Research ArticleExperimental Studies
Open Access

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

KAZUMASA YOSHIDA, KENSUKE NISHI, SHUHEI ISHIKURA, KAZUHIKO NAKABAYASHI, RYO YAZAKI, TAKASHI OHSHIMA, MASAHIKO SUENAGA, SENJI SHIRASAWA and TOSHIYUKI TSUNODA
Anticancer Research August 2022, 42 (8) 3993-4001; DOI: https://doi.org/10.21873/anticanres.15895

Abstract

Background/Aim: In a screen of compounds to selectively suppress the growth of cancer spheroids, which contained mutant (mt) KRAS, NPD10621 was discovered and associated derivatives were investigated. Materials and Methods: Spheroid areas from HCT116-derived HKe3 spheroids expressing wild type (wt) KRAS (HKe3-wtKRAS) and mtKRAS (HKe3-mtKRAS) were treated with 12 NPD10621 derivatives and measured in three-dimensional floating (3DF) cultures. Several cancers were treated with NPD1018 (pyra-metho-carnil: PMC) in 3DF cultures. In a nude mouse assay, 50% cell growth inhibition (GI50) values were determined. Results: From these 12 derivatives, PMC was the most effective inhibitor of HKe3-mtKRAS spheroid growth with the least toxicity. Furthermore, PMC-mediated growth suppression was observed in all tested cancer cell lines, independent of tissue context, driver gene mutations, and drug resistance, suggesting that the PMC target(s) was crucial for cancer growth in a context-independent manner. The GI50 value of PMC in nude mice assay was 7.7 mg/kg and nude mice that were administered 40 mg/kg PMC for 7 days did not show any abnormal blood cell count values. Conclusion: PMC is a low-toxicity compound that inhibits the growth of different tumor cell types.

Key Words:

The human gene encoding KRAS GTPase is among the most frequently mutated cancer drivers (1). Mutant KRAS (mtKRAS), with activating missense mutations, constitutively activates numerous signaling pathways implicated in cell proliferation and survival, and therefore promotes cancer development, metastasis, and therapy resistance (2, 3). Oncogenic KRAS mutations are frequently identified in hard-to-treat cancers such as pancreatic cancers (86%-96% with oncogenic KRAS mutations) (4), colorectal cancers (CRCs) (40%-54%), and non-small cell lung cancers (15%-20%) (5, 6). Hence, considerable efforts have been focused on developing drugs targeting the activated KRAS or KRAS-related tumor-promoting pathways.

Recently, AMG510 (sotorasib), which directly targets the KRAS G12C mutation, was developed (7) and approved by the United States Food and Drug Administration. However, the G12C mutation accounts for approximately 10% of mutations in oncogenic mtKRAS (5, 7-12); therefore, oncogenic mtKRAS remains an “undruggable” target. In addition, while molecular-targeted drugs against KRAS-related signaling factors (such as BRAF and EGFR) are clinically approved and effective, intrinsic or acquired drug resistance is an important unsolved issue in the present cancer treatment. Therefore, novel agents, which overcome KRAS-targeted therapy limitations, are highly anticipated.

Different natural compounds have served as important discovery sources for effective anticancer agents, while canonical anticancer agents from natural products, such as camptothecin (topoisomerase I inhibitor) and paclitaxel (mitotic inhibitor), are highly toxic and cause severe side effects (13). We previously developed a novel drug-screening system using three-dimensional (3D) floating (3DF) cultures to identify low-toxicity inhibitors of the mtKRAS-mediated oncogenic pathway. 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 (17-19).

In this study, we report the identification of NPD1018 (pyra-metho-carnil: PMC), from natural product libraries, as an effective low-toxicity inhibitor of HKe3-mtKRAS tumor growth in 3DF cultures and a nude mouse xenograft model. PMC has the potential to become a useful anticancer agent, as it exhibits strong efficacy toward many cancer cell types independent of tissue context, driver gene mutations, and drug resistance.

Materials and Methods

Compounds. A natural product compound library was provided by RIKEN NPDepo (Saitama, Japan). Chemical distances were determined using the Jaccard similarity index (16, 20). PMC (IUPAC Name: 1-{3-[(3,5-dimethylpyrazol-1-yl)methyl]-4-methoxyphenyl]-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole) was synthesized by Kyushu-University, Graduate School of Pharmaceutical Sciences and Namiki Shoji Co., Ltd. (Tokyo, Japan).

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). Human bladder cancer KK47 cells, cisplatin-resistant KK47 cells (KK47/DDP20), patient-derived melanoma cells, and a vemurafenib-resistant subline were obtained and maintained as previously described (22, 23).

View this table:
Table I.

Efficacy of growth suppression by PMC on several types of cancer spheroids.

3DF cell culture. Cells were seeded in 96-well plates with round-bottoms and ultralow attachment surfaces (product number 7007; Corning Inc., Corning, NY, USA) and treated with derivatives at day 0 as described previously (14-16).

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). Relative growth rates were calculated by comparing control spheroid areas on day 3.

Tumorigenicity assays. Four-week-old female SCID Hairless Outbred (SHO) mice (Crlj:SHO-PrkdcscidHrhr) were purchased from Charles River Laboratories (Yokohama, Japan). For implantation, HCT116 cells were trypsinized and re-suspended in a 1:1 mixture of phosphate-buffered saline and Matrigel (BD Bioscience, Bedford, MA, USA). Further, a 100 μl aliquot containing 1.5×106 HCT116 cells was subcutaneously injected into the flank of mice as previously described (17).

Gene effect scores. The DepMap portal was used to determine KRAS gene effect scores, which was calculated using Chronos algorisms (24).

Data presentation. All the experiments were performed in triplicate and data are presented as mean±standard deviation.

Results

NPD10621 derivatives inhibit HKe3-mtKRAS growth in 3DF cultures. During the first screening of compounds from natural products, NPD10621 was identified as a candidate drug that specifically inhibited HKe3-mtKRAS spheroid growth but not that of HKe3-wtKRAS spheroids (data not shown). Using the chemical distances, 12 NPD10621 derivatives were selected from RIKEN natural product libraries (20) (Table II). Further, cells grown in 3DF cultures were treated with 16.6 μM and 50.0 μM derivatives (#103–#114) to examine their effects on cell proliferation. While the HKe3-mtKRAS spheroid areas treated with dimethyl sulfoxide (DMSO) were 2.53-fold larger than control HKe3-wtKRAS spheroids treated with DMSO on day 3, HKe3-mtKRAS spheroid areas treated with 16.6 μM #107 and #113 were 1.69- and 1.85-fold larger on day 6, respectively, when compared with control spheroids on day 3 (Figure 1A). This suggested that these derivatives suppressed cancer cell growth.

View this table:
Table II.

List of NPD10621 derivatives.

Figure 1.
Figure 1.

NPD10621 derivatives inhibit HKe3-mutant (mt) KRAS spheroid growth. The relative spheroid area of HKe3-mt KRAS (A and B) and HKe3-wild type (wt) KRAS (C and D) cells treated with NPD10621 derivatives on day 3 and 6 compared with HKe3-wt KRAS treated with dimethyl sulfoxide (DMSO control) on day 3. The NPD10621 derivative concentration was 16.6 μM (A and C) and 50 μM (B and D).

The areas of HKe3-mtKRAS spheroid treated with 50.0 μM #103, #105, #107, #109, #111, #112, and #113 were 0.19, 0.09-, 0.17-, 0.54-, 1.71-, 1.91-, and 1.63-fold larger on day 6, respectively, when compared with those of HKe3-wtKRAS spheroids treated with DMSO on day 3 (Figure 1B).

The areas of HKe3-wtKRAS spheroids treated with 16.6 μM #107, #109, #110, #111, #112, and #114 were on day 6 1.63-, 2.36-, 1.96-, 2.19-, 2.13-, and 2.30-fold larger, respectively, when compared with those of HKe3-wtKRAS spheroids treated with DMSO on day 3 (Figure 1C). The areas of HKe3-wtKRAS spheroid treated with 50.0 μM #106, #109, #110, #111, #113, and #114 were on day 3 1.59, 2.17-, 3.33-, 2.40-, 1.64-, and 1.91-fold larger, respectively, when compared with those of HKe3-wtKRAS spheroids treated with DMSO alone on day 3 (Figure 1D). These observations suggested that the toxicity of these derivatives was low in normal cells. To select the best compounds from these derivatives, we scored toxicity in the normal model (HKe3-wtKRAS) and growth suppression efficacy in the cancer model (HKe3-mtKRAS) at low or high doses (Table III). When combined, these results indicated that #113 (NPD1018) had the highest score suggesting that it is a good candidate for further analyses. We renamed NPD1018 as PMC from its three functional groups: pyrazole, methoxyphenyl, and β-carboline.

View this table:
Table III.

Scoring method.

PMC suppress growth of several cancer spheroids with or without KRAS mutations. The parental HKe3 cells were derived from HCT116 cells by disrupting the KRAS G13D mutation by homologous recombination. PMC selectively suppressed HCT116 spheroid growth in a dose-dependent manner (Figure 2A). To examine PMC efficacy against other cell lines, 3DF cultures were treated with 5, 15, 45, and 90 μM (high concentration) PMC (Figure 2B and Table I). Notably, PMC suppressed not only cells with KRAS mutations, such as Calu-6 (colon) and SW620 (colon) cells, but also cells without KRAS mutations (Figure 2B). These cells had other driver mutations, such as BRAF [WiDr (colon) and SK-MEL28 (skin) cells] or PTEN [MDA-MB-468 (breast) and LNCaP (prostate) cells], suggesting that PMC could effectively inhibit the growth of cancer cells with different driver mutations. Indeed, PMC suppressed growth of all examined cell lines, independent of tissue or gene mutations (Table I). Furthermore, PMC suppressed the growth of cisplatin-resistant bladder cancer KK47 spheroids and patient-derived melanoma spheroids with vemurafenib resistance (Figure 2C) (22, 23). The effect of PMC on growth suppression was also independent of KRAS dependency (high KRAS dependency: KRAS gene effect <–0.5) (Table I). These results suggested that the PMC target(s) was independent of tissue context and driver gene mutations and was not closely associated with KRAS dependency.

Figure 2.
Figure 2.

Pyra-metho-carnil (PMC) inhibits cancer cell growth in three-dimensional floating (3DF) cultures. A: HCT116 or HKe3 cells were grown in 3DF cultures and treated with dimethyl sulfoxide (DMSO) or PMC (5, 15, and 45 μM) on day 0. Spheroid images of four replicates on day 6 are shown (upper panel). Relative spheroid areas on day 3 and 6 compared with HKe3 spheroid areas treated with DMSO on day 3 are shown (lower panel). B and C: Spheroid images of the indicated cancer cells treated with DMSO (control) or PMC (45 μM) on day 6 in 3DF cultures.

The effects of PMC on in vivo human colorectal cancer cell (HCT116) tumorigenicity. HCT116 cells were subcutaneously injected into flanks of nude mice to examine the effects of PMC on HCT116 cell tumorigenicity. PMC was administered from day 0. In control mice, tumor volume was 2,362 mm3 on day 7. In contrast, in mice treated with 10 mg/kg, 40 mg/kg, and 80 mg/kg PMC, tumor volumes were 829 mm3, 525 mm3, and 201 mm3, respectively, on day 7 (Figure 3A). The 50% cell growth inhibition (GI50) value was 7.7 mg/kg. Furthermore, mice that administered 40 mg/kg PMC for 7 days showed no abnormal blood cell count values (Figure 3B), suggesting that PMC inhibited in vivo tumor growth with low toxicity.

Figure 3.
Figure 3.

The effects of pyra-metho-carnil (PMC) on human colorectal cancer (HCT116) cell tumorigenicity in vivo. A: HCT116 cells were subcutaneously injected into the flanks of nude mice. Relative tumor volumes are shown for mice treated with and without PMC. Derivatives were administered each day from day 0. B: Mice were administered DMSO (control) or 40 mg/kg PMC each day for 7 days. Blood biochemistry parameters were unchanged. WBC: White blood cell; RBC: red blood cell; HGB: hemoglobin; HCT: hematocrit; MCV: mean corpuscular volume; MCH: mean corpuscular hemoglobin; MCHC: mean corpuscular hemoglobin concentration; PLT: platelets.

Discussion

In this study, we identified PMC as a potent drug for the treatment of several cancers. Furthermore, PMC showed low toxicity and suppressed mtKRAS CRC growth in vivo. The PMC structure consists of β-carboline with methoxyphenyl and pyrazole at the C1 position. β-carbolines such as harmine are often identified in medicinal plants and exert antiproliferative effects toward several cancers (25, 26). In addition, β-carboline hybrids with pyrazole at the C3 position exhibit similar anticancer properties toward A549 (lung), DU145 (prostate), MCF-7 (breast), and Hela (cervix) cells through targeting topoisomerase I (27). PMC was effective in several in vitro cancer cell lines regardless of driver gene mutations (Figure 2 and Table I), suggesting that the direct PMC target(s) is not associated with hub genes that are canonically associated with mtKRAS-related signals. Moreover, PMC was effective in cell lines with wt KRAS and low KRAS dependency (KRAS gene effect >–0.5) (Table I). Furthermore, PMC was similarly effective toward parental and drug-resistant cells, suggesting the PMC target(s) was not involved in drug-resistance mechanisms (Figure 2C). PMC was also effective in vivo with low toxicity (Figure 3), suggesting that PMC was different for canonical anticancer drugs targeting cell proliferation with cytotoxicity. The identification of PMC targets will be crucial for the identification of the Achilles’ heel of cancers. We are currently performing pull-down assays to determine PMC targets.

Acknowledgements

The Authors thank Yuriko Isoyama and Yumiko Hirose for technical assistance. This work was supported by Grant-in-Aid for Scientific Research (C) (KAKENHI, Grant Number 15K06847, 18K07215, 21K07161, 22K07221) from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan and the Fukuoka Foundation for Sound Health Cancer Research Fund.

Footnotes

  • Authors’ Contributions

    K.Y., Kensuke N, and T.T. performed experiments, analyzed the data, and wrote the first manuscript draft. S.I., Kazuhiko N., R.Y., and M.S. participated in study design, data collection, and analysis. T.O. and S.S. conceived the idea, designed the study, interpreted the data, provided important intellectual content, and obtained final approval for manuscript submission.

  • Conflicts of Interest

    The Authors declare no conflicts of interest in relation to this study.

  • Received May 20, 2022.
  • Revision received June 6, 2022.
  • Accepted June 7, 2022.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).

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Cancer Spheroid Proliferation Is Suppressed by a Novel Low-toxicity Compound, Pyra-Metho-Carnil, in a Context-independent Manner
KAZUMASA YOSHIDA, KENSUKE NISHI, SHUHEI ISHIKURA, KAZUHIKO NAKABAYASHI, RYO YAZAKI, TAKASHI OHSHIMA, MASAHIKO SUENAGA, SENJI SHIRASAWA, TOSHIYUKI TSUNODA
Anticancer Research Aug 2022, 42 (8) 3993-4001; DOI: 10.21873/anticanres.15895
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