Skip to main content
Download PDF

An extract from the frass of swallowtail butterfly (Papilio machaon) larvae inhibits HCT116 colon cancer cell proliferation but not other cancer cell types

BMC Genomics volume 24, Article number: 735 (2023) Cite this article

Abstract

Background

The frass of several herbivorous insect species has been utilised as natural medicines in Asia; however, the metabolite makeup and pharmaceutical activities of insect frass have yet to be investigated. Oligophagous Papilionidae insects utilise specific kinds of plants, and it has been suggested that the biochemicals from the plants may be metabolised by cytochrome P450 (CYP) in Papilionidae insects. In this study, we extracted the components of the frass of Papilio machaon larvae reared on Angelica keiskei, Oenanthe javanica or Foeniculum vulgare and examined the biological activity of each component. Then, we explored the expression of CYP genes in the midgut of P. machaon larvae and predicted the characteristics of their metabolic system.

Results

The components that were extracted using hexane, chloroform or methanol were biochemically different between larval frass and the host plants on which the larvae had fed. Furthermore, a fraction obtained from the chloroform extract from frass of A. keiskei-fed larvae specifically inhibited the cell proliferation of the human colon cancer cell line HCT116, whereas fractions obtained from the chloroform extracts of O. javanica- or F. vulgare-fed larval frass did not affect HCT116 cell viability. The metabolites from the chloroform extract from frass of A. keiskei-fed larvae prevented cell proliferation and induced apoptosis in HCT116 cells. Next, we explored the metabolic enzyme candidates in A. keiskei-fed larvae by RNA-seq analysis. We found that the A. keiskei-fed larval midgut might have different characteristics from the O. javanica- or F. vulgare-fed larval metabolic systems, and we found that the CYP6B2 transcript was highly expressed in the A. keiskei-fed larval midgut.

Conclusions

These findings indicate that P. machaon metabolites might be useful as pharmaceutical agents against human colon cancer subtypes. Importantly, our findings show that it might be possible to use insect metabolic enzymes for the chemical structural conversion of plant-derived compounds with complex structures.

Peer Review reports

Background

Over one million species of insects have been recorded, and half of them are categorised as herbivorous insects that utilise plants [1, 2]. Herbivorous insects are categorised into three types: (1) monophagous insects that utilise only one species of host plant, (2) oligophagous insects that utilise a narrow range of host plants, and (3) polyphagous insects that utilise a broad range of host plants [2]. Polyphagous insects have the advantage of being able to feed on a balanced diet at any time and in any place, while monophagous and oligophagous insects can specialise in utilising toxic metabolites from their host plants to protect themselves from predators. Heliconian butterflies, which are monophagous insects, produce cyanogenic glucoside, which is metabolised from their host plants in the Passifloraceae family [3]. Furthermore, troidine swallowtails, which belong to Papilionidae and feed on plants of the Aristolochiaceae family, sequester aristolochic acid from their host plants [4]. These monophagous and oligophagous insects are therefore difficult for predators to eat because they are unpalatable [5].

Frass from herbivorous insects has been used as a source of natural medicine in several countries. Insect frass contains plant-derived ingredients whose chemical structures were changed by insect metabolism. Thus, ingredients from insect frass may have enhanced biological activity compared with the biological activity of the metabolites that the insects originally consumed from plants. Some medicinal herbs and frass from a stick insect, Eurycnema versifasciata, are used to treat a variety of ailments, including diarrhoea, asthma, upset stomach and muscular pain, in Malaysia and China [6, 7]. In addition, ingredients included in the Bombyx mori larval frass are thought to stimulate the adrenal gland and to decrease blood cholesterol content [8]. These examples demonstrate that the frass of several phytophagous insect species has been used as pharmaceutical agents in several countries to treat human diseases. The therapeutic effects of insect frass may depend on the insects’ host plants, with the metabolites found in frass being converted into more effective metabolites than those found in the host plant tissues through the process of insect metabolism [9]. However, the process by which insect metabolism changes the biological activity of host plant metabolites have been obscured.

Cytochrome P450 (CYP), a drug-metabolising enzyme that is highly conserved in most organisms, has potential for biotechnological use because of its high diversity of catalysed reactions, and because high productivity is needed for such biotechnological applications [10, 11]. Most Papilionidae family members, including polyphagous and oligophagous insects, feed on furanocoumarin-containing plants [12]. Furthermore, oligophagous Papilionidae can metabolise furanocoumarins more efficiently than polyphagous insects by means of their CYP enzymes [13, 14]. Thus, CYP enzymes from oligophagous Papilionidae are a potential resource for biotechnology; however, their metabolic function in processing host plant metabolites, other than furanocoumarins, has yet to be investigated.

Larvae of Papilio machaon, which belongs to the Papilionidae family, are specialists of Apiaceae family plants, such as Angelica keiskei, Oenanthe javanica and Foeniculum vulgare (commonly known as fennel). These plants have been used as traditional medicine for a wide range of ailments, with their extracts showing various biological activities, including antitumour, anti-inflammatory, antioxidant and antiviral effects [15,16,17]. Therefore, we speculated that the metabolites of P. machaon larval frass, which includes host plant-derived metabolites, may contain biological activities of interest [18]. In this study, we aimed to evaluate the biological activity of the metabolites from the frass of P. machaon larvae fed on A. keiskei, O. javanica or F. vulgare using a human cancer cell line (HCT116), and we analysed the metabolic process of these metabolites in P. machaon larvae (Fig. 1).

Fig. 1
figure 1

Outline of this study. P. machaon larvae ate three kinds of host plants, and then the metabolites were extracted from the frass of each host plant group using three kinds of organic solvents, and the fractions were separated using open column chromatography. The fractions were examined for biological activity using human cancer cell lines

Full size image

Results

Fraction derived from the chloroform extract of frass of P. machaon larvae fed with A. keiskei specifically inhibits HCT116 cell viability

First, we produced extracts from the frass of P. machaon larvae fed on A. keiskei, O. javanica, or F. vulgare using n-hexene, chloroform, and methanol (Fig. 1). Then, we compared the metabolic products from both P. machaon frass and the leaves of A. keiskei, O. javanica, or F. vulgare using thin-layer chromatography (TLC) plate chromatograms (Fig. 2, Table S1). Staining the TLC plate chromatograms with sodium phosphomolybdate solution revealed two spots with calculated Relative to front (Rf) values ranging from 0.55 to 0.40 in the n-hexane extract (Hex. ext.) of frass from O. javanica-fed larvae (Fig. 2A lane 1, blue arrowheads), and we detected five spots with Rf values ranging from 0.55 to 0.31 in the Hex. ext. of frass from A. keiskei-fed larvae (Fig. 2A lane 3, orange arrowheads), and one spot with an Rf value of 0.36 in the Hex. ext. of frass from F. vulgare-fed larvae (Fig. 2A lane 5, grey arrowheads). Seven spots with Rf values from 0.61 to 0.13 were detected in the chloroform extract (Chl. ext.) of frass from O. javanica-fed larvae (Fig. 2B lane 1, blue arrowheads), and we detected five spots with Rf values ranging from 0.57 to 0.39 in the Chl. ext. of frass from A. keiskei-fed larvae (Fig. 2B lane 3, orange arrowheads), and five spots of Rf values from 0.59 to 0.18 in the Chl. ext. of frass from F. vulgare-fed larvae (Fig. 2B lane 5, grey arrowheads). Three spots with Rf values from 0.76 to 0.30 were detected in the methanol extract (Met. ext.) of frass from O. javanica-fed larvae (Fig. 2C lane 1, blue arrowheads), and we detected five spots with Rf values from 0.85 to 0.38 in the Met. ext. of frass from A. keiskei-fed larvae (Fig. 2C lane 3, orange arrowheads), and five spots of Rf values from 0.86 to 0.38 in the Met. ext. of frass from F. vulgare-fed larvae (Fig. 2C lane 5, grey arrowheads). We did not find these spots in the Hex., Chl. and Met. extracts of leaves from the A. keiskei, O. javanica, and F. vulgare plants (Fig. 2A and C, lane 2,4,6). In a previous study, the Chl. ext. of frass from Poncirus trifoliata-fed Papilio xuthus larvae inhibited cell viability of the human pancreatic cancer cell line MIA PaCa2 [19].

Fig. 2
figure 2

Comparison of the metabolites included in each extract using TLC analysis. The different spots between extracts from frass and extracts from host plants are shown as arrowheads for Hex. ext. (A), Chl. ext. (B) and Met. ext. (C)

Full size image

Thus, we chose the Chl. extracts produced from the frass of P. machaon larvae that had been fed on A. keiskei, O. javanica or F. vulgare for use in subsequent investigations. These extracts were separated using open column chromatography to obtain an active fraction for use in testing the biological activity of the extracts against the human cancer cell line HCT116. Next, we evaluated the cell viability under exposure to these fractions using the HCT116 cell line by WST-1 or WST-8 assay. The Chl. ext. of frass from P. machaon larvae that fed on O. javanica was separated into three fractions, and these fractions did not decrease viability in the HCT116 cells (Fig. 3A and B). Similarly, the Chl. ext. of frass from P. machaon larvae that fed on F. vulgare, which was separated into four fractions, did not decrease viability in the HCT116 cells (Fig. 3C and D).

Fig. 3
figure 3

Cell viability of HCT116 cells treated with fractions. (A, B) Fractions separated from Chl. ext. of O. javanica-fed larval frass (A) and their effect on HCT116 viability (B). (C, D) Fractions separated from Chl. ext. of F. vulgare-fed larval frass (C) and their effect on HCT116 viability (D). Cell viability was measured using the WST-8 assay. Error bars represent the mean ± SD from three biological replicates

Full size image

However, the Chl. ext. of frass from P. machaon larvae that fed on A. keiskei was separated into eleven fractions, and TLC analysis showed the different spots in the Fraction (Fr.) 1, 3, and 9 (Fig. S1A, orange arrowheads). Of these fractions, we found that Fr. 1 showed significantly decreased viability (10.4%) in HCT116 cells (Fig. 4A and B). Additionally, we measured proliferation of HCT116 cells by using the bromodeoxyuridine (BrdU) assay. We observed significantly decreased BrdU uptake in HCT116 cells treated with 5 µg/mL of Fr. 1 from the A. keiskei-fed larval frass (Fig. 4C).

Fig. 4
figure 4

Biological activity on HCT116 by fractions separated from A. keiskei-fed larval frass. (A) Thirteen fractions separated from Chl. ext. using open column chromatography. (B) Cell viability of HCT116 cells treated with Fr. 1, Fr. 3 and Fr. 9. (C) BrdU assay in HCT116 cells after treatment with Fr. 1. The absorbance indicates inhibition of cell proliferation. (D) Morphological observation of HCT116 cells treated with 5 µg/mL of Fr. 12 or Fr. 13. DMSO (0.5%, v/v) was used as a negative control (NC), and 5 µM cisplatin (CDDP) was used as a positive control (PC). (E) Cell viability of A549 (lung cancer), HeLa (cervical cancer), HepG2 (hepatic cancer), MIA PaCa2 (pancreatic cancer), TGBC1TKB (gallbladder cancer) and HFSKF-II (human fibroblasts derived from foetal skin) cells treated with 5 µg/mL of Fr. 12. Cell viability was determined by WST-1 assay. Error bars represent the mean ± SD from three biological replicates. Significant differences from the control were analysed with Student’s t test, *P <0.05, **P <0.01, ***P <0.001. Scale bars = 100 μm

Full size image

Then, we separated Fr. 1 by open-column chromatography into two fractions (Fr.12 and 13). TLC analysis showed a different spot in Fr.12 compared with Fr.13 (Fig. S1B, red and black arrowheads). Next, we observed cell morphological changes in HCT116 cells treated with Fr. 12 or 13, and we found the cell density was decreased in HCT116 cells treated with Fr. 12 compared with Fr. 13, and the negative control (Fig. 4D). Subsequently, we evaluated the viability several human cancer cell lines using Fr. 12. The results indicated that Fr.12 significantly affected cell viability only in the HCT116 cell line, but not in the A549, HeLa, HepG2, MIA PaCa2, and TGBC1TKB cell lines. Interestingly, Fr. 12 did not affect the viability of HFSKF-II cells, a normal human fibroblast cell line (Fig. 4E). TLC analysis revealed the specific spot in Fr.12 compared with Fr.13 (Fig. S1B, red arrowhead). Therefore, Fr. 12 only strongly affected the colon cancer cell line.

Examination of metabolites in larval food plants using the TUATinsecta database

Fr.12, which was extracted from the frass of P. machaon larvae that were fed A. keiskei, inhibited HCT116 cell viability, whereas the fractions from the frass of larvae that were fed O. javanica or F. vulgare had no effect on the HCT116 cell viability. Thus, we predicted that the chloroform extract frass from A. keiskei-fed larvae contained compounds that differ from compounds in the frass of larvae fed with O. javanica or F. vulgare. To examine the compounds of each food plant—O. javanica, F. vulgare, or A. keiskei—we utilised the TUAT-insecta database, which integrates information for insect-host plant, its ingredients and ingredient-biological function. We can get plant-metabolite information or related biological activity when we input the host plant’s name [20]. This interrogation revealed that the metabolites of A. keiskei, O. javanica and F. vulgare are mainly classified into 3 groups based on their chemical structures: phenolics, terpenes/steroids and alkaloids. Notably, chalcones, including phenolics and flavonoids, are found only in A. keiskei (Table 1).

Table 1 Grouping the metabolites from food plants using the TUATinsecta database
Full size table

Mechanism of action of metabolites from the frass of P. machaon larvae fed with A. keiskei on HCT116 cells

To investigate the mechanism by which Fr. 12 decreases HCT116 cell viability, we employed transcriptome analysis using total RNA of the HCT116 cells after 48 h of treatment with 5 µg/mL of Fr. 12. Then, differentially expressed genes (DEGs) were identified based on the DEG analysis (false discovery rate, FDR < 0.05). All DEGs were shown in Table S2. Gene enrichment analysis showed that the DEGs were related to microtubule cytoskeleton organisation (GO: 0000226), a Gene Ontology (GO) term involved in cell division (Fig. 5). Additionally, we found that 19 genes out of the top 50 DEGs were those involved in HCT116 cell proliferation (15 genes), the cell cycle (6 genes) and apoptosis (9 genes) (Table 2 and Table S2). Therefore, our transcriptome analysis showed that Fr. 12 may induce inhibition of cell proliferation and apoptosis in HCT116 cells.

Table 2 The function of the top 50 DEGs in HCT116 cells treated with Fr. 12. DEGs
Full size table
Fig. 5
figure 5

Gene enrichment analysis of DEGs in HCT116 cells treated with Fr. 12. Gene enrichment analysis of differentially expressed transcripts in HCT116 cells treated with Fr. 12 using Metascape. A bar graph for enriched terms across the input transcript lists; different coloured bars indicate different P values

Full size image

Exploration of metabolic enzyme candidates that convert metabolites, including those from larval food plants, to active agents

Extracts from the frass of P. machaon larvae fed with A. keiskei inhibited HCT116 cell viability. This indicates that metabolic enzymes in P. machaon convert metabolites, including those from its food plants, to active agents. Thus, we compared metabolic enzymes among the larvae that were fed A. keiskei, O. javanica or F. vulgare by using midgut and fat body transcriptome analysis. Importantly, CYP enzymes play a critical role in the metabolism of plant secondary metabolites [42]. Therefore, we focused on CYPs as metabolic enzymes that convert metabolites, including those of food plants, to active agents. First, we examined upregulated CYPs using their TPM value (Table S3). Next, we validated whether these CYP genes were upregulated or not by real-time quantitative PCR (RT-qPCR) as a biological replication, and we found that the expression of CYP6B2 was upregulated in the midgut of A. keiskei-fed larvae compared with expression in the midgut of F. vulgare-fed larvae (Fig. 6A-C). Finally, we concluded that the CYP6B2 transcript was upregulated in the midgut of A. keiskei-fed larvae.

Fig. 6
figure 6

mRNA expression of CYP transcripts in the P. machaon larval midgut. The CYP transcripts expressed in the larval midgut fed on A. keiskei or F. vulgare were validated by RT-qPCR (n = 3). Relative Quantification (RQ) values represent the relative expression level compared to the larval midgut fed on F. vulgare as 1. rpL31 was used as endogenous control. The error bars represent the relative minimum / maximum expression levels relative to the mean RQ value from three biological replicates. (A) CYP6B2, (B) CYP6B5, (C) CYP6B6.

Full size image

Discussion

In this study, we examined the biological activity of the metabolites in the frass extracts from P. machaon larvae fed A. keiskei, O. javanica or F. vulgare using a human cancer cell line (HCT116) and we analysed the metabolic processing of these metabolites in the larvae. P. machaon metabolites included in the chloroform extract from frass of A. keiskei-fed larvae inhibited cell viability and proliferation only in HCT116 cells. Notably, the fractions separated from the chloroform extract of A. keiskei leaves did not affect HCT116 viability (Fig. S2). Therefore, our results suggest that the metabolites from the frass might reflect a change in chemical structure and biological activity of A. keiskei metabolites when processed by the P. machaon larval metabolic system. A. keiskei contains several bioactive chalcones that are prenylated or geranylated at the 5’-position [15]. Xanthoangelol (5’-geranylated chalcone) induces apoptosis in the human leukaemia cell line K562 more strongly than isobavachalcone (5’-prenylated chalcone), whereas these compounds have no effect on human umbilical vein endothelial cells (HUV-EC) [43]. Xanthoangelol also suppresses the cell viability of the human leukaemia cells HL60, melanoma cells (CRL1579, AZ521) and human stomach cancer cells (KATO III); however, it does not inhibit A549 cell viability [44, 45]. Taken together, these findings indicate that the anticancer activity of 5’-prenylated chalcones changes depending on their modification. Therefore, we speculated that a P. machaon larval metabolite produced from an A. keiskei chalcone may have inhibited cell viability and proliferation, but only in the HCT116 cell line. The metabolism of chalcones in herbivorous insects awaits further investigation.

It has been reported that the 3’-prenylated chalcone xanthohumol is metabolised by CYP enzymes, and that its metabolites are excreted as faeces in rats [46, 47]. In this study, we found that the expression of CYP6B2 transcript was upregulated in the midgut of A. keiskei-fed larvae compared with expression in the midgut of F. vulgare-fed larvae, and chalcones, including phenolics and flavonoids, were found only in A. keiskei. The CYP enzymes of the CYP6B subfamily from Papilionidae, including CYP6B1 and CYP6B3 from P. polyxenes, CYP6B17 and CYP6B21 from P. glaucus, and CYP6B33 from P. multicaudata, metabolise furanocoumarins [13, 14, 48, 49]. Of these, CYP6B1 enzyme metabolises more than 20 times more xanthotoxin (furanocoumarin) than that of CYP6B3 enzyme [49]. CYP6B1 and CYP6B3 mRNA are highly expressed in the larval midgut when xanthotoxin is administered to P. polyxenes larvae [50]. Although some Papilionidae that use furanocoumarin-containing plants have the CYP6B2 gene, the metabolic products of CYP6B2 enzyme have not been identified [51]. Our results suggest that CYP6B2 enzyme in P. machaon larvae might relate to metabolise furanocoumarins and chalcones from the A. keiskei host plant. Therefore, chalcones from A. keiskei might be metabolised by CYP enzymes in P. machaon larvae as well as in mammals. However, we could not clarify how these P. machaon CYP enzymes metabolize plant-derived ingredients and decrease only HCT116 cell viability in this study.

In a future study, we will examine the function of these P. machaon CYP enzymes and determine whether P. machaon CYPs can be used as a bioprocess tool to change the chemical structure of plant ingredients.

Conclusion

The frass of P. machaon larvae reared on A. keiskei contain a metabolite that prevents cell division and induces apoptosis in human colon HCT116 cells. When we investigated the mechanism by which this metabolite was produced, the CYP6B2 enzyme appeared to be related to the metabolism of A. keiskei tissues containing chalcones. The metabolites from the plant undergo conversion of their biological activity through the P. machaon larval metabolic system (Fig. 7). These results indicate that the metabolites from P. machaon may be useful as pharmaceutical agents against human colon cancer. Additionally, it might be possible to use insect metabolic enzymes for the chemical structural conversion of plant-derived compounds with complex structures. Taken together, our results contribute to the future construction of a novel biotechnology system that entails creating potential pharmaceutical candidates using metabolic enzymes from P. machaon larvae.

Fig. 7
figure 7

The predicted metabolic process for the metabolites derived from host plants. P. machaon larvae eat host plants, and their metabolic enzymes, CYPs, change the structure of compounds included in the host plant tissues. The metabolites from the host plants undergo conversion of their biological activity through the P. machaon larval metabolic process

Full size image

Methods

Insects and sample collection

P. machaon larvae and eggs were collected at the Fuchu campus of the Tokyo University of Agriculture and Technology. P. machaon larvae were reared on the leaves of A. keiskei (open-pollinated variety), O. javanica (population), or F. vulgare (open-pollinated variety). These plants cultivated at the Fuchu campus of the Tokyo University of Agriculture and Technology. Larvae were maintained at 25 °C under a 16-h light/8-h dark cycle. The larval frass and host plant leaves were collected and stored at -30 °C until use.

Extraction of metabolites from larval frass and leaves

We placed each sample on a glass petri dish and freeze-dried it with a lyophiliser (VD-250 F, TAITECH Co., Ltd., Saitama, Japan) for 24 h. We pulverised each sample and weighed it using a Mettler balance. We transferred the weighed sample to a 1000 mL Erlenmeyer flask and added 3 volumes of n-hexane (Hex; Wako Pure Chemical Industries, Ltd., Osaka, Japan). We allowed the mixture to sit for 24 h at room temperature (RT) and then filtered it. We transferred the pellet to a 1000 mL Erlenmeyer flask, added 3 volumes of Hex, left it for 24 h at RT, and then filtered it. We repeated this operation one more time and transferred the filtrate to an eggplant flask for evaporation. We transferred the pellet to a 1000 mL Erlenmeyer flask and added 3 volumes of chloroform (CHCl3; Wako Pure Chemical Industries, Ltd.) to the pellet. Then, we performed the same operation with Hex. We then transferred the pellet to a 1000 mL Erlenmeyer flask and added 3 volumes of methanol (MeOH; Wako Pure Chemical Industries, Ltd.) to the pellet. Then, we performed the same operation with Hex. We concentrated these filtrates, including Hex, CHCl3 or MeOH, by a rotary evaporator (N-1110 N, Tokyo Scientific Instruments Co., Ltd., Tokyo, Japan). Finally, we collected each extract in a screw tube (No. 7, Marem Co., Ltd., Osaka, Japan), kept the extracts in a vacuum desiccator for 3–5 days to completely remove each organic solvent and then weighed the obtained extract. We shielded the screw tubes containing the extracts from light with aluminium foil and stored them at 4 °C until use. We referred to the extract with n-hexane as Hex. ext., the extract with CHCl3 as Chl. ext., and the extract with MeOH as Met. ext. Table S4 shows the weight of samples before and after freeze-drying and the weight of their extracts.

Thin-layer chromatography (TLC)

To separate and compare the metabolites from the larval frass or the host plants, each extract dissolved with extraction solvent at 10 mg/mL was applied to a silica gel TLC aluminium plate (Merck®, Darmstadt, Germany) in 1 µL (chloroform extract) or 2 µL (hexane and methanol extract) using a glass capillary. A mixed solvent was used as the eluent: chloroform-methanol (15:1, v/v) for the hexane extract, chloroform-methanol (10:1, v/v) for the chloroform extract and acetic acid-butanol-H2O (1:8:1, v/v) for the methanol extract. For detection of spots, we first used UV light to mark the spots (256 nm; left, 366 nm; right). Next, the plates were stained with sodium phosphomolybdate n-hydrate in ethanol (9%, v/v) to detect spots without UV exposure. We used the position of each spot as a relative to the front (Rf) value. The Rf value was calculated as follows: distance from baseline travelled by the solute divided by distance from baseline travelled by the solvent (solvent front).

Separation of metabolites using open column chromatography

Chloroform extracts were separated on a silica gel (Kanto Chem., Tokyo, Japan) column (column size 30 × 300 mm and 170 mm height silica gel) with an eluting solvent of chloroform-methanol (10:1, v/v). These fractions dissolved in 300 mL solvent were collected in 26 mL glass test tubes to yield 11 fractions from the frass of A. keiskei-fed larvae, 3 fractions from the frass of O. javanica-fed larvae, 4 fractions from the frass of F. vulgare-fed larvae and 6 fractions from A. keiskei leaves based on the TLC profile. Fr. 1, separated from the chloroform extract from the frass of A. keiskei-fed larvae, inhibited HCT116 viability. Thus, Fr. 1 was further separated using a silica gel (FUJI SILYSIA Chemical LTD., Aichi, Japan) column (column size 15 × 300 mm, 170 mm height silica gel) and by eluting solvent of chloroform-methanol (18:1, v/v). The fractions dissolved in 60 mL of solvent were collected to yield 2 fractions based on the TLC profile.

Cell culture

HCT116 (RCB2979), A549 (RCB0098), HeLa (RCB0007), HepG2 (RCB1886), MIA PaCa2 (RCB2094), TGBC1TKB (RCB1129) and HFSKF-II (RCB0698) cells were obtained from the Riken Cell Bank (Riken Tsukuba, Japan). A549, HCT116, HeLa, HepG2 and MIA PaCa2 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Nacalai Tesque, Kyoto, Japan) supplemented with 10% foetal bovine serum (FBS, Gibco, USA). TGBC1TKB cells were maintained in DMEM supplemented with 5% FBS. HFSKF-II was maintained in Ham’s F-12 (Nacalai Tesque, Kyoto, Japan) supplemented with 15% FBS. One hundred units/mL penicillin‒streptomycin (Fujifilm Wako Pure Chemical Corp., Osaka, Japan) was added to DMEM and Ham’s F-12. Cell lines were cultured at 37 °C in a humidified chamber containing 5% CO2.

Cell viability assays

For the cell viability assays, cells were seeded in 96-well plates (HepG2: 700 cells per well; A549, HCT116, HeLa and TBC1TKB: 1000 cells per well; MIA PaCa2 and HFSKF-II: 2000 cells per well; experiment performed in triplicate). Then, after 24 h of incubation, medium containing each fraction dissolved in dimethyl sulfoxide (DMSO; Wako Pure Chemical Industries, Ltd.) was added to each well at final concentrations of 0.05, 0.5 and 5 µg/mL, whereas medium containing only DMSO was added to each control well at a final concentration of 0.5% (v/v). Forty-eight hours later, 10 µL/well WST-1 reagent (Takara Bio., Shiga, Japan) or WST-8 reagent (Doujin Chemical, Kumamoto, Japan) was added. After incubation for 4 h, the absorbance at 450 and 620 nm was measured using a microplate reader, iMark™ (Bio-Rad Laboratories Inc., Hercules, California, U.S.A.) or Gene5 (BioTek Instruments, Winooski, Vermont, U.S.A). The value of the absorbance at 620 nm, which was the control wavelength, was subtracted from the value of the absorbance at 450 nm. As a medium blank, 10 µL of WST-1 or WST-8 reagent was added to a well containing only medium without cells, and the average of the absorbance of this well was subtracted from all other values obtained. Finally, the cell viability was calculated using the following formula.

$$\begin{gathered} Cell{\text{ }}viability{\text{ }}\left( \% \right){\text{ }} = \hfill \\\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\left( {\left[ {Ab{s_{sample}} - Ab{s_{blank}}} \right]/\left[ {{\text{ }}Ab{s_{control}} - Ab{s_{blank}}} \right]} \right){\text{ }} \times {\text{ }}100 \hfill \\ \end{gathered}$$

Cell proliferation assay

For the BrdU cell proliferation assay, 1000 HCT116 cells per well were cultured in a 96-well plate for 24 h. Then, the medium including Fr. 1 dissolved in DMSO was diluted with medium, and the medium was added to a well to a final concentration of 0.2, 1 and 5 µg/mL. After incubation for 24 h, 20 µL of BrdU reagent-containing medium was added to the measuring well, whereas 20 µL of BrdU reagent-free medium was added to a well as background. Finally, after culturing for 24 h, cell proliferation was evaluated using a BrdU assay kit (Abcam, Cambridge, U.K). according to the manufacturer’s protocols. The absorbance of background wells was subtracted from all other values obtained.

Cell morphological observation

To assess the morphological changes in HCT116 cells after treatment with Fr. 12 and Fr. 13, the cells were cultured in a 24-well plate for 24 h. Then, Fr. 12, Fr. 13 (5 µg/mL), 0.5% (v/v) DMSO as a negative control or 5 µM cisplatin (CDDP; Fujifilm Wako Pure Chemical Corp., Osaka, Japan) as a positive control. Forty-eight hours later, the cell morphology was observed under a microscope, EVOS® FLoid® Cell Imaging Station (Thermo Fisher Scientific, Waltham, Massachusetts, U.S.A.)

Comparisons of host plant components

To compare the chemicals contained in each host plant, we utilised TUAT-insecta [20], which is a database integrating information on herbivorous insects and their host plants with information on biological activity and chemicals. Information on A. keiskei, O. javanica and F. vulgare was obtained using their scientific names to search the TUAT-insecta database.

RNA sequencing for HCT116 cells treated with Fr. 12

We purified total RNA from HCT116 cells treated with Fr. 12 (5 µg/mL, n = 3) or 0.1% (v/v) DMSO (n = 3) using TRIzol reagent (Thermo Fisher Scientific, USA) and the PureLink® RNA Extraction Kit (Thermo Fisher Scientific, Japan) according to the manufacturer’s protocol. Then, we used an Agilent TapeStation 2200 (Agilent Technologies, Santa Clara, CA) to assess the RNA quality. cDNA library construction of total RNA from these samples (100 ng) was carried out using the TruSeq® Stranded mRNA Library Preparation Kit (Illumina, Inc., San Diego, CA) according to the manufacturer’s instructions. Finally, the libraries (100 bp, paired-end) were sequenced using the Illumina NovaSeq6000 platform.

Gene enrichment analysis in HCT116 cells treated with Fr. 12

To analyse the differentially expressed genes (DEGs) in HCT116 cells treated with Fr. 12, FASTQ files were assessed by TrimGalore! version 0.6.6 (https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/). Then, these transcript data were mapped to the human transcript reference (GRCh38.p14) obtained from NCBI (accessed on 5th September 2022) using Salmon version 1.9.0 (http://salmon-tddft.jp/download.html) to obtain transcripts per kilobase million (TPM). Read count data were obtained from TPM by Salmon using the tximport package version 1.24.0 (https://github.com/thelovelab/tximport) [52]. All statistical analyses were performed using R version 4.2.1 (https://www.r-project.org/) with TMM normalization and voom package version 3.16. We extracted DEGs from HCT116 cells treated with Fr. 12 using a False Discovery Rate (FDR) < 0.05 [53]. Finally, we performed a functional analysis using the gene list of DEGs with the Metascape online tool (https://metascape.org/) with a default parameters [54]. The most statistically significant term within a cluster were chosen by Metascape to represent the cluster. Three samples in each group were used for RNA sequencing analysis.

RNA-Seq analysis in P. machaon larval midgut and fat body

Total RNA from the midgut and fat body of P. machaon 5th larvae reared on A. keiskei (n = 1), O. javanica (n = 1) or F. vulgare (n = 1) were purified, and RNA-seq analysis was performed as mentioned above. All expressed genes were calculated using P. machaon reference transcripts downloaded from NCBI (ilPapMach1.1, accessed on 5th September 2022). To explore the CYP genes, we used the reference transcript data as follows: (1) translated the reference transcript into amino acid sequences using TransDecoder (https://github.com/TransDecoder/TransDecoder/releases) version 5.5.0; (2) the translated transcripts that having CYP domain (ID: PF00067, Protein Family (Pfam) database) extracted by HMMER program (http://hmmer.org) for obtaining CYP candidate IDs; (3) CYP multi-fasta file was created from the translated transcripts by extracting CYP candidate IDs list using makeblastdb with -parse_seqIds option in blastdbcmd functions of BLAST package (Basic Local Alignment Search Tool, version 2.13.0); (4) performed the functional gene annotation for identified CYP transcripts by BLAST search by blastp program with a cut-off E-value of 1e-10 using UniProt as a reference database; (4) Finally, we integrated the TPM from the Salmon results and the annotated these CYPs using TIBCO Spotfire v12.2.0 (TIBCO Spotfire, Inc., Palo Alto, CA; http://spotfire.tibco.com/better-world-donation-program/).

Evaluating mRNA expression of CYP6B2, CYP6B5 and CYP6B6 in P. machaon larval midgut by RT-qPCR

Total RNA from the midgut of P. machaon 5th larvae reared on A. keiskei (n = 3), or F. vulgare (n = 3) were purified, then one microgram of total RNA was treated with DNase I (Invitrogen, Van Allen Way, Carlsbad, CA, USA), and then 500 ng of DNase-treated total RNA was used as a template for cDNA synthesis using a PrimeScript™ 1st strand cDNA Synthesis Kit (Takara co. Ltd., Tokyo, Japan) in accordance with the manufacturer’s instructions. Real-time quantitative PCR (RT‒qPCR) was performed in 20 µL reaction volumes with 0.13 µL of cDNA template and 0.4 µM each specific primers (Table 3) along with a KAPA SYBR Fast qRT‒PCR Kit (Nippon Genetics Co., Ltd., Tokyo, Japan) in accordance with the manufacturer’s instructions. RT‒qPCR was performed on a Step One Plus Real-Time PCR System (Applied Biosystems Foster City, CA) by following the Delta–Delta Ct method. rpL31 was utilized as an endogenous reference against which RNA expression levels were standardized, and all data were calibrated against universal reference data. The relative expression level in comparison to a reference sample is represented by relative quantification (RQ) values. All sample sets were assayed in triplicate as biological replicates.

Table 3 Primers for RT-qPCR were used in this study
Full size table

Data Availability

The RNA sequencing datasets generated and/or analysed during the current study are available in the Sequence Read Archive, DNA Data Bank of Japan repository, under the following accession IDs: HCT116 treated with Fr. 12 (SRA accession numbers: DRR428503, DRR428504, and DRR428505), HCT116 treated with DMSO (SRA accession numbers: DRR428506, DRR428507, and DRR428508), P. machaon larval midgut and fat body from larvae eating A. keiskei, (DRR428498, and DRR428497), P. machaon larval midgut and fat body from larvae eating O. javanica (DRR428502, and DRR428501), and P. machaon larval midgut and fat body from larvae eating F. vulgare (DRR428500, and DRR428499).

Data Availability

The RNA sequencing datasets generated and/or analysed during the current study are available in the Sequence Read Archive, DNA Data Bank of Japan repository, under the following accession IDs: HCT116 treated with Fr. 12 (SRA accession numbers: DRR428503, DRR428504, and DRR428505), HCT116 treated with DMSO (SRA accession numbers: DRR428506, DRR428507, and DRR428508), P. machaon larval midgut and fat body from larvae eating A. keiskei, (DRR428498, and DRR428497), P. machaon larval midgut and fat body from larvae eating O. javanica (DRR428502, and DRR428501), and P. machaon larval midgut and fat body from larvae eating F. vulgare (DRR428500, and DRR428499).

Abbreviations

CYP:

Cytochrome P450

TLC:

Thin-layer chromatography

DEG:

Differentially Expressed Gene

GO:

Gene Ontology

KEGG:

Kyoto Encyclopedia of Genes and Genomes

PCA:

Principal component analysis

TPM:

Transcripts per million

FDR:

False Discovery Rate

BrdU:

Bromodeoxyuridine

Rf:

Relative to the front

RT-qPCR:

Real-time quantitative PCR

rpL31:

60 S ribosomal protein L31

RQ:

Relative quantification

References

  1. Adler PH, Foottit RG. Introduction. Insect Biodiversity, edited by Foottit RG, Adler PH, 1st ed. Wiely; 2017;1:1–7.

  2. Bernays EA, Chapman RF. Patterns host-plant use. Host-plant selection by phytophagous insects, edited by Bernays EA, Chapman RF, 1st ed. Chapman & Hall; 1994.

  3. Engler-Chaouat HS, Gilbert LE. De novo synthesis vs. sequestration: negatively correlated metabolic traits and the evolution of host plant specialization in cyanogenic butterflies. J Chem Ecol. 2007;33:25–42.

    Article  CAS  PubMed  Google Scholar 

  4. Klitzke CF, Brown KS Jr. The occurrence of aristolochic acids in neotropical troidine swallowtails (Lepidoptera: Papilionidae). Chemoecology. 2000;10(2):99–102.

    Article  CAS  Google Scholar 

  5. Brower LP, Brower JV. Birds, butterflies, and plant poisons: a study in ecological chemistry. Zoologica: Sci Contrib New York Zoological Soc. 1964;37(13):137–59.

    Google Scholar 

  6. Choen SF. Stick and leaf insect (Phasmida: Insecta) biodiversity in the natural reserves of Singapore. Proceedings of the Nature Reserves Survey Seminar Gardens’ Bulletin Singapore, 1997;49:297–312.

  7. DeFoliart GR. Chapter 25. Other Countries in Southeastern Asia. The Human Use of Insects as a Food Resource: A Bibliographic Account in Progress. 2002. https://insectsasfood.russell.wisc.edu/the-human-use-of-insects-as-a-food-resource/.

  8. Singh KP, Jayasomu RS. Bombyx mori - A review of its potential as a medicinal insect. Pharm Biol. 2002;40(1):28–32.

    Article  Google Scholar 

  9. Dossey AT. Insects and their chemical weaponry: new potential for drug discovery. Nat Prod Rep. 2010;27(12):1737–57.

    Article  CAS  PubMed  Google Scholar 

  10. Urlacher VB, Girhard M. Cytochrome P450 monooxygenases in biotechnology and synthetic biology. Trends Biotechnol. 2019;37(8):882–97.

    Article  CAS  PubMed  Google Scholar 

  11. Bernhardt R, Urlacher VB. Cytochromes P450 as promising catalysts for biotechnological application: chances and limitations. Appl Microbiol Biotechnol. 2014;98(14):6185–203.

    Article  CAS  PubMed  Google Scholar 

  12. Berenbaum M. Coumarins and caterpillars: a case for coevolution. Evolution. 1983;37(1):163–79.

    Article  CAS  PubMed  Google Scholar 

  13. Li W, Schuler MA, Berenbaum MR. Diversification of furanocoumarin-metabolizing cytochrome P450 monooxygenases in two papilionids: specificity and substrate encounter rate. Proc Natl Acad Sci USA. 2003;100(Suppl 2):14593–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Mao W, Schuler MA, Berenbaum MR. Cytochrome P450s in Papilio multicaudatus and the transition from oligophagy to polyphagy in the Papilionidae. Insect Mol Biol. 2007;16(4):481–90.

    Article  CAS  PubMed  Google Scholar 

  15. Kil Y, Pham ST, Seo EK, Jafari M. Angelica Keiskei, an emerging medicinal herb with various bioactive constituents and biological activities. Arch Pharm Res. 2017;40(6):655–75.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Lu C, Li X. A review of Oenanthe javanica (blume) DC. As traditional medicinal plant and its therapeutic potential. Evidence-Based Complement Altern Med. 2019;2019:6495819.

    Article  Google Scholar 

  17. Badgujar SB, Patel VV, Bandivdekar AH. Foeniculum vulgare mill: a review of its botany, phytochemistry, pharmacology, contemporary application, and toxicology. Biomed Res Int. 2014;2014:842674.

    Article  PubMed  PubMed Central  Google Scholar 

  18. Bull DL, Ivie GW, Beier RC, Pryor NW, Oertli EH. Fate of photosensitizing furanocoumarins in tolerant and sensitive insects. J Chem Ecol. 1984;10(6):893–911.

    Article  CAS  PubMed  Google Scholar 

  19. Nakane W, Nakamura H, Nakazato T, Kaminaga N, Nakano M, Sakamoto T, Nishiko M, Bono H, Ogiwara I, Kitano Y, Iwabuchi K, Kinoshita K, Simpson RJ, Tabunoki H. Construction of TUATinsecta database that integrated plant and insect database for screening phytophagous insect metabolic products with medicinal potential. Sci Rep. 2020;10(1):17509.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. TUATinsecta database. Tokyo University of Agriculture and Technology, Tokyo. 2019. http://togodb.org/db/tuat_insecta. Accessed 4 Oct 2022.

  21. Youn CK, Lee J, Hariharasudhan G, Kim HB, Kim JB, Lee S, Lim SC, Yoon SP, Park SG, Chang IY, You HJ. HspBP1 is a dual function regulatory protein that controls both DNA repair and apoptosis in Breast cancer cells. Cell Death Dis. 2022;13(4).

  22. Han G, Wei Z, Cui H, Zhang W, Wei X, Lu Z, Bai X. NUSAP1 gene silencing inhibits cell proliferation, migration and invasion through inhibiting DNMT1 gene expression in human Colorectal cancer. Exp Cell Res. 2018;367(2):216–21.

    Article  CAS  PubMed  Google Scholar 

  23. Faria JA, Corrêa NC, de Andrade C, de Angelis Campos AC, Dos Santos Samuel, de Almeida R, Rodrigues TS, de Goes AM, Gomes DA, Silva FP. SET domain-containing protein 4 (SETD4) is a newly identified cytosolic and nuclear lysine methyltransferase involved in breast cancer cell proliferation. J Cancer Sci Ther 2013;21;5(2):58–65.

  24. Mo Q, Xu K, Luo C, Zhang Q, Wang L, Ren G. BTNL9 is frequently downregulated and inhibits proliferation and Metastasis via the P53/CDC25C and P53/GADD45 pathways in Breast cancer. Biochem Biophys Res Commun. 2021;553:17–24.

    Article  CAS  PubMed  Google Scholar 

  25. Charong N, Patmasiriwat P, Zenklusen JC. Localization and characterization of ST7 in cancer. J Cancer Res Clin Oncol. 2011;137:89–97.

    Article  CAS  PubMed  Google Scholar 

  26. Singh K, Mogare D, Giridharagopalan RO, Gogiraju R, Pande G, Chattopadhyay S. p53 target gene SMAR1 is dysregulated in Breast cancer: its role in cancer cell migration and invasion. PLoS ONE. 2007;2(8).

  27. Taye N, Alam A, Ghorai S, Chatterji, Deya G, Parulekar A, Mogare D, Singh S, Sengupta P, Chatterjee S, Bhat MK, Santra MK, Salunkhe PB, Finston SK, Chattopadhyay S. SMAR1 inhibits Wnt/β-catenin signaling and prevents Colorectal cancer progression. Oncotarget. 2018;9(30):21322.

    Article  PubMed  PubMed Central  Google Scholar 

  28. Xu Y, Zhu K, Chen J, Lin L, Huang Z, Zhang J, Chen Y. SASS6 promotes proliferation of esophageal squamous carcinoma cells by inhibiting the p53 signaling pathway. Carcinog (New York). 2021;42(2):254–62.

    Google Scholar 

  29. Du L, Jing J, Wang Y, Xu X, Sun T, Shi Y, Wang W, Tian B, Han C, Zhao X, Chang H. Knockdown of SASS6 reduces growth of MDA-MB-231 triple-negative Breast cancer cells through arrest of the cell cycle at the G2/M phase. Oncol Rep. 2021;456:101.

    Article  Google Scholar 

  30. Liu SS, Zheng HX, Jiang HD, He J, Yu Y, Qu YP, Yue L, Zhang Y, Li Y. Identification and characterization of a novel gene, c1orf109, encoding a CK2 substrate that is involved in cancer cell proliferation. J Biomed Sci. 2012;19(1):1–11.

    Article  Google Scholar 

  31. Long Q, Sun J, Lv J, Liang Y, Li H, Li X. PTPN13 acts as a Tumor suppressor in clear cell renal cell carcinoma by inactivating akt signaling. Exp Cell Res. 2020;396(1):112286.

    Article  CAS  PubMed  Google Scholar 

  32. Pellon-Maison M, Montanaro MA, Lacunza E, Garcia-Fabiani MB, Soler-Gerino MC. Glycerol-3-phosphate acyltranferase-2 behaves as a cancer testis gene and promotes growth and tumorigenicity of the Breast cancer MDA-MB-231 cell line. PLoS ONE. 2014;9(6).

  33. Pangon L, Ng I, Giry-Laterriere M, Currey N, Morgan A, Benthani F, Tran PN, Al-Sohaily S, Segelov E, Parker BL, Cowley MJ, Wright DC, Heaps LSt, Carey L, Rooman I, Kohonen-Corish MRJ. JRK is a positive regulator of β-catenin transcriptional activity commonly overexpressed in colon, breast and Ovarian cancer. Oncogene. 2016;35(22):2834–41.

    Article  CAS  PubMed  Google Scholar 

  34. Cai Z, Zhai T, Muhanhali D, Ling Y. TNRC6C functions as a Tumor suppressor and is frequently downregulated in papillary thyroid cancer. Int J Endocrinol. 2021;2021:1.

    Article  Google Scholar 

  35. Strzeszewska-Potyrała A, Staniak K, Czarnecka-Herok J, Rafiee M-R, Herok M, Mosieniak G, Krijgsveld J, Sikora E. Chromatin-Directed Proteomics identifies ZNF84 as a p53-Independent Regulator of p21 in genotoxic stress response. Cancers. 2021;13(9):2115.

    Article  PubMed  PubMed Central  Google Scholar 

  36. Zhang L, Xu B, Qiang Y, Huang H, Wang C, Li D, Qian J. Overexpression of deubiquitinating enzyme USP28 promoted non-small cell Lung cancer growth. J Cell Mol Med. 2015;19(4):799–805.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Chen L, Xu Z, Li Q, Feng Q, Zheng C, Du Y, Yuan R, Peng X. USP28 facilitates Pancreatic cancer progression through activation of Wnt/β-catenin pathway via stabilising FOXM1. Cell Death Dis. 2021;12(10):887.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Ko MJ, Seo YR, Seo D, Park SY, Seo JH, Jeon EH, Kim SW, Park KU, Koo DB, Kim S, Bae JH, Song DK, Cho CH, Kim KS, Lee YH. RPL17 promotes Colorectal cancer proliferation and stemness through ERK and NEK2/β-catenin signalling pathways. J Cancer. 2022;13(8):2570–83.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Chen Y, Zhang B, Bao L, Jin L, Yang M, Peng Y, Kumar A, Wang JE, Wang C, Zou X, Xing C, Wang Y, Luo W. ZMYND8 acetylation mediates HIF-dependent Breast cancer progression and Metastasis. J Clin Invest. 2018;128(5):1937–55.

    Article  PubMed  PubMed Central  Google Scholar 

  40. Dou C, Mo H, Chen T, Liu J, Zeng Y, Li S, Guo C, Zhang C. ZMYND8 promotes the growth and Metastasis of hepatocellular carcinoma by promoting HK2-mediated glycolysis. Pathol Res Pract. 2021;219:153345.

    Article  CAS  PubMed  Google Scholar 

  41. Liu Y, Li R, Yin K, Ren G, Zhang Y. The crucial role of SEMA3F in suppressing the progression of oral squamous cell carcinoma. Cell Mol Biol Lett. 2017;22(1):32.

    Article  PubMed  PubMed Central  Google Scholar 

  42. Cui S, Wang L, Ma L, Geng X. P450-Mediated detoxification of botanicals in insects. Phytoparasitica. 2016;44(5):585–99.

    Article  CAS  Google Scholar 

  43. Wang HM, Zhang L, Liu J, Yang ZL, Zhao HY, Yang Y, Shen D, Lu K, Fan ZC, Yao QW, Zhang YM, Teng YO, Peng Y. Synthesis and anti-cancer activity evaluation of novel prenylated and geranylated chalcone natural products and their analogs. Eur J Med Chem. 2015;92:439–48.

    Article  CAS  PubMed  Google Scholar 

  44. Akihisa T, Kikuchi T, Nagai H, Ishii K, Tabata K, Suzuki T. 4-Hydroxyderricin from Angelica Keiskei roots induces caspase-dependent apoptotic cell death in HL60 human Leukemia cells. J Oleo Sci. 2011;60(2):71–7.

    Article  CAS  PubMed  Google Scholar 

  45. Takaoka S, Hibasami H, Ogasawara K, Imai N. Chalcones from Angelica Keiskei induce apoptosis in Stomach cancer cells. J Herbs Spices Med Plants. 2008;14(3–4):166–74.

    Article  CAS  Google Scholar 

  46. Yilmazer M, Stevens JF, Deinzer ML, Buhler DR. In vitro biotransformation of xanthohumol, a flavonoid from hops (Humulus lupulus), by rat liver microsomes. Drug Metab Dispos. 2001;29(3):223–31.

    CAS  PubMed  Google Scholar 

  47. Nookandeh A, Frank N, Steiner F, Ellinger R, Schneider B, Gerhäuser C, Becker H. Xanthohumol metabolites in faeces of rats. Phytochemistry. 2004;65(5):561–70.

    Article  CAS  PubMed  Google Scholar 

  48. Wen Z, Pan L, Berenbaum MR, Schuler MA. Metabolism of linear and angular furanocoumarins by Papilio polyxenes CYP6B1 co-expressed with NADPH cytochrome P450 reductase. Insect Biochem Mol Biol. 2003;33(9):937.

    Article  CAS  PubMed  Google Scholar 

  49. Wen Z, Rupasinghe S, Niu G, Berenbaum MR, Schuler MA. CYP6B1 and CYP6B3 of the Black Swallowtail (Papilio polyxenes): adaptive evolution through subfunctionalization. Mol Biol Evol. 2006;23(12):2434–43.

    Article  CAS  PubMed  Google Scholar 

  50. Petersen RA, Zangerl AR, Berenbaum MR, Schuler MA. Expression of CYP6B1 and CYP6B3 cytochrome P450 monooxygenases and furanocoumarin metabolism in different tissues of Papilio polyxenes (lepidoptera: Papilionidae). Insect Biochem Mol Biol. 2001;31(6–7):679.

    Article  CAS  PubMed  Google Scholar 

  51. Sato A, Okamura Y, Murakami M. Diversification and selection pattern of CYP6B genes in Japanese Papilio butterflies and their association with host plant spectra. PeerJ 2020:8e10625.

  52. Soneson C, Love MI, Robinson MD. Differential analyses for RNA-seq: transcript-level estimates improve gene-level inferences. F1000Research 2015;4.

  53. Su W, Sun J, Shimizu K, Kadota K. TCC-GUI: a Shiny-based application for differential expression analysis of RNA-Seq count data. BMC Res Notes. 2019;12(1):133.

    Article  PubMed  PubMed Central  Google Scholar 

  54. Zhou Y, Zhou B, Pache L, Chang M, Khodabakhshi AH, Tanaseichuk O, Benner C, Chanda SK. Metascape provides a biologist-oriented resource for the analysis of systems-level datasets. Nat Commun. 2019;10(1):1523.

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We are very grateful to Mr. Satoshi Uchimoto from Tokyo University of Agriculture and Technology for maintaining the host plants that were eaten by the swallowtail butterfly larvae and to Dr. Takeru Nakazato from the Database Center for Life Science (DBCLS) for help with the analysis of the RNA sequencing data. We also thank Dr. Kikuo Iwabuchi, Tokyo University of Agriculture and Technology for helpful discussion. We thank Ms. Hiroe Mori for drawing the Fig. 7 illustration.

Funding

This work was supported by JSPS KAKENHI grant numbers 21J21584 to MN, JSPS KAKENHI grants 18H02212 to HT, and JSPS KAKENHI grants 20K20571 to HT. This work was also supported by the Center of Innovation for Bio-Digital Transformation (BioDX), an open innovation platform for industry–academia cocreation (COI-NEXT), Japan Science and Technology Agency (JST, COI-NEXT, JPMJPF2010), provided to H.B. and H.T. The ROIS-DS-JOINT grant 004RP2017 and 016RP2018 were provided to H.B. and H.T.

Author information

Authors and Affiliations

  1. Cooperative Major in Advanced Health Science, Graduate School of Bio-Applications and System Engineering, Tokyo University of Agriculture and Technology, Tokyo, Fuchu, 183-8509, Japan

    Miho Nakano & Hiroko Tabunoki

  2. Department of Science of Biological Production, Graduate School of Agriculture, Tokyo University of Agriculture and Technology, Tokyo, Japan

    Takuma Sakamoto & Hiroko Tabunoki

  3. Department of Applied Biological Science, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai- cho, Fuchu-shi, Tokyo, 183-8509, Japan

    Yoshikazu Kitano

  4. Laboratory of Bio-DX, Genome Editing Innovation Center, Hiroshima University, 3-10-23 Kagamiyama, Higashi-Hiroshima City, 739-0046, Japan

    Hidemasa Bono

  5. Laboratory of Genome Informatics, Graduate School of Integrated Sciences for Life, Hiroshima University, 3- 10-23 Kagamiyama, Higashi-Hiroshima City, 739-0046, Japan

    Hidemasa Bono

  6. Department of Biochemistry and Chemistry, La Trobe Institute for Molecular Science (LIMS), School of Agriculture, Biomedicine and Environment, La Trobe University, Melbourne, VIC, 3086, Australia

    Richard J. Simpson

  7. Institute of Global Innovation Research, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo, 183-8509, Japan

    Richard J. Simpson & Hiroko Tabunoki

Authors
  1. Miho Nakano
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Takuma Sakamoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yoshikazu Kitano
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hidemasa Bono
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Richard J. Simpson
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Hiroko Tabunoki
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

M.N., R.J.S., and H.T. conceived and designed the study. M.N., H.B., and T.S. performed the experiments and collected data. Y.K. helped analyse the ingredients. Writing—original draft: M.N. and H.T. All authors discussed the data and helped with manuscript preparation. H.T. supervised the project. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Hiroko Tabunoki.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Nakano, M., Sakamoto, T., Kitano, Y. et al. An extract from the frass of swallowtail butterfly (Papilio machaon) larvae inhibits HCT116 colon cancer cell proliferation but not other cancer cell types. BMC Genomics 24, 735 (2023). https://doi.org/10.1186/s12864-023-09841-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s12864-023-09841-0

Keywords

BMC Genomics

ISSN: 1471-2164

Contact us