrosis, and several other age-related conditions. This would be consistent with the ubiquitous expression and multiple functions of TGF-β and its receptors. TGF-β is involved in many of the lesion observed in diabetes. The role of TGF-β in neoplasia is complex and context dependent. At early stages of neoplasia it acts as a tumor suppressor, but it aggravates cancer in the late phases, and contributes to metastatic disease . TherefoInflammation is thought to be a major contributor to aging, in a process sometimes referred to as inflammaging . ChronicThe NF-κB family includes NF-κB1 (p50), NF-κB2 (p52), RelA (p65), and RelB, and the NF-κB activation pathways have been recently reviewed . Interestingly, s-Klotho added to cultures reverses the effects of Klotho knockdown, and prevents NF-κB activation . Blockade of insulin/IGF-1 pathways releases inhibition of the FOXOs, resulting in their nuclear migration and the expression of several genes encoding antioxidant enzymes, such as manganese superoxide dismutase . In acco2+ pathways. In canonical signaling, multiple Wnt ligands are able to bind to the cognate receptor, denoted Frizzled, to initiate a response. In the absence of a Wnt ligand the pathway is inactive. This is due to the constant phosphorylation of cytoplasmic β-catenin, as mediated by a protein complex, which results in its elimination by proteasomal degradation. However, following the binding of a Wnt ligand to Frizzle and coreceptor LRP, the degradation of β-catenin is terminated, and it can migrate into the nucleus to activate target genes through interactions with the T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) transcription factors. This canonical pathway regulates mainly cell proliferation, involving cyclin D1, c-myc and many other proteins; whereas the non-canonical pathways regulate cell polarity and motility . Wnt is a signaling cascade involved in embryogenesis, stem cell biology, cell fate specification, polarity, mitosis and migration, as recently reviewed . Wnt dysmotility . The proKlotho blocks Wnt activation by binding to several Wnt ligands, including Wnt1, Wnt3, Wnt4, and Wnt5a  treatment protects against type 1 diabetes (T1D) in mice . It induIn vitro, Klotho stimulated human β-cell survival, replication and insulin secretion  are hypoglycemic, despite having low insulin levels  plaques, as well as neuronal aggregation of Tau protein forming neurofibrillary tangles (NFTs) . It has One potential mechanism of disease involves deficient autophagy, which is a process that normally degrades and removes proteins and other substances, preventing their toxic accumulation in the cell. Klotho ameliorates autophagy, and this might protect against neurodegenerative conditions like Alzheimer’s . Klotho The hippocampus plays a major role in memory. It is one of the few regions of the brain that sustains neurogenesis in adulthood, and there is loss of neurons in that area in Alzheimer’s disease . RemarkaTo our knowledge, Klotho-based therapy has not been applied in humans with neurodegenerative diseases. Nevertheless, compared to subjects with low levels of Klotho, those with high levels (CSF or plasma) have superior cognitive functions, and a lower incidence of dementia . It has Klotho gene variant denoted KL-VS (2 amino acid substitutions in the protein) appears to provide some protection against Alzheimer’s disease, but only in the heterozygous state . Indeed, these are some of the few drugs that have been shown to enhance plasma Klotho in clinical trials . For insKlotho and SIRT1, which have both been linked to increased longevity  and everolimus are inhibitors of mTOR in clinical use, which have both been shown to increase Klotho . Rapamycm levels . These im levels . The incm levels . The benm levels .Several clinical anti-diabetic drugs have been shown to increase Klotho expression in mice, e.g., GLP-1RAs, DPP-4 inhibitors, metformin and peroxisome proliferator-activated receptors γ (PPAR-γ) agonists . Some wein vitro and/or in vivo, troglitazone, ciglitazone and rosiglitazone increased Klotho mRNA expression. They observed increased Klotho protein in the kidneys. A PPAR-γ antagonist had the opposite effects.PPAR-γ is a transcription factor regulating insulin sensitivity and adipogenesis, and agonistic drugs find applications in diabetes and lipid disorders. PPAR-γ agonists are another class of drugs that enhance Klotho expression . There iSee also Numerous drugs, recombinant proteins (or peptides) and gene therapies have been tested in preclinical disease models for their ability to boost Klotho levels . SpecifiRecombinant Klotho protein therapy has been successfully applied to the treatment of renal, cardiovascular and neurodegenerative disease, as well as diabetes and cancer . GeneralCommon food/diet components , and traditional medicines and/or their extracted compounds , have been examined and successfully induced Klotho . Some ofOf major interest, physical exercise and sports are simple ways to increase circulating Klotho, as reviewed by others . A singlSee also The BBB (blood-brain barrier) blocks entry of the majority of drugs into the brain, and represents a challenge for the therapy of neurodegenerative conditions, as reviewed . Some smOther drugs may be shuttled by specific transporters. Large drugs  generally do not cross, or transfer very inefficiently, but transfer can be improved by engineering constructs for receptor-mediated transport. In addition, neuropilin-1 appears to facilitate entry into the brain of some cell-penetrating peptides, larger molecules, or viruses .RAAS blocking drugs represent an interesting example, because they can be divided into BBB-crossing and non-crossing types. For example, the AT1-receptor blockers losartan, irbesartan, olemesartan and eprosartan do not penetrate through the BBB, whereas valsartan, telmisartan and candesartan do cross . Memory Some antidiabetic drugs (or at least members of these drug families) that enhance circulating Klotho levels in rodents can cross the BBB. This includes some GLP-1RAs , metformAs shown in In some cases, drugs that do not cross the BBB can ameliorate Alzheimer’s disease, or other neurodegenerative conditions, by indirect mechanisms. The improvement of hypertension, hyperlipidemia, diabetes or renal function can all have a major impact on the brain. In young and aged mice, systemic delivery of a Klotho-derived peptide that does not cross the BBB improved cognition and reduced neurodegenerative pathology . Others Klotho or FGF23 in humans result in a hyperphosphatemic syndrome, characterized by massive tissue calcification and systemic inflammation. In chronic renal disease of various etiology, as well as diabetes, Klotho production is decreased. It has been proposed that declining renal function, and associated low Klotho, accelerate or aggravate many of the pathologies found in old age. This may involve, for example, the precipitation of CPPs in tissue , or a therapeutic molecule. In particular, the function(s) of Klotho in the brain are not well defined, but neuroprotective effects are apparent. Klotho levels in the circulation or CSF could indicate susceptibility to disease. Multiple drugs appear to increase Klotho, but studies in humans are scant. Does Klotho contribute to the therapeutic effects of some drugs used to treat hypertension, hyperlipidemia, diabetes or transplant rejection? Could some of these drugs ameliorate neurodegenerative diseases by enhancing Klotho? This is all unknown. Indeed, there have been practically no clinical investigations of Klotho-related treatment, outside of preclinical work. Another important aspect is the role of Klotho as a tumor suppressor molecule. This is well documented in animals models, but needs to be developed in the clinical context. The slim clinical applications of Klotho research can perhaps be explained by the limited understanding of its mechanisms of action, especially outside the kidney. However, knowledge of the molecular and physiological aspects of Klotho has progressed considerably in recent years, as outlined in this review.For therapy, several approaches are feasible, as described in this manuscript. Clinical research focused on any of these approaches would be an important contribution to the field. The most direct approach is the administration of recombinant Klotho or peptides. In view of the relatively short half-life of these products, there is a need to produce long-acting constructs, or slow-release formulations. There is little evidence from preclinical work that Klotho administration is toxic, at least when restoring normal levels. However, the long-term effects of supra-normal levels are not known. Gene therapy is also feasible, but much more challenging. In addition to some clinical prescription drugs, there are also commonly available over-the-counter medicines, supplements, or nutraceuticals that increase Klotho. This is based on animal work, and it would be of much interest to examine the possibility of Klotho enhancement in humans. In the case of neurodegenerative diseases it is relevant that some drugs, including some traditional medicines, cross the BBB and might increase Klotho in the brain. Finally, exercise and the long-term maintenance of fitness are simple methods to increase Klotho. In conclusion, most of the studies on Klotho have been performed in animal disease models, and a considerable amount of work has to be performed to introduce Klotho therapy into the clinic. This review provides a framework of mechanisms and findings that favor the clinical investigation of Klotho-based treatments.


The medical application of cannabidiol (CBD) has been gathering increasing attention in recent years. This non-psychotropic cannabis-derived compound possesses antiepileptic, antipsychotic, anti-inflammatory and anxiolytic properties. Recent studies report that it also exerts antineoplastic effects in multiple types of cancers, including melanoma. In this in vitro study we tried to reveal the anticancer properties of CBD in malignant melanoma cell lines  administered alone, as well as in combination with mitoxantrone (MTX) or cisplatin (CDDP). The effects of CBD on the viability of melanoma cells were measured by the MTT assay; cytotoxicity was determined in the LDH test and proliferation in the BrdU test. Moreover, the safety of CBD was tested in human keratinocytes (HaCaT) in LDH and MTT tests. Results indicate that CBD reduces the viability and proliferation of melanoma-malignant cells and exerts additive interactions with MTX. Unfortunately, CBD produced antagonistic interaction when combined with CDDP. CBD does not cause significant cytotoxicity in HaCaT cell line. In conclusion, CBD may be considered as a part of melanoma multi-drug therapy when combined with MTX. A special attention should be paid to the combination of CBD with CDDP due to the antagonistic interaction observed in the studied malignant melanoma cell lines. Melanoma is a malignant tumor of pigment-producing cells, usually localized in the skin. The incidence rates of this cancer remain high and still increase, mostly in elderly patients ,2. AlthoCannabis sativa, which interact with the mammalian endocannabinoid system [Cannabidiol (CBD) is one of the 120 known phytocannabinoids, i.e., compounds extracted from the plant d system . In reced system ,10. Numed system ,12.CBD has been successfully implemented in patients with various cancers, usually in metastatic stages, e.g., breast, prostate, oesophageal, with spectacular anti-tumor effects in patients with glioma. However, clinical evidence is limited to case reports ,13. PrecTaking into account the facts that CBD can be effective in the treatment of various cancers and is efficacious in some skin disorders, we attempted to establish if CBD reduces the viability of various human melanoma cell lines  and if CBD can be considered as a part of multi-drug chemotherapy when combined with two chemotherapeutic drugs commonly used in experimental in vitro studies, i.e., cisplatin (CDDP) and mitoxantrone (MTX).Cisplatin (CDDP) is one of the most potent antitumor agents whose cytotoxic activity is mediated by its interaction with DNA to form DNA adducts, which activate several signal transduction pathways , leading finally to cell apoptosis ,37,38,39Due to the various molecular mechanisms of the action of the studied drugs , it is expected that the two-drug combinations of CBD with CDDP or CBD with MTX should exert synergistic interactions in terms of the anti-proliferative effects of these drugs in various human melanoma cell lines . The assessment of the interaction between CBD and CDDP or CBD and MTX was performed by means of the isobolographic analysis, which is considered to be a gold standard in evaluation of types of drug–drug interaction in cancer studies ,46,47.50) values for CBD, CDDP and MTX in various melanoma cell lines are presented in Cannabidiol (CBD), cisplatin (CDDP) and mitoxantrone (MTX) reduced the viability of various human melanoma cell lines  in a concentration-dependent manner, when applied separately , Table 1The following step was the quantification of the cytotoxicity of CBD in malignant melanoma cell lines and normal keratinocytes in the LDH test. The diagrams show that the cytotoxicity of CBD in various malignant melanoma cell lines grows in a concentration-dependent manner, while CBD has no significant impact on normal human keratinocytes .Next, the BrdU test was performed to determine the effect of CBD on the proliferation of malignant melanoma cells  and normal human keratinocytes (HaCaT). As presented below, CBD inhibited the proliferation of all tested melanoma cell lines, where the effect was the strongest in the cell line obtained from a primary tumor—the FM55P cell line . CBD in 50 values. The obtained results presented the concentration-dependent reduction in cancer cell viability  from the IC50 add values  from the IC50 add values  from the IC50 add values , and the most resistant was SK-MEL 28 cell line (IC50 = 7.75 ± 0.29 µg/mL). Similar results were obtained by Burch et al. in B16 mice melanoma cells, however, the range of used CBD concentrations was much higher, up to 200 µg/mL [Our study shows that CBD reduces melanoma cells viability in concentration-dependent manner. The most vulnerable cell line to the effects of CBD was the melanoma cell line obtained from primary tumor—FM55P (IC00 µg/mL . Simmerm00 µg/mL .It should be stressed that in this study we determined the anti-proliferative effects of CBD and CDDP and MTX in two closely related cell lines—primary-derived melanoma (FM55P) and metastatic-derived melanoma (FM55M2). The difference between these two cell lines was reported earlier, documenting various (quite different) types of interactions when combined naturally occurring coumarins with CDDP . It seemOn the other hand, the combination of CBD and MTX produces additive interactions, with the most potent effect in FM55P cell line. These results stand in line with data obtained from the study performed on canine urothelial carcinoma cells, where CBD presented synergistic effects with MTX . The potIn LDH test we found that CBD reduces HaCaT cells viability in concentrations higher than those needed to significantly reduce the viability of melanoma cell lines. Other authors presented that CBD did not affect the viability of HaCaT cells in concentrations up to 20 μM (6.29 µg/mL) during exposition up to 24 h . Our expThe majority of currently available drugs contain CBD combined with THC in various dose ratios. The study of Armstrong et al. investigated in vitro the effectiveness of CBD administered together with THC in equal ratio, in doses of 0.5 μM up to 2.5 μM in SK-MEL 28, A375 and CHL-1 cell lines . Co-admiCBD can potentially be used in phytoradiotherapy, where it enhances the therapeutic efficacy of radiotherapy and mitigate its side effects ,58. Howe50 add values with statistical comparison between theoretically calculated IC50 add values (accepted as additive) with experimentally derived IC50 mix values, respectively. Details concerning the isobolographic analysis have been published elsewhere [Isobolographic analysis is the method of choice allowing precisely classifying the type of interactions observed experimentally between two or more drugs used in mixture ,60,61,62lsewhere ,64,65,662. The cells were grown to 80% confluence.Primary (FM55P) and metastatic (FM55M2) malignant melanoma cells were purchased from European Collection of Cell Cultures (ECACC) and cultured in RPMI—1640 Medium . Another two cell lines A375  and SK-MEL 28  were purchased from the American Type Culture Collection (ATCC) and cultured in Dulbecco’s Modified Eagle’s Medium—high glucose (DMEM) (Sigma-Aldrich) and Eagle’s minimal essential medium (EMEM), respectively. All culture media were supplemented with 10% Fetal Bovine Serum  and 1% of penicillin/streptomycin (Sigma-Aldrich). Cultures were kept at 37 °C in a humidified atmosphere of 95% air and 5% CO2+ and Mg2+. Mitoxantrone (MTX—Sigma-Aldrich) was dissolved in DMSO as stock solutions. The cannabidiol  was dissolved in ethanol as stock solutions in concentration of 5 mg/mL. The drugs were dissolved to the respective concentrations with culture medium before use.Cisplatin (CDDP—Sigma-Aldrich) was dissolved in phosphate buffered saline (PBS) with Ca4 cells/mL, 3 × 104 cells/mL, 2 × 104 cells/mL and 2 × 104 cells/mL, respectively. Next day, the culture medium was removed and cells were exposed to serial dilutions of CBD, CDDP and MTX in fresh culture medium. Cell viability was assessed after 72 h by means of MTT test, in which the yellow tetrazolium salt (MTT) is metabolized by viable cells to purple formazan crystals. Cells were incubated for 3 h in the MTT solution . Formazan crystals were solubilized overnight in sodium dodecyl sulfate (SDS) buffer (10% SDS in 0.01 N HCl) and the product was determined spectrophotometrically by measuring absorbance at 570 nm wavelength using microplate spectrophotometer . Each treatment was performed in triplicate and each experiment was repeated 3 times.SK-MEL 28, A375, FM55P and FM55M2 cells were placed on 96-well plates  at a density of 2 × 104/mL) SK-MEL 28 (3 × 104/mL), FM55P (2 × 104/mL), FM55M2 (2 × 104/mL) cells were placed on a 96-well plate (Nunc) (100 μL/well). On the next day, the cancer cells were treated with increased concentrations of CBD for 48 h, followed by 10 µL/well BrdU Labeling Solution (100 µM) was added and cells were reincubated for additional 24 h at 37 °C. Then, the culture medium was removed and cells were fixed in FixDenat solution (200 µL/well) . The working solution of anti-BrdU antibody coupled with horseradish peroxidase (anti-BrdU-POD) were subsequently added (100 µL/well)  and detected using tetramethylobenzidine substrate (TMB) (100 µL/well) . A total of 1 M sulfuric acid was added (25 µL/well) to stop enzymatic reaction, and quantitation was performed spectrophotometrically at 450 nm using microplate spectrophotometer .Cell Proliferation Elisa, BrdU Kit  was used following manufacturer’s instructions. Optimized amounts of A375 (2 × 104/mL) SK-MEL 28 (3 × 104/mL), FM55P (2 × 104/mL), FM55M2 (2 × 104/mL) and normal human keratinocytes HaCaT (1 × 104/mL) cells were placed on 96-well plates (Nunc). Next day, cells were washed in PBS, and then exposed to increasing concentrations of CBD in fresh culture medium. The cytotoxicity was estimated based on the measurement of cytoplasmic lactate dehydrogenase (LDH) activity released from damaged cells after 72 h exposure to CBD. LDH assay was performed according to manufacturer’s instruction (Cytotoxicity Detection KitPLUS LDH) (Roche). Briefly, 50 µL of cell medium was collected from each well, then 50 µL of reaction mixture (freshly prepared) was added and incubated for 30 min at RT. Finally, 25 µL of Stop solution was added to each well on the 96-well plate. Absorbance was measured at two different wavelengths, one being the “measurement wavelength” (492 nm) and the other “reference wavelength” (690 nm) using microplate spectrophotometer . Maximum LDH release (positive control) was achieved by the addition of Lysis buffer to untreated control cells. The average values of the culture medium background were subtracted from all values of experimental wells and the percentage of death cells was calculated in relation to the maximum LDH release.Optimized amounts of A375  of CBD and CDDP, MTX were calculated [50 values, as recommended earlier [50 add) for the mixture of CBD with CDDP or MTX, which theoretically should inhibit 50% of cell viability, were calculated as demonstrated earlier [50 mix at the fixed ratio of 1:1 was based on the concentration of the mixture of CBD and CDDP or MTX that inhibited 50% of cell viability in melanoma cell lines measured in vitro by the MTT assay. In this study, the experimental evaluation of interaction between two drugs in mixture was performed when the drugs were applied in a constant fixed ratio combination of 1:1 [50 value) so as to determine the effects exerted by the drugs in mixture. If one of the studied drugs is inactive in this experimental approach, the type II isobolographic analysis must be conducted, which considerably differs from the type I isobolographic analysis [Log-probit analysis was used to determine the percentage of inhibition of cell viability per concentration of CBD, CDDP and MTX when administered singly in the A375, SK-MEL 28, FM55P and FM55M2 cell lines measured in vitro by the MTT assay. Subsequently, from the log-probit concentration–response lines, median inhibitory concentrations . The IC50 and IC50 mix values for CBD and CDDP or MTX administered alone or in combination at the fixed ratio of 1:1 were calculated by computer-assisted log-probit analysis [50 mix values for the mixture of CBD with CDDP and CBD with MTX were statistically compared with their respective theoretical additive IC50 add values by the use of unpaired Student’s t-test with Welch correction, as described earlier [GraphPad Prism 8.0 Statistic Software was used for statistical analysis. One-way analysis of variance (ANOVA test) for multiple comparisons followed by Tukey’s significance test was used. Data are expressed as the mean ± standard error (SEM)  responsible for such antagonistic interactions in various human malignant melanoma cell lines.