Abstract
Background/Aim: Among compounds from natural products selectively suppressing the growth of cancer spheroids, which have mutant (mt) KRAS, NP910 was selected and its derivatives explored. Materials and Methods: The area of HKe3 spheroids expressing wild type (wt) KRAS (HKe3-wtKRAS) and mtKRAS (HKe3-mtKRAS) were measured in three-dimensional floating (3DF) cultures treated with 18 NP910 derivatives. The 50% cell growth inhibition (GI50) was determined by long-term 3DF (LT3DF) culture and nude mice assay. Results: We selected NP882 (named STAR3) as the most effective inhibitor of growth of HKe3-mtKRAS spheroids with the least toxicity among NP910 derivatives. GI50s of STAR3 in LT3DF and nude mice assay were 6 μM and 30.75 mg/kg, respectively. However, growth suppression by STAR3 was observed in 50% of cell lines independent of KRAS mutation, suggesting that the target of STAR3 was not directly associated with KRAS mutation and KRAS-related signals. Conclusion: STAR3 is a low-toxicity compound that inhibits growth of certain tumour cells.
KRAS is the most frequently mutated oncogene among human cancers (1), with high rates of activating missense mutations in pancreatic cancers (86 to 96%) (2), in colorectal cancers (CRC) (40 to 54%), and in non–small cell lung cancer (NSCLC) (15 to 20%) (3, 4). Once activated, mutated RAS remains “on” persistently, thereby enhancing downstream signalling and leading tumourigenesis. Recently, AMG510, which targets specifically KRAS G12C mutant, was developed and was found to be effective in clinical trials in some patients with NSCLC (5). However, the KRAS G12C mutation occur in about 13% of NSCLCs (6, 7), in 3 to 5% of CRCs, and in 1 to 2% of various other solid cancers (3, 5, 8-10). Therefore, the range of use of AMG510 is limited, and the KRAS mutation is still an “undruggable” target.
Canonical anticancer agents, including alkylating drugs, platinum compounds, antimetabolites, topoisomerase inhibitors, and microtubule inhibitors, are highly toxic and cause serious side effects such as myelosuppression (11, 12). Recently, molecularly targeted drugs are expected to be the new drugs with low toxicity (13); however, drug resistance is unavoidable (14). Therefore, new types of compounds are indispensable for cancer treatment.
Recently, we screened genes that were up-regulated by mtKRAS in a three-dimensional (3D) matrigel culture and found that the phosphodiesterase 4B (PDE4B) levels were higher in clinical tumour samples from CRC patients in comparison to those from healthy control (15). We examined several PDE4 inhibitors, such as pan PDE4 inhibitor, resveratrol, and PDE4 selective inhibitor, apremilast (16, 17), revealing that these compounds are selective for cancer spheroids with mtKRAS, and exhibit high efficacy and low toxicity. Notably, resveratrol, which has similar PDE4-inhibitory activity to that of rolipram (16), is present in various natural products, suggesting that some compounds from natural products will become low-toxicity anticancer agents.
To screen compounds from natural products, we established 3D floating (3DF) culture using HKe3-wtKRAS (normal model) and HKe3-mtKRAS (cancer model) spheroids. Using this system as pilot screening, we identified a 5-bromouridine (BrUrd) (18); uridine derivatives may be able to target HKe3-mtKRAS spheroids selectively and may exhibit low-level toxicity to wtKRAS.
This study, utilized natural product libraries from RIKEN Natural Products Depository (NPDepo) and found several core compounds, including NP910 (19) and explored the efficacy of NP910 derivatives using 3DF culture. We selected NP882 (19) as lead compound and further examined its properties.
Materials and Methods
Compounds. Compounds library from natural products were kindly provided by RIKEN NPDepo (Saitama, Japan). Chemical distances were determined by the Jaccard similarity index (20).
Cell culture. HKe3, HKe3-wtKRAS, and HKe3-mtKRAS cultures were established and maintained as described previously (21, 22). DLD-1, SW620, SW837, A549, MIA PaCa2, Hs766T, HepG2, HEC1-B, CCK-81, Hs700T, SK-M EL-28, A498, DU145, MCF-7, HeLa, Caov-3 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained at 37˚C in 5% CO2 atmosphere in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin as described previously (21, 22).
Short-term 3D floating cell culture. Cells were seeded in 96-well plates with round-bottom and ultralow attachment surfaces (product number 7007; Corning Inc., Corning, NY, USA) and treated with a drug at day 0. Cells were maintained at 37˚C in 5% CO2 atmosphere as described previously (18, 22, 23).
Long-term 3D floating cell culture. Cells were seeded in 96-well plates and treated with a drug at day 0. The drug added every three days and the area of spheroids was measured every three to four days until day 27 as previously described (18).
Measurement of area of spheroids. Photomicrographs of cells were taken and analyzed using an IN Cell Analyzer 1000 (GE Healthcare, Little Chalfont, UK) and IN Cell Developer Toolbox (GE Healthcare). The relative growth rate was calculated from a comparison of the area of control spheroids at day three.
Assay for tumourigenicity. Four-week-old female SHO mice (Crlj:SHO-PrkdcscidHrhr) were purchased from Charles River Laboratories (Yokohama, Japan). Cells for implantation were trypsinized and re-suspended in a 1:1 mixture of phosphate-buffered saline and Matrigel (BD Bioscience, Bedford, MA, USA). A 100 μl volume containing 1.5×106 HCT116 cells was subcutaneously injected into the flank of mice as described previously (17).
Statistical analyses. All experiments were performed in triplicate. Data are presented as means±standard deviations. Statistical analyses were performed using unpaired two-tailed Student’s t-test in Microsoft Excel. p-Values of less than 0.05 were considered statistically significant.
Results
NP910 derivatives inhibit the growth of HKe3-mtKRAS cells grown in 3DF cultures. During the first screening of compounds from natural products, NP910 was identified as a candidate drug that inhibits the growth of HKe3-mtKRAS spheroids but not HKe3-wtKRAS spheroids (data not shown). Eighteen NP910 derivatives were selected from RIKEN libraries of natural products using chemical distance (11) (Table I). Cells were treated with 16.6 μM and 50.0 μM of 18 NP910 derivatives in 3DF culture to examine the effects of NP910 derivatives on cell proliferation. The area of HKe3-mtKRAS spheroids treated with 16.6 μM of NP882 at day 6 was 1.27-fold larger in comparison to that of HKe3-wtKRAS spheroids treated with DMSO at day 6 (Figure 1A), suggesting that NP882 displays the highest efficacy for suppressing the growth of cancer cells. The area of HKe3-mtKRAS spheroids treated with 50.0 μM of NP910, NP770, NP917, and NP882 at day 6, was 1.19-, 0.11-, 0.16-, and 0.86-fold smaller in comparison to that of HKe3-wtKRAS spheroids treated with DMSO at day 6, respectively (Figure 1B). The area of HKe3-wtKRAS spheroids treated with 16.6 μM of NP910, NP770, NP917, and NP882 at day 6 were 1.53-, 1.68-, 1.99-, and 2.01-fold larger in comparison to that of HKe3-wtKRAS spheroids treated with DMSO at day 6, respectively (Figure 1A), suggesting that NP917 and NP882 will show the lowest toxicity for normal cells. To select the best compounds from these derivatives, we scored the toxicity in the normal model (Hke3-wtKRAS) and the efficacy of growth suppression in the cancer model at a low or high dose. Furthermore, we used the growth rate from day 3 to day 6 in the cancer model for scoring (Table II). These results together suggest that NP882 (named STAR3) is a good candidate for further analyses.
List of NP910 derivatives.
NP910 derivatives inhibit the growth of HKe3-mutant (mt) KRAS spheroids. The relative area of spheroids of HKe3-wild type (wt) KRAS and HKe3-mtKRAS treated with NP910 derivatives at day three and day six compared to that of HKe3-wtKRAS treated with dimethyl sulfoxide (DMSO, control) at day 3. The concentration of NP910 derivatives was 16.6 μM (A) or 50 μM (B).
Scoring method.
The long-term effects of STAR3 for HKe3- wtKRAS and mtKRAS spheroids. A long-term 3DF culture was established to determine the cytotoxicity of STAR3 during long-term exposure. The area of HKe3-wtKRAS spheroids treated with STAR3 at day 27 was similar to DMSO control (Figure 2A), suggesting that none of the drugs is cytotoxic to cells with wtKRAS. Treatment of HKe3-mtKRAS spheroids by serially-diluted STAR3 showed that the concentration required to achieve a 50% maximal inhibition in cell proliferation is approximately 6 μM (Figure 2B).
The long-term effect of STAR3 for HKe3-wild type (wt) KRAS and mutant (mt) KRAS spheroids A: Relative area of HKe3-wtKRAS spheroids treated with STAR3 from day 3 to day 27 compared to that for HKe3-wtKRAS treated with dimethyl sulfoxide (DMSO, control) at day 3. B: Relative area of HKe3-mtKRAS spheroids with STAR3 from day 3 to day 27 against HKe3-mtKRAS with dimethyl sulfoxide (DMSO) control at day 3.
Effect of STAR3 on in vivo tumourigenicity of human colorectal cancer HCT116 cells. HCT116 cells were injected subcutaneously into the nude mice to address the effects of STAR3 on tumourigenicity of HCT116 cells, STAR3 was administered on day 0. In control mice, tumour volume was 2362 mm3 on day 7. In contrast, in mice treated with 10 mg/kg, 40 mg/kg and 50 mg/kg STAR3 tumour volume was 1985 mm3, 750 mm3, and 514 mm3, at day 7, respectively (Figure 3). GI50 was 30.75 mg/kg. HCT116 cell tumour volume was dramatically reduced in in mice treated with STAR3 compared to those administered DMSO, suggesting that STAR3 inhibits tumour growth in vivo.
Effect of STAR3 on the in vivo tumourigenicity of human colorectal cancer HCT116 cells. HCT116 cells were injected subcutaneously into the flanks of nude mice. Relative tumour volumes are shown for mice treated with and without STAR3.
Efficacy of STAR3 on several cell lines. To examine the efficacy of STAR3 against other cell lines, 3DFCs were performed using 2, 6, 18, 54, and 162 (high concentration) μM of STAR3 (Table III). The number of cell lines in which STAR3 suppressed cell growth was 9 (50%). The number of mtKRAS cell lines whose growth was suppressed by STAR3, was 6 (60%). On the other hand, the number of wtKRAS cell lines whose growth was suppressed by STAR3 was 3 (37.5%). However, One-tailed Fisher’s exact test did not show any significant difference between mtKRAS and wtKRAS cell lines. These results suggest that the target of STAR3 is not closely associated with KRAS-related signals.
The effect of STAR3 in several cell lines with or without KRAS mutation.
Discussion
In this study, we identified STAR3 as a potent drug for cancer with mtKRAS. In long-term 3DFC, GI50 was 6 μM, and drug resistance was not observed. Furthermore, STAR3 showed low toxicity and suppressed the growth of mtKRAS CRC in vivo. However, STAR3 was not effective in all cancer cell lines with or without mtKRAS, suggesting that the direct target is not associated with KRAS-related driver genes. There is a possibility that STAR3 will act through KRAS-unrelated genes, which are specific for cell lines. Identification of targets of STAR3 is needed for determining biomarkers and further developing STAR3.
Recent studies have reported the certain natural compounds that are effective against the KRAS-linked up-stream and down-stream signalling pathways that are directly or indirectly linked with cell proliferation, division, and apoptosis (24, 25). We are now developing another compound (named STAR2) that shows low toxicity, and is effective against all mtKRAS cells.
Acknowledgements
The Authors would like to thank Yuriko Isoyama and Yumiko Hirose for their technical assistance. This work was supported by Grant-in-Aid for Scientific Research (C) (KAKENHI, Grant Number 15K06847, 18K07215) from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan and the Fukuoka Foundation for Sound Health Cancer Research Fund.
Footnotes
This article is freely accessible online.
Authors’ Contributions
S.H., M.N., K.N. and T.T. performed experiments, analysed the data, and wrote a manuscript draft. S.I., K.N., R.Y., and M.S. participated in the study design, data collection, and analysis. T.O. and S.S. conceived the idea and designed the study, interpreted the data, provided important intellectual content, and obtained the final approval of the submitted manuscript.
Conflicts of Interest
The Authors declare no competing interests in relation to this study.
- Received May 22, 2021.
- Revision received June 4, 2021.
- Accepted June 5, 2021.
- Copyright © 2021 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
