Growth Suppression of Cancer Spheroids With Mutated KRAS by Low-toxicity Compounds from Natural Products

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 PDE4inhibitory 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.

Application of the Luminescence Syncytium Induction Assay to Identify Chemical Compounds That Inhibit Bovine Leukemia Virus Replication

“…Indeed, BSI-625 was most effective between 0 and 0.5 h of co-cultivation, and its inhibitory effect remained at 16 h post co-cultivation. BSI-625 and compounds with analogous skeletal structures have been reported as potential anticancer agents that inhibit mutant K-Ras-related signaling [ 46 ]. To date, no relationship between BLV and mutant K-Ras has been reported; however, Tax or G4 of BLV enhance transformation by Ha-Ras [ 47 , 48 ].…”

Cancer Spheroid Proliferation Is Suppressed by a Novel Low-toxicity Compound, Pyra-Metho-Carnil, in a Context-independent Manner

“…A natural product compound library was provided by RIKEN NPDepo (Saitama, Japan). Chemical distances were determined using the Jaccard similarity index (16,20). Cell culture.…”

“…Cell culture. HKe3, HKe3-wtKRAS, and HKe3-mtKRAS cultures were established and maintained as previously described (14,16,21). Other cells (Table I), including HCT116 cells were purchased from the American Type Culture Collection (Manassas, VA, USA).…”

“…In this system, two HKe3-derived cell lines were used to examine if the compounds could selectively suppress cancer cell spheroid growth (14): one expressing wt KRAS (HKe3-wtKRAS; normal cell model) and another expressing KRAS G13D mutant (HKe3-mtKRAS; cancer cell model) with the same genetic backgrounds other than KRAS mutation. Using this system and natural product libraries, we previously identified several new compounds displaying selective growth-suppressive effects in cancer cells harboring mtKRAS but not in normal cells (15,16). We also used this system to validate the anti-tumor effects of MK615 (Japanese apricot extract), apremilast (PDE4 inhibitor), and UHA6052 (resveratrol-derivative) against human CRC cells harboring mtKRAS (17)(18)(19).…”

“…2 O 264,32 0,395 3DF cell culture. Cells were seeded in 96-well plates with roundbottoms and ultralow attachment surfaces (product number 7007; Corning Inc., Corning, NY, USA) and treated with derivatives at day 0 as described previously(14)(15)(16).Spheroid area measurements. Photomicrographs of the cells were taken and analyzed using an IN Cell Analyzer 1000 (GE Healthcare, Little Chalfont, UK) and an IN Cell Developer Toolbox (GE Healthcare).…”