Streptospherin A, a New Cancer Stem Cell Inhibitor, Produced by Streptomyces sp. KUSC-240

Abstract

Cancer stem cells (CSCs) are a major cause of tumor recurrence; therefore, using CSC inhibitors is a potential strategy for cancer chemotherapy. We used our previously reported screening system that targets CSCs to identify a new scarce compound, streptospherin A (1), isolated from Streptomyces sp. KUSC-240. The planar structure of 1 was determined by high-resolution-MS (HR-MS) and NMR analysis. Nuclear Overhauser effect spectroscopy correlation and J-resolved heteronuclear multiple bond connectivity analysis revealed the partial relative configuration on the tetrahydropyran ring. Streptospherin A (1) inhibited CSC sphere formation and suppressed the growth of CSCs. These results suggest that streptospherin A (1) is a potential anticancer agent that may target CSCs.

Introduction

Cancer stem cells (CSCs) are a highly malignant tumor fraction and 1 of the main causes of tumor recurrence. CSCs initiate tumor progression in vivo and exhibit resistance to conventional anticancer agents. Thus, CSCs are potential targets for cancer treatment.^1,2) CSCs can form spheres in vitro 3-dimensional (3D) culture systems; therefore, finding CSC sphere formation inhibitors is a promising strategy for targeting CSCs.

To find potential therapeutic agents against CSCs, we previously established a new screening strategy using colorectal cancer HT29 cells with a 3D culture system. By culturing the cells in CSC medium, we obtained spheres with CSC-like features of elevated stemness with characteristic markers (Sox2, Nanog, and Oct4) and resistance to anticancer agents (camptothecin and paclitaxel).³⁾ By using this CSC culture method, we searched for compounds that overcome the tolerance of CSCs to conventional drugs.

In this study, we found that a microbial broth, Streptomyces sp. KUSC-240, inhibited CSC sphere formation. Activity-guided fractionation identified a new scarce compound, named streptospherin A (1). We describe the isolation, structural determination, and biological activity of 1.

Results and Discussion

Fermentation and Isolation

Streptomyces sp. KUSC-240 was cultured in GPF production medium at 28°C for 4 d. After fermentation, the culture medium (10 L) was extracted with the same volume of acetone, filtered, and concentrated in vacuo. The remaining culture broth was extracted with EtOAc twice (10 L × 2) to give a brown residue (1534.24 mg). The extract was subjected to silica gel column chromatography (64–210 mm Wakosil 60, FUJIFILM Wako Pure Chemical Corp., Osaka, Japan) using a CHCl₃-MeOH stepwise system. The active fraction (CHCl₃ : MeOH = 100 : 2; 123.0 mg) was fractionated further with a Sep-Pak C18 cartridge (Waters Corp., Milford, MA, U.S.A.) using a MeCN-H₂O stepwise system. The MeCN : H₂O = 4 : 6 fraction (46.41 mg) was purified by preparative HPLC on a COSMOSIL Cholester column (Nacalai Tesque Inc., Kyoto, Japan; φ10 × 250 mm; 5 mL/min) with a MeCN-H₂O system with a linear gradient of 30 : 70–100 : 0 over 30 min to give an active fraction (retention time [t_R]: 16.2 min; 5.69 mg). Further purification was performed by HPLC on a COSMOSIL Cholester column (φ4.6 × 250 mm; 1 mL/min) with MeCN : H₂O = 40 : 60 (t_R: 22.3 min; 1.24 mg), followed by MeOH : H₂O = 70 : 30 (t_R: 15.5 min) to give a pure new compound, streptospherin A (1; 0.45 mg; Fig. 1).

Fig. 1. Structure of Streptospherin A (1)

Streptospherin A (1): Pale-yellow oil; ${\left[\alpha \right]}_{\mathrm{D}}^{20}$ –14.3 (c = 0.10, MeOH); UV λ_max (MeOH) nm (log ε): 218 (3.78), 242 (sh), 290 (3.63), 332 (3.20); IR (KBr) cm^–1: 3389, 2959, 2930, 2869, 1709, 1621, 1460, 1417, 1375, 1269, 1240, 1214, 1159, 1065, 975, 875; electrospray ionization-MS (ESI-MS) m/z: 447.2356 (Calcd for C₂₃H₃₆O₇Na⁺: 447.2353). ¹H-NMR and ¹³C-NMR data are summarized in Table 1.

Table 1. ¹H-NMR Data (500 MHz) and ¹³C-NMR Data (125 MHz) for Streptospherin A (1) in CD₃OD

Position	δH (mult., J in Hz)	δC
1	1.08 (3H, d, 6.4)	16.9
2	3.55 (1H, q, 6.4)	74.0
3	—	100.1
4	1.27 (1H, m)	38.7
	1.87 (1H, m)
5	3.97 (1H, dddd, 4.6, 4.6, 11.3, 11.3)	65.6
6	1.13 (1H, m)	39.6
	1.78 (1H, m)
7	4.09 (1H, ddd, 2.1, 7.6, 11.6)	72.0
8	3.66 (1H, m)	50.8
9	1.81 (2H, m)	33.2
10	1.22 (2H, q, 7.6)	21.8
11	0.87 (3H, t, 7.6)	14.6
1′	—	208.6
2′	—	115.2
3′	—	162.9
4′	—	111.9
5′	—	162.3
6′	—	121.1
7′	7.51 (1H, s)	131.8
8′	2.08 (3H, s)	8.1
9′	2.40 (1H, dd, 6.7, 13.8)	40.4
	2.52 (1H, dd, 6.7, 13.8)
10′	1.91 (1H, m)	29.8
11′/12′	0.93 (6H, d, 6.7)	22.6
		22.9

Structure Elucidation

Streptospherin A (1) was obtained as a pale-yellow oil with high purity (Supplementary Fig. S1). According to high-resolution (HR) ESI-MS (Supplementary Fig. S2), ¹H-NMR, and ¹³C-NMR data, the molecular formula of 1 was found to be C₂₃H₃₆O₇ (m/z: 447.2356, Calcd for C₂₃H₃₆O₇Na⁺: 447.2353). UV absorptions were detected at λ = 218, 242, 290, and 332 nm. ¹H, ¹³C, and heteronuclear multiple quantum coherence NMR analyses measured in CD₃OD (Supplementary Figs. S3–S5) revealed the presence of 23 carbons, which were assigned to 1 carbonyl, 6 sp² carbons (1 is proton-bearing), 1 fully substituted sp³ carbon, 5 sp³ methines including 3 oxygenated methines, 5 methylenes, and 5 methyl carbons. The full planar structure was elucidated by 1D- and 2D-NMR spectra. The ¹H–¹H correlation spectroscopy spectrum revealed the proton–proton correlations from H-1 to H-2, from H-4 to H-11, and from H-10′ to H-12′ (Fig. 2a and Supplementary Fig. S6). The linkage of C6′-C-9′-C-10′ was determined from heteronuclear multiple bond connectivity (HMBC) correlations from H-9′ to C-5′/C-6′/C-7′/C-10′. HMBC correlations from H-7′ to C-1′/C-3′/C-5′/C-9′, from H-8′ to C-3′/C-4′/C-5′, and from H-9′ to C-5′/C-6′/C-7′ established a carbonyl (C-1′)-linked (C-2′) benzene ring with oxygen (C-3′ and C-5′), methyl (C-4′), and isobutyl (C-6′) group. The linkage of C-1′ and C-8 was determined by the HMBC correlation from H-8 to C-1′. The linkage of C-4-C-3-C-2 was also proved by the HMBC correlation from H-4 to C-3 and from H-1 to C-3 (Fig. 2a and Supplementary Fig. S7). The chemical shift of C-3 (δ_C 100.1, fully substituted sp³ carbon) suggested that C-3 is a hemiacetal carbon, and the connection of C-3 and C-7 through oxygen to form a tetrahydropyran ring with a chair conformation was established by the nuclear Overhauser effect (NOE) observation not only between H-4 and H-6 but also between H-5 and H-7 due to their diaxial relationships. From these structural analyses, the planar structure of 1 was determined (Fig. 2a).

Fig. 2. Structure Elucidation of Streptospherin A (1)

(a) Key COSY and HMBC correlation of 1. (b) NOESY correlation of 1. (c) Newman projection at the C-3 position of 1. (d) Relative configuration of 1.

The NOE spectroscopy (NOESY) spectrum revealed the correlations from H-2 to H-4a, H-4a to H-6a, and H-5 to H-7 (Fig. 2b and Supplementary Fig. S8). J-resolved HMBC was performed to confirm the relative configuration at the C-3 position.⁴⁾ Small ¹H-¹³C long-range coupling (³J_C–H of less than 3 Hz) between H-4a and C-2 and no coupling signal (³J_C–H of almost 0 Hz) between H-4e and C-2 were observed (Supplementary Fig. S9). These results indicated the gauche relationships of C-2 with H-4a and H-4e (Fig. 2c), determining the axial orientation of the hydroxy group at the C-3 position. NOESY correlations and J-resolved HMBC analysis revealed the partial relative configuration as (3R^*, 5S^*,and 7S^*) (Fig. 2d). The determination of the whole absolute configuration of 1 using synthetic chemistry is underway in our laboratory.

Luminacins^5,6) and migracins⁷⁾ were isolated in 2000 and 2013, respectively (Supplementary Fig. S10). These compounds have a skeleton partially in common with 1. In contrast, the hemiacetal group with a side chain at the C-3 position and the methyl group at the C-8′ position are characteristic in 1. There are no reports of luminacins and migracins as CSC inhibitors, although these compounds are reported to inhibit capillary tube formation or cancer cell migration.

Effect of Streptospherin A on CSCs

We previously demonstrated that 3D-cultured HT29 spheres in 3D Tumorsphere Medium XF (XF medium, PromoCell GmbH, Heidelberg, Germany) exhibited high CSC markers and resistance to a high dose of anticancer agents such as 10 μM camptothecin. We also found the potent inhibitory effect of polyether ionophore lenoremycin against CSC spheres at the concentration of 10 nM.³⁾ Therefore, we observed the effect of 1 on spheres in XF medium under a microscope at the concentrations of 3.0, 10, and 30 μg/mL. As a result, we found that 30 μg/mL (71 μM) of 1 significantly inhibited sphere formation (Fig. 3a). Cell growth analysis with an ATP assay also showed that 1 inhibited the cell growth of spheres in XF medium at an IC₅₀ of 42.3 μM (Fig. 3b). These results indicated that streptospherin A (1) targeted CSCs and inhibited cell growth.

Fig. 3. Inhibitory Activity of Streptospherin A (1) against CSCs

(a) CSC sphere formation under an optical microscope. (b) Cell growth using an ATP assay.

Conclusion

CSCs are a major cause of tumor recurrence. We previously established a screening system targeting CSCs by using 3D-cultured HT29 spheres grown in CSC medium. In this study, we isolated a new scarce compound, streptospherin A (1), from Streptomyces sp. KUSC-240 as an inhibitor of CSC sphere formation. We determined the planar structure and partial relative stereochemistry of 1 by HR-ESI-MS and NMR spectrum analyses. Streptospherin A (1) also inhibited CSC sphere growth, indicating that 1 can target CSCs. The total synthesis of 1 and isolation of derivatives are currently underway in our laboratory, and we expect to perform detailed biological assays and structure–activity relationship studies.

Experimental

General Experimental Procedures

NMR spectra were recorded on a JEOL ECA500 spectrometer (JEOL Ltd., Tokyo, Japan) or a Varian Inova-500 (Varian Medical Systems Inc., Palo Alto, CA, U.S.A.) at 500 MHz (¹H-NMR) or at 125 MHz (¹³C-NMR) in CD₃OD (residual solvent peak or solvent peak: ¹H δ 3.31 ppm, ¹³C δ 49.0 ppm). Optical rotation was measured on a JASCO P-2200 polarimeter (JASCO Corp., Tokyo, Japan). The UV spectrum was recorded on a Hitachi U-3210 spectrophotometer (Hitachi Ltd., Tokyo, Japan). The IR spectrum was measured on a JASCO FTIR-4100 spectrometer (JASCO Corp.). HR-ESI-MS was recorded on a Shimadzu LCMS-IT-TOF (Shimadzu Corp., Kyoto, Japan).

Fermentation of Streptomyces sp. KUSC-240

Streptomyces sp. KUSC-240 was isolated from Minakami-machi (Tsukiyono-machi), Tone-gun, Gunma, Japan. This strain was identified as a member of Streptomyces from the 16S rRNA (ribosomal RNA) gene sequence analysis and deposited under an accession number ABP-04045 in NITE NPMD (Chiba, Japan). Streptomyces sp. KUSC-240 cultured on a GS agar medium (glucose 2.0%, soybean flour 0.5%, CaCO₃ 0.2%, and agar 2.0% [pH 7.2]) was inoculated into 500 mL Erlenmeyer flasks containing 100 mL of the GSF seed medium (glucose 2.0%, soybean flour 0.5%, FeSO₄ 0.01%, and CaCO₃ 0.2% [pH 7.2]). The flasks were incubated on a rotary shaker (160 rpm) at 28°C for 2 d. The seed culture (2 mL) was transferred into 500 mL Erlenmeyer flasks containing 100 mL of the GPF production medium (glucose 2.0%, pharmamedia 1.0%, FeSO₄ 0.01%, and CaCO₃ 0.2% [pH 7.2]). The flasks were incubated on a rotary shaker (160 rpm) at 28°C for 4 d.

Cell Culture

Colorectal cancer cell line HT29 cells were purchased from the American Type Culture Collection. HT29 cells were maintained in Roswell Park Memorial Institute-1640 (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) supplemented with 10% heat-inactivated fetal bovine serum at 37°C in a humidified atmosphere containing 5% CO₂.

Sphere Formation Assay

Cells were resuspended in 3D Tumorsphere Medium XF (PromoCell GmbH) and cultured on poly(2-hydroxyethyl methacrylate) (poly-HEMA, Sigma-Aldrich/Merck KGaA, Darmstadt, Germany)-coated 6-well plates. A half volume of fresh medium was added every 3 d. After 7 d of culture, spheres were collected and dispersed with accutase (Nacalai Tesque Inc.), resuspended in the XF medium, and reseeded on poly-HEMA-coated 96-well plates. Simultaneously, the compound was added. After 3 d of treatment, spheres were observed under a microscope. Cell growth was measured by an ATP assay using the CellTiter-Glo 3D Cell Viability Assay kit (Promega Corp., Madison, WI, U.S.A.).

Acknowledgments

This study was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan (Grant Numbers: 17H06401 to HK and MI, 19H02840, 22H04901, 23H04882, 24H00493 all awarded to HK, and 23K13896 to HI), the Project for Promotion of Cancer Research and Therapeutic Evolution (JP24ama221540 and JP25ama221540 to HK), and the Platform Project for Supporting Drug Discovery and Life Science Research (JP24ama121034 and JP25ama121034 to HK) from the Japan Agency for Medical Research and Development (AMED), Japan.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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