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Sea cucumber triterpene glycosides are a class of secondary metabolites that possess distinctive chemical structures and exhibit a variety of biological and pharmacological activities. The application of MS-based approaches for the study of triterpene glycosides allows rapid evaluation of the structural diversity of metabolites in complex mixtures. However, the identification of the detected triterpene glycosides can be challenging. The objective of this study is to establish the first spectral library containing the mass spectra of sea cucumber triterpene glycosides using ultraperformance liquid chromatography-quadrupole time-of-flight mass spectrometry. The library contains the electrospray ionization tandem mass spectra and retention times of 191 triterpene glycosides previously isolated from 15 sea cucumber species and one starfish at the Laboratory of the Chemistry of Marine Natural Products of the G.B. Elyakov Pacific Institute of Bioorganic Chemistry. In addition, the chromatographic behavior and some structure-related neutral losses in tandem MS are discussed. The obtained data will accelerate the accurate dereplication of known triterpene glycosides and the annotation of novel compounds, as we demonstrated by the processing of LC-MS/MS data of Eupentacta fraudatrix extract.

Two new natural compounds, sulfated polyhydroxysteroid, microdiscusol G (1), and polyhydroxysteroid bioside, microdiscusoside A (2), along with eight previously known polar steroid substances 3-10, were isolated from the Arctic starfish Asterias microdiscus. The structures of 1 and 2 have been elucidated by extensive 1D and 2D NMR spectroscopy and HRESIMS techniques. The 28-sulfooxy-24-methylcholestane side chain in 1 has been found among starfish steroid metabolites for the first time. Steroid saponins 9 and 10 exhibited cytotoxic effects against normal cells JB6 Cl41 and cancer cells HT-29, MDA-MB-231, THP-1, and Raji and effectively suppressed cell proliferation and colony formation of cancer cells HT-29 and MDA-MB-231 in non-toxic concentrations. Compounds 5, 7, and 8 were inactive or less active in the same experiments.

New asterosaponin, acanthaglycoside G (1), along with three previously known steroidal oligoglycosides (2‒4), were isolated from the ethanolic extract of the starfish Acanthaster planci, collected off the coast of Vietnam. The structure of 1 was mainly elucidated by extensive NMR and ESIMS techniques as sodium 6-O-{β-D-fucopyranosyl-(1→2)-β-D-quinovopyranosyl-(1→4)-[β-D-quinovopyranosyl-(1→2)]-β-D-quinovopyranosyl-(1→3)-β-D-quinovopyranosyl}-6α-hydroxy-5α-pregn-9(11)-en-20-one-3β-yl sulfate. Compounds 3 and 4 showed slight cytotoxic activities against cancer RPMI-7951, HT-29, and MDA-MB-231 cell lines, but effectively inhibited in non-toxic concentrations colony formation of HT-29 and MDA-MB-231 cells and cell migration of MDA-MB-231 cells. Compounds 1 and 2 were inactive or less active, respectively.

Nanoflow liquid chromatography coupled with a captive spray ionization time-of-flight tandem mass spectrometer (nLC/CSI-QTOF-MS/MS) was used in the structural determination of polar steroid compounds of starfish Lethasterias fusca. A total of 207 compounds including 106 asterosaponins, 81 glycosides of polyhydroxysteroids, and 14 polyhydroxylated steroids were detected and characterized by MS and MS/MS. Twenty compounds among them were unambiguously identified using authentic standard compounds, isolated earlier from this and other starfish species. The other compounds were tentatively characterized by accurate mass measurement and comparing retention times and characteristic MS/MS fragmentation patterns with reference standards. Moreover, fragmentation behaviors of a series of pure standards of starfish polar steroids and polyhydroxysteroid compounds detected in L. fusca have been extensively investigated and characteristic fragmentation pathways were described and used for the characterization of unknown compounds.

This study was aimed to determine the efficacy of combination of fucoidan from the brown algae Fucus evanescens (FeF) or its derivatives with thornasteroside A (ThA) or asteropsiside A (AsA) from the starfish Asteropsis carinifera in combating human melanoma cells. In vitro MTS and soft agar methods were performed to determine effect of FeF, its derivatives, ThA, AsA or their combination on proliferation and colony formation of SK-MEL-28 cells in 2D and 3D culture. Desulfation of FeF, but not deacetylation, led decreasing of its Mw and anti-proliferative activity. The combinatorial effect of FeF with ThA and AsA depended on the sequences of treatment by compounds. There was additive anticancer effect of FeF with ThA or AsA during simultaneous treatment of cells. ThA and AsA were not active against SK-MEL-28 cells after their pre-treatment with FeF. Potential synergism of action was identified only when SK-MEL-28 cells were pre-treated with ThA and AsA and then by FeF. This process going through the regulation of MEK1/2/ERK1/2/MSK1 pathway and expression of the cell cycle proteins as determined by Western Blot. Thus, the combination of fucoidan with the asterosaponins opens up the prospects for the development of effective combined chemotherapeutic methods for melanoma treatment.

Three new triterpene glycosides, pacificusosides A–C (1–3), and three previously known triterpene glycosides, cucumariosides C1 (4), C2 (5), and A10 (6), were isolated from the alcoholic extract of the Far Eastern starfish Solaster pacificus. The structures of 1–3 were elucidated by extensive NMR and ESIMS techniques and chemical transformations. Compound 1 has a novel, unique structure, containing an aldehyde group of side chains in its triterpene aglycon. This structural fragment has not previously been found in the sea cucumber triterpene glycosides or starfish steroidal glycosides. Probably, pacificusoside A (1) is a product of the metabolism of the glycoside obtained through dietary means from a sea cucumber in the starfish. Another two new triterpene glycosides (2, 3) have closely related characteristics to sea cucumber glycosides. The cytotoxicity of compounds 1–6 was tested against human embryonic kidney HEK 293 cells, colorectal carcinoma HT-29 cells, melanoma RPMI-7951 cells, and breast cancer MDA-MB-231 cells using MTS assay. Compounds 4–6 revealed the highest cytotoxic activity against the tested cell lines, while the other investigated compounds had moderate or slight cytotoxicity. The cytotoxic effects of 2–6 were reduced by cholesterol like the similar effects of the previously investigated individual triterpene glycosides. Compounds 3, 4, and 5 almost completely suppressed the colony formation of the HT-29, RPMI-7951, and MDA-MB-231 cells at a nontoxic concentration of 0.5 µM.

Asterosaponins are a class of steroid oligoglycosides isolated from starfish with characteristic structures and diverse biological activities. In this review, we have attempted to combine the most important data concerning asterosaponins and give a list of these secondary metabolites with their structural peculiarities. The purpose of this review is to provide a brief but as complete as possible principal information about their chemical structures, taxonomic distribution in the marine environment, distribution in different geographical areas and depths, some properties, biological activities, and functions. Some other rare steroid metabolites from starfish, closely related in structures and probably biogenesis to asterosaponins, are also discussed.

Two new natural compounds, sulfated polyhydroxysteroid, microdiscusol G (

Four new conjugates, esters of polyhydroxysteroids with long-chain fatty acids (1&ndash, 4), were isolated from the deep-water Far Eastern starfish Ceramaster patagonicus. The structures of 1&ndash, 4 were established by NMR and ESIMS techniques as well as chemical transformations. Unusual compounds 1&ndash, 4 contain the same 5&alpha, cholestane-3&beta, 6&beta, 15&alpha, 16&beta, 26-pentahydroxysteroidal moiety and differ from each other in the fatty acid units: 5'Z,11'Z-octadecadienoic (1), 11'Z-octadecenoic (2), 5'Z,11'Z-eicosadienoic (3), and 7'Z-eicosenoic (4) acids. Previously, only one such steroid conjugate with a fatty acid was known from starfish. After 72 h of cell incubation, using MTS assay it was found that the concentrations of compounds 1, 2, and 3 that caused 50% inhibition of growth (IC50) of JB6 Cl41 cells were 81, 40, and 79 &micro, M, respectively, for MDA-MB-231 cells, IC50 of compounds 1, 2, and 3 were 74, 33, and 73 &micro, for HCT 116 cells, IC50 of compounds 1, 2, and 3 were 73, 31, and 71 &micro, M, respectively. Compound 4 was non-toxic against tested cell lines even in three days of treatment. Compound 2 (20 &micro, M) suppressed colony formation and migration of MDA-MB-231 and HCT 116 cells.

Colorectal cancer is one of the most frequent types of malignancy in the world. The search for new approaches of increasing the efficacy of cancer therapy is relevant. This work was aimed to study individual, combined anticancer effects, and molecular mechanism of action of sulfated laminaran AaLs of the brown alga Alaria angusta and protolinckiosides A (PL1), B (PL2), and linckoside L1 (L1) of the starfish Protoreaster&nbsp, lincki using a 3D cell culture model. The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), soft agar, 3D spheroids invasion, and Western blotting assays were performed to determine the effect and mechanism of the action of investigated compounds or their combinations on proliferation, colony formation, and the invasion of 3D HCT 116 spheroids. AaLs, PL1, PL2, and L1 individually inhibited viability, colony growth, and the invasion of 3D HCT 116 spheroids in a variable degree with greater activity of linckoside L1. AaLs in combination with L1 exerted synergism of a combined anticancer effect through the inactivation of protein kinase B (AKT) kinase and, consequently, the induction of apoptosis via the regulation of proapoptotic/antiapoptotic proteins balance. The obtained data about the efficacy of the combined anticancer effect of a laminaran derivative of brown algae and polyhydroxysteroid glycosides of starfish open up prospects for the development of new therapeutic approaches for colorectal cancer treatment.

Two new sulfated steroidal glycosides were isolated from the EtOH extract of the tropical starfish Pentaceraster regulus. Regulusoside S1 was proposed to be the sodium salt of (24R)-3-O-(2-O-methyl-β-D-xylopyranosyl)-24-ethyl-5α-cholestane-3β,6α,8,15α,16β,29-hexaol 16-O-sulfate. Regulusoside S2 was its analog and contained an additional β-D-xylopyranose residue in a side chain on aglycon C-29. The structures of the isolated compounds were established by 2D NMR spectroscopy and tandem mass spectrometry with electrospray ionization (ESI).

Glycoconjugated and other polar steroids of starfish have unique chemical structures and show a broad spectrum of biological activities. However, their biological functions remain not well established. Possible biological roles of these metabolites might be indicated by the studies on their distribution in the organism&ndash, producer. In order to investigate the localization of polar steroids in body components of the Far Eastern starfish Lethasterias fusca, chemical constituents of body walls, gonads, stomach, pyloric caeca, and coelomic fluid were studied by nanoflow liquid chromatography/mass spectrometry with captive spray ionization (nLC/CSI&ndash, QTOF&ndash, MS). It has been shown that the levels of polar steroids in the studied body components are qualitatively and quantitatively different. Generally, the obtained data confirmed earlier made assumptions about the digestive function of polyhydroxysteroids and protective role of asterosaponins. The highest level of polar steroids was found in the stomach. Asterosaponins were found in all body components, the main portion of free polyhydroxysteroids and related glycosides were located in the pyloric caeca. In addition, a great inter-individual variability was found in the content of most polar steroids, which may be associated with the peculiarities in their individual physiologic status.

Six new polyhydroxylated steroids conjugated with taurine, microdiscusols A-F, were isolated from the alcoholic extract of the Arctic starfish Asterias microdiscus. Three of them have been found to have additional sulfate groups in tetracyclic cores. The structures of new compounds were established by 1D and 2D NMR and HRESIMS techniques. The found steroid taurine conjugates resemble, by their structures, salts of bile acids and alcohols of lower vertebrates.

A new asterosaponin aphelasteroside F (1) was isolated from the ethanolic extract of the Far Eastern starfish Aphelasterias japonica along with the previously known ophidianoside F (2). The structure of 1 was elucidated by extensive use of NMR and ESI-MS techniques, and chemical transformations. Compound 1 contains a new type of asterosaponin carbohydrate chain linked to C-6 of the steroid aglycone aphelaketotriol. Compounds 1 and 2 at non-toxic concentrations slightly inhibited cell proliferation and colony formation of the cancer melanoma cell lines SK-Mel-28, SK-Mel-5, and RPMI-7951.

Two polar steroid compounds, taurochenodeoxycholic acid sodium salt (1) and a rare cyclic steroid glycoside luzonicoside A (2), were isolated from the tropical starfish Leiaster sp. and identified by extensive NMR and ESIMS techniques. The isolation of primary bile acid 1 is the first report from a representative of the class Asteroidea and on the whole in invertebrates. Its presence confirms the hypothesis about the digestive role of some polar steroids in starfish and possibly demonstrates the parallel evolution of fat emulsifying agents in vertebrates and some starfish. Compound 2 was also obtained from starfish belonging to this genus for the first time. This finding indicates that cyclic steroid glycosides are more widely distributed in starfish than the two species of the genus Echinaster from which they were isolated earlier.

Three new polyhydroxysteroid glycosides, regulusosides A-C (1-3) along with the previously known five steroid compounds (4-8) were isolated from the tropical starfish Pentaceraster regulus. The structures of compounds 1-3 were established by extensive NMR and ESI-MS techniques. New glycosides have the same aglycon, (24S)-5α-cholestane-3β,5,6β,8,15α,24-hexol, and various disaccharide moieties: β-D-xylopyranosyl-(l-2)-3-0-methyl-α-L- arabinofuranosyl (1), β-D-xylopyranosyl-(l-2)-c-L-arabinofuranosy (2), and 2-0-methyl-β-D-xylopyranosyl-(-2)-3-0-methyl-α-L-arabinofuranosy (3), attached at C-24 of the side chain. Compounds 1, 2 and 6 exhibit potential immunomodulatory properties, inducing intracellular ROS (reactive oxygen species) formation in the RAW 264.7 murine macrophage cells.

Four new steroidal glycosides, protolinckiosides A - D (1 - 4, resp.), were isolated along with four previously known glycosides, 5 - 8, from the MeOH/EtOH extract of the starfish Protoreaster lincki. The structures of 1 - 4 were elucidated by extensive NMR and ESI-MS techniques as (3β,4β,5α,6β,7α,15α,16β,25S)-4,6,7,8,15,16,26-heptahydroxycholestan-3-yl 2-O-methyl-β-d-xylopyranoside (1), (3β,5α,6β,15α,24S)-3,5,6,8,15-pentahydroxycholestan-24-yl α-l-arabinofuranoside (2), sodium (3β,6β,15α,16β,24R)-29-(β-d-galactofuranosyloxy)-6,8,16-trihydroxy-3-[(2-O-methyl-β-d-xylopyranosyl)oxy]stigmast-4-en-15-yl sulfate (3), and sodium (3β,6β,15α,16β,22E,24R)-28-(β-d-galactofuranosyloxy)-6,8,16-trihydroxy-3-[(2-O-methyl-β-d-xylopyranosyl)oxy]ergosta-4,22-dien-15-yl sulfate (4). The unsubstituted β-d-galactofuranose residue at C(28) or C(29) of the side chains was found in starfish steroidal glycosides for the first time. Compounds 1 - 4 significantly decreased the intracellular reactive oxygen species (ROS) content in RAW 264.7 murine macrophages at induction by proinflammatory endotoxic lipopolysaccharide (LPS) from E. coli.

Four new polyhydroxylated steroids 1&ndash, 4 were isolated along with two previously known related steroids 5 and 6 from the methanolic extract of the starfish Anthenoides laevigatus collected off the coastal waters of Vietnam. Structures of new compounds were substantially elucidated by one-dimensional (1D) and two-dimensional (2D) NMR spectroscopy and HRESIMS techniques. Heptaol 1 and hexaol 2 contain the common 5&alpha, cholestane skeleton, while hexaol 3 and heptaol 4 have the rare among starfish steroid compounds 5&beta, cholestane skeleton. Compounds 1, 5, and 6 do not show cytotoxic effects against normal JB6 Cl41 and cancer HT-29 and MDA-MB-231 cells, however they inhibit cell proliferation and colony formation of cancer HT-29 and MDA-MB-231 cells.

New marine glycoconjugates&mdash, the steroidal glycosides designated as anthenosides V&ndash, X (1&ndash, 3)&mdash, and the seven previously known anthenosides E (4), G (5), J (6), K (7), S1 (8), S4 (9), and S6 (10) were isolated from the extract of the tropical starfish Anthenea aspera. The structures of 1&ndash, 3 were elucidated by extensive NMR and ESIMS techniques. Glycoside 1 contains a rare 5&alpha, cholest-8(14)-ene-3&alpha, 7&beta, 16&alpha, hydroxysteroidal nucleus. Compounds 2 and 3 were isolated as inseparable mixtures of epimers. All investigated compounds (1&ndash, 10) at nontoxic concentrations inhibited colony formation of human melanoma RPMI-7951, breast cancer T-47D, and colorectal carcinoma HT-29 cells to a variable degree. The mixture of 6 and 7 possessed significant anticancer activity and induced apoptosis of HT-29 cells. The molecular mechanism of the proapoptotic action of this mixture was shown to be associated with the regulation of anti- and proapoptotic protein expression followed by the activation of initiator and effector caspases.

Two new steroid glycosides, granulatosides D (

Two new steroid glycosides, granulatosides D (1) and E (2), belonging to the group of bi- and monoglycosides of polyhydroxysteroids, respectively, were isolated from the ethanolic extract of the starfish Choriaster granulatus along with thirteen previously known glycosides of polyhydroxysteroids (3–15) and one steroid heptaol (16). The structures of the new compounds were elucidated by extensive NMR and ESIMS techniques. Cytotoxic and immunomodulatory activities of compounds 1, 3–8, and 10–16 using murine splenocytes and peritoneal macrophages were studied. At a dose of 0.1 μM new glycoside 1 showed immunomodulatory properties, increasing the intracellular ROS (reactive oxygen species) level in peritoneal murine macrophages by 20% and decreasing intracellular ROS level by 21% in pre-treated with endotoxic lipopolysaccharide from E. coli (LPS) peritoneal macrophages.

Six new polyhydroxysteroidal glycosides, anthenosides S1 - S6 (1 - 6), along with a mixture of two previously known related glycosides, 7 and 8, were isolated from the methanolic extract of the starfish Anthenea sibogae. The structures of 1 - 6 were established by NMR and HR-ESI-MS techniques as well as by chemical transformations. All new compounds have a 5α-cholest-8(14)-ene-3α,6β,7β,16α-tetrahydroxysteroidal nucleus and differ from majority of starfish glycosides in positions of carbohydrate moieties at C(7) and C(16) (1 - 4, 6) or only at C(16) (5). The 4-O-methyl-β-d-glucopyranose residue (2) and Δ24 -cholestane side chain (3) have not been found earlier in the starfish steroidal glycosides. The mixture of 7 and 8 slightly inhibited the proliferation of human breast cancer T-47D cells and decreased the colony size in the colony formation assay.

Three new sulfated steroid monoglycosides, leptaochotensosides A–C (1–3), and a new sulfated polyhydroxylated steroid (4) were isolated from the alcoholic extract of the Far Eastern starfish Leptasterias ochotensis. The structures of compounds 1–4 were established by extensive nuclear magnetic resonance (NMR) and electrospray ionization mass spectrometry (ESIMS) analyses and chemical transformations. Although the isolated compounds did not show any apparent cytotoxicity against melanoma RPMI-7951 and breast cancer T-47D cell lines, leptaochotensoside A (1) demonstrated inhibition of T-47D cell colony formation in a soft agar clonogenic assay at nontoxic doses. In addition, this compound decreased the epidermal growth factor (EGF)-induced colony formation of mouse epidermal JB6 Cl41 cells. The cancer preventive action of 1 is realized through regulation of mitogen-activated protein kinase (MAPK) signaling pathway.

Seven new asterosaponins, pentaregulosides A–G (1–7), including two furostane-type steroid oligoglycosides (2, 3), along with four previously known compounds (8–11) were isolated from the ethanolic extract of the starfish Pentaceraster regulus, collected off the coast of Vietnam. The structures of 1–7 were elucidated by extensive NMR and ESIMS techniques as well as chemical transformations. The aglycons of compounds 1 and 3 have not previously been observed in starfish steroid oligoglycosides, while the aglycons of compounds 2 and 4–6 are very rare for this structural group. Compound 1 exhibited cytotoxic activity with an IC50 value of 6.4 ± 0.3 μM against RAW 264.7 murine macrophages. In contrast, nontoxic asterosaponins 3, 4, and 5 showed a potential immunomodulatory action at a concentration of 5 μM, reducing by 40%, 28%, and 55%, respectively, reactive oxygen species formation in the RAW 264.7 cells, co-stimulated with the pro-inflammatory endotoxic lipopolysaccharide from E. coli.

A new steroidal glycoside called planciside D was isolated from the tropical starfish Acanthaster planci and assigned the structure (24S)-3-O-(2-O-methyl-β-D-xylopyranosyl)-28-O-[α-L-fucopyranosyl-(1→2)- β-Dxylopyranosyl]-24-methylcholest-4-ene-3β,6β,8,15α,16β,28-hexaol. Planciside D was a triglycoside containing two carbohydrate chains, which are rarely found in starfish. Its structure was established using 2D NMR spectroscopy and tandem mass spectrometry with electrospray ionization (ESI).

Six new asterosaponins, leptasteriosides A-F (3-8), one new and one previously known asterogenins (1, 2) were isolated from the alcoholic extract of the Far Eastern starfish Leptasterias ochotensis. The structures of 1-8 were elucidated by extensive NMR and ESI-MS techniques. Compounds 2-8 showed slight or moderate cytotoxic activities against cancer cell lines RPMI-7951 and T-47D. The asterosaponins 3-5 demonstrated a significant inhibition of RPMI-7951 and T-47D cell colony formation in soft agar clonogenic assay in nontoxic doses.

Ten new polyhydroxysteroidal glycosides, anthenosides L–U (1–10), with rare positions of carbohydrate fragment attachments, were isolated from the starfish Anthenea aspera. The structures of 1–10 were established by NMR and ESIMS techniques as well as by chemical transformations. The unoxidized Δ22-24-nor-cholestane (1), (24S)-Δ22-24-methylcholestane (2–5), and Δ22-cholestane (7) side chains of the steroidal aglycons, 3-O-methyl-β-d-galactofuranosyl residue (2, 8), and 5α-cholest-8(14)-ene-3α,7β,16α-trihydroxysteroidal nucleus (9, 10) have not been found previously in starfish polar steroidal compounds. The mixture of glycosides 9 and 10 showed hemolytic activity with an EC50 = 8 μM. Compound 4 at a dose of 10 μM exhibited a potential immunomodulatory action, decreasing by 24% the intracellular ROS content in RAW 264.7 murine macrophages, induced by pro-inflammatory endotoxic lipopolysaccharide from E. coli.

Four new steroidal glycosides, protolinckiosides A - D (1 - 4, resp.), were isolated along with four previously known glycosides, 5 - 8, from the MeOH/EtOH extract of the starfish Protoreaster lincki. The structures of 1 - 4 were elucidated by extensive NMR and ESI-MS techniques as (3β,4β,5α,6β,7α,15α,16β,25S)-4,6,7,8,15,16,26-heptahydroxycholestan-3-yl 2-O-methyl-β-d-xylopyranoside (1), (3β,5α,6β,15α,24S)-3,5,6,8,15-pentahydroxycholestan-24-yl α-l-arabinofuranoside (2), sodium (3β,6β,15α,16β,24R)-29-(β-d-galactofuranosyloxy)-6,8,16-trihydroxy-3-[(2-O-methyl-β-d-xylopyranosyl)oxy]stigmast-4-en-15-yl sulfate (3), and sodium (3β,6β,15α,16β,22E,24R)-28-(β-d-galactofuranosyloxy)-6,8,16-trihydroxy-3-[(2-O-methyl-β-d-xylopyranosyl)oxy]ergosta-4,22-dien-15-yl sulfate (4). The unsubstituted β-d-galactofuranose residue at C(28) or C(29) of the side chains was found in starfish steroidal glycosides for the first time. Compounds 1 - 4 significantly decreased the intracellular reactive oxygen species (ROS) content in RAW 264.7 murine macrophages at induction by proinflammatory endotoxic lipopolysaccharide (LPS) from E. coli.

Despite significant advances in the understanding, prevention, and treatment of cancer, the disease continues to affect millions of people worldwide. Chemoradiation therapy is a rational approach that has already proven beneficial for several malignancies. However, the existence of toxicity to normal tissue is a serious limitation of this treatment modality. The aim of the present study is to investigate the ability of polar steroids from starfish Patiria (=Asterina) pectinifera to enhance the efficacy of radiation therapy in colorectal carcinoma cells. The cytotoxic activity of polar steroids and X-ray radiation against DLD-1, HCT 116, and HT-29 cells was determined by an MTS assay. The effect of compounds, X-ray, and their combination on colony formation was studied using the soft agar method. The molecular mechanism of the radiosensitizing activity of asterosaponin P1 was elucidated by western blotting and the DNA comet assay. Polar steroids inhibited colony formation in the tested cells, and to a greater extent in HT-29 cells. Asterosaponin P1 enhanced the efficacy of radiation and, as a result, reduced the number and size of the colonies of colorectal cancer cells. The radiosensitizing activity of asterosaponin P1 was realized by apoptosis induction through the regulation of anti- and pro-apoptotic protein expression followed by caspase activation and DNA degradation.