Insects as a Prospective Source of Biologically Active Molecules and Pharmaceuticals—Biochemical Properties and Cell Toxicity of Tenebrio molitor and Zophobas morio Cell-Free Larval Hemolymph
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Citation: Kneži´c, T.; Avramov, M.; Tati´c, V.; Petrovi´c, M.; Gadjanski, I.; Popovi´c, Ž.D. Insects as a Prospective Source of Biologically Active Molecules and Pharmaceuticals— Biochemical Properties and Cell Toxicity of Tenebrio molitor and Zophobas morio Cell-Free Larval Hemolymph. Int. J. Mol. Sci. 2024,25, 7491. https://doi.org/10.3390/ ijms25137491 Academic Editor: Xesús Feás Received: 30 April 2024 Revised: 3 July 2024 Accepted: 4 July 2024 Published: 8 July 2024 Copyright: © 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). International Journal of Molecular Sciences Article Insects as a Prospective Source of Biologically Active Molecules and Pharmaceuticals—Biochemical Properties and Cell Toxicity of Tenebrio molitor and Zophobas morio Cell-Free Larval Hemolymph Teodora Kneži´c 1, Miloš Avramov 2, Vanja Tati´c 2, Miloš Petrovi´c 3, Ivana Gadjanski 1,* and Željko D. Popovi´c 2,* 1Center for Biosystems, BioSense Institute, University of Novi Sad, 21000 Novi Sad, Serbia; [email protected] 2Department of Biology and Ecology, Faculty of Sciences, University of Novi Sad, 21000 Novi Sad, Serbia; [email protected] (M.A.); [email protected] (V.T.) 3Department of Plant and Environmental Protection, Faculty of Agriculture, University of Novi Sad, 21000 Novi Sad, Serbia; [email protected] *Correspondence: [email protected] (I.G.); [email protected] (Ž.D.P.) Abstract: Insects are of great interest as novel sources of alternative proteins and biologically active compounds, primarily anticancer agents. Protein-rich insect larval hemolymph is a prospective candidate for pharmaceutical and food industry-related research. In this study, selected biochemical properties and cell toxicity of larval hemolymph from two mealworm species, Tenebrio molitor and Zophobas morio, were analyzed. Total proteins and carbohydrates, antioxidant capacity, and the level of lipid peroxidation were determined. Human cancer (U-87) and normometabolic (MRC-5) cells were treated with different concentrations of larval hemolymph proteins, and the effects on cell viability were assayed 24, 48, and 72 h after treatments. Z. morio hemolymph was shown to be richer in total proteins, showing a higher antioxidant capacity and lipid peroxidation level than T. molitor hemolymph, which was richer in total carbohydrates. Cytotoxicity assays showed that T. molitor and Z. morio hemolymphs differently affect the viability of U-87 and MRC-5 cells in cell type-, dose-, and time-dependent manners. Hemolymph from both species was more cytotoxic to U-87 cells than to MRC-5 cells, which was particularly prominent after 48 h. Additionally, a more potent cytotoxic effect of Z. morio hemolymph was observed on both cell lines, likely due to its higher antioxidant capacity, compared to T. molitor hemolymph. Keywords: insect hemolymph; yellow mealworm; superworm; biochemical properties; antioxidative status; cell toxicity; alternative proteins 1. Introduction In food science, there is widespread discussion regarding the integration of alternative proteins into new food products. The challenges of producing considerably more food for a growing global population with concurrent reduction in the environmental footprint of our agricultural systems must be addressed. With the world population projected to reach approximately 9.8 billion people by 2050, both the accessibility and affordability of alternative proteins are to be taken into account if they are to play a substantial role in addressing these challenges [ 1 ]. Showing great potential for future food systems, edible insects are considered an environmentally friendly choice as alternative sources of proteins. Their primary benefit is reflected in a good nutritional profile: high percentage of protein with high-quality amino acids, fatty acids (e.g., omega-3), fibers, vitamins (e.g., vitamin B12), and minerals such as calcium and iron [ 1 , 2 ], as well as the highly efficient conversion of ingested matter into biomass. Secondly, the production of insects for food and feed Int. J. Mol. Sci. 2024,25, 7491. https://doi.org/10.3390/ijms25137491 https://www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2024,25, 7491 2 of 20 significantly reduces the environmental footprint by generating less greenhouse gasses and ammonia emissions, as well as by using considerably less water, energy, and land in comparison with conventional animal farming [ 3 , 4 ]. Additionally, many insects possess the potential for recycling agricultural waste products, which they can use as feeding substrates and transform them into nutritious food and feedstuff, which is then returned to the production cycle. Therefore, insects are ideal candidates for integration into the framework of the circular economy concept [ 5 ], while the consumption of insects positively contributes to both the environment and human health. It is estimated that there are more than 2000 insect species used for human consumption, mainly in tropical countries [ 6 , 7 ]. However, according to the Food and Agriculture Organization (FAO) and Wageningen University & Research (WUR), insects can be considered a viable option for both food and feed, not only in tropical regions but in other global regions as well [ 8 ]. In recent years, the subject of the global exploitation of insects as food and feed has been the focus of exploration and study in a plethora of scientific publications [7,9–11]. Today, mealworms represent one of the more prevalent groups of insects that are used as alternative sources of protein worldwide. Although the yellow mealworm Tenebrio molitor and superworm Zophobas morio (Coleoptera: Tenebrionidae) are known as pests of secondary storage products such as grain, flour, and bran, and have therefore been considered unsuitable for human and animal consumption for a very long time [ 12 , 13 ], their notorious status began to change in the early 1980s. It was at that time that the National Aeronautics and Space Administration (NASA) announced that they were exploring the use of insects as an alternative protein source during their space missions [ 14 ]. Since then, mealworms have been one of the focal points of research towards discovering promising alternatives for conventional sources of proteins for both human and animal consumption [ 2 , 15 – 21 ]. Consequently, in 2022, T. molitor was approved by the European Food Safety Authority (EFSA) as a novel food, i.e., insect species safe for human consumption, which allowed for the mass upscaling of production in numerous European countries [ 22 ]. Contrary to its smaller relative, the superworm does not yet have an official edible status, but because of its high-quality nutritional profile and numerous beneficial properties that have been discovered, recent research suggests that, in the near future, species belonging to the genus Zophobas may also be accepted as novel food [20,23,24]. In addition to their nutritional qualities, insects are known to have other beneficial effects on human health. Recent publications, such as the one by Stull et al. (2018), have demonstrated that edible cricket powder supported the growth of the probiotic bacterium Bifidobacterium animalis, thereby improving human gut health and reducing systemic inflammation [ 25 ]. Also, de Carvalho et al. (2019) have shown that T. molitor flour has a potential prebiotic effect [ 26 ], while protein hydrolysates made from edible powders of T. molitor and Acheta domesticus (house cricket) have significant antioxidative power [ 27 ], potentially higher than fresh orange juice and olive oil [ 28 , 29 ]. Similarly, Zieli´nska et al. (2017) suggested that, together with other edible insect species, Z. morio larvae represent a valuable and unexploited source of proteins whose peptides have high antioxidative activity. The results obtained in this study also showed that selected edible insects, after in vitro digestion, have higher antioxidant activity compared to some protein hydrolysates obtained from plants or other animal products [ 30 ]. As for other mealworm species, it has been shown that peptides generated from a protein found in the lesser mealworm Alphitobius diaperinus represent dipeptidyl peptidase IV (DPP IV) inhibitors that play a role in glucose metabolism, and thus in the management of type 2 diabetes [ 31 ]. Although the research was conducted on animal model organisms, it was shown that T. molitorand Z. morio-based full-fat meals, as functional feed additives, increased the growth performance of broiler chickens and changed the traits of their immune system [ 32 ], while defatted Z. morio larvae meals can lead to immunomodulation in the gilt-head seabream fish Sparus aurata [33]. When it comes to T. molitor and Z. morio specifically, different larval extracts of these insects have been shown to exhibit remarkable anticancer activity [ 24 , 34 – 39 ]. However, the
Int. J. Mol. Sci. 2024,25, 7491 3 of 20 effect of larval hemolymph, as the main depot of various biologically active microand macromolecules such as proteins, hormones, and other metabolites, has not been sufficiently examined on different normometabolic and cancer cells. Such research was recently carried out by Mahmoud et al. (2020), who showed that the larval hemolymph and fat body of the flesh fly Sarcophaga argyrostoma (Diptera: Sarcophagidae) had a cytotoxic effect on the MDA-MB-231 cell line, that is breast adenocarcinoma cells [ 40 ]. Also, hemolymph from the stinkbug Aspongopus chinensis Dallas (Hemiptera: Heteroptera: Pentatomidae) has been proven to have antiproliferative and antimetastatic effects on murine (4T1) and human (HCC1937) breast cancer cell lines [ 41 ]. Further, Elfar et al. (2023) tested the anticancer effects of hemolymph extracts from three bee species (Apis mellifera,Chalicodoma siculum, and Xylocopa pubescens) on human liver cancer (HepG2) and human cervical cancer (HeLa) cells, where all hemolymph extracts resulted in the inhibition of cell viability against the tested cancer cell lines in a dose-dependent manner [ 42 ]. Finally, the findings obtained in the most recent study conducted by Osman et al. (2024) concluded that hemolymph from the American cockroach Periplaneta americana, tested on Solid Ehrlich carcinoma-bearing mice, has remarkable anticancer effects [43]. Although there has been recent research devoted to the antimicrobial and antiinflammatory activity of Z. morio hemolymph rich in antimicrobial peptides [ 44 – 46 ], there is a lack of studies not only on the anticancer activity of hemolymph from this species and its relative T. molitor but also regarding their effects on different normometabolic cell lines. The latter research would aid the case for Z. morio to be granted the status of edible insect, while furthering the potential of T. molitor as novel food. Taken altogether, the potential of insects as novel sources of alternative proteins and biologically active compounds merits further exploration. In particular, there is a constant need for suitable replacements to synthetic antioxidants that are added to food as protective substances against free radicals, such as reactive oxygen species (ROS), in part due to strict regulations of their use. Natural compounds, potentially found in insect hemolymph, would therefore be more desirable substitutes for consumers to enhance the quality of food. Besides this aspect, insect hemolymph is a prospective candidate for the discovery of novel pharmaceuticals and anticancer agents. For those reasons, in this study, selected biochemical parameters of T. molitor and Z. morio larval hemolymph, as well the effects of cell-free hemolymph from these insect species on cancer and normometabolic cell lines, were analyzed. Biochemical analyses include assaying total protein and carbohydrate content, as well as antioxidative capacity and levels of lipid peroxidation as indicators of oxidative stress. The effects of different concentrations of T. molitor and Z. morio hemolymph proteins on the viability of U-87 glioblastoma cells and MRC-5 lung fibroblasts were quantified with the MTT assay 24, 48, and 72 h after treatment. 2. Results 2.1. Biochemical Analyses The results obtained from the quantitative analysis of the biochemical properties of larval hemolymph from the two selected insect species have shown that protein content is higher in Z. morio samples in comparison to T. molitor samples (Figure 1A). On the other hand, hemolymph from T. molitor larvae contained nearly twice the concentration of total carbohydrates compared to Z. morio larval hemolymph (Figure 1B). When it comes to the analysis of hemolymph antioxidative status, the results of ferric reducing antioxidant power (FRAP) and lipid peroxidation (MDA) assays have shown that the hemolymph of Z. morio has a higher antioxidative capacity, as well as level of lipid peroxidation, compared to T. molitor hemolymph. Using the antioxidative power of vitamin C as the standard, larval hemolymph from Z. morio was shown to have more than 30% higher antioxidative capacity than the hemolymph from T. molitor (Figure 2A). When it comes to the amount of malondialdehyde (MDA) produced, as a measure of lipid
Int. J. Mol. Sci. 2024,25, 7491 4 of 20 peroxidation levels, hemolymph from Z. morio was demonstrated to contain double the amount of MDA compared to its T. molitor counterpart (Figure 2B). Int.J.Mol.Sci.2024,25,74914of20 Figure1.TotalproteinconcentrationsdeterminedbyBradfordassay(A)andtotalcarbohydrate concentrationsdeterminedbyanthronereaction(B)fromthelarvalhemolymphofselectedinsect species—T.molitor(TM)andZ.morio(ZM).Valuesarereportedasthemeanoftriplicatemeasurementsperformedonhemolymphobtainedfrom100larvaeofeachinsectspecies,respectively,with theerrorbarsrepresentingthestandarddeviationofthemeasurements.Asterisks(*)abovethebars denotestatisticallysignificantdifferencesinthemeasuredvaluesofthebiochemicalparametersbetweenthetwodifferentinsectspecies,determinedbyone-wayANOVAfollowedbyTukey’spost hoctestforasignificancelevelofp<0.05. Whenitcomestotheanalysisofhemolymphantioxidativestatus,theresultsofferric reducingantioxidantpower(FRAP)andlipidperoxidation(MDA)assayshaveshown thatthehemolymphofZ.moriohasahigherantioxidativecapacity,aswellasleveloflipid peroxidation,comparedtoT.molitorhemolymph.UsingtheantioxidativepowerofvitaminCasthestandard,larvalhemolymphfromZ.moriowasshowntohavemorethan 30%higherantioxidativecapacitythanthehemolymphfromT.molitor(Figure2A).When itcomestotheamountofmalondialdehyde(MDA)produced,asameasureoflipidperoxidationlevels,hemolymphfromZ.moriowasdemonstratedtocontaindoublethe amountofMDAcomparedtoitsT.molitorcounterpart(Figure2B). Figure2.AntioxidativecapacityofT.molitor(TM)andZ.morio(ZM)larvalhemolymphmeasuredbythe antioxidativepowerofmgofvitaminCpermgoftotalprotein,determinedbyFRAPassay(A),aswell aslipidperoxidationlevelsmeasuredinnmolofproducedMDApermgofprotein,determinedbyMDA assay(B).Valuesarereportedasthemeanoftriplicatemeasurementsperformedonhemolymphobtainedfrom100larvaeofeachinsectspecies,respectively,withtheerrorbarsrepresentingthestandard deviationofthemeasurements.Asterisks(*)abovethebarsdenotestatisticallysignificantdifferencesin themeasuredvaluesofthebiochemicalparametersbetweenthetwodifferentinsectspecies,determined byone-wayANOVAfollowedbyTukey’sposthoctestforsignificancelevelofp<0.05. 2.2.CytotoxicityAssays ThedatainFigure3showthatthehighestconcentrationofT.molitorhemolymph proteins(2000µg/mL)decreasedcellviabilitybelowthe80%viabilitythresholdalready 24haftertreatment.AsignificantcytotoxiceffectonU-87cellsalsooccurs48haftertreatmentwith1000µg/mLofT.molitorhemolymphtotalproteins,withanearly70%reductionincellviabilitycomparedtoafter24h.Cellviabilitywasfurthernegativelyaffected Figure 1. Total protein concentrations determined by Bradford assay (A) and total carbohydrate concentrations determined by anthrone reaction (B) from the larval hemolymph of selected insect species—T. molitor (TM) and Z. morio (ZM). Values are reported as the mean of triplicate measurements performed on hemolymph obtained from 100 larvae of each insect species, respectively, with the error bars representing the standard deviation of the measurements. Asterisks (*) above the bars denote statistically significant differences in the measured values of the biochemical parameters between the two different insect species, determined by one-way ANOVA followed by Tukey’s post hoc test for a significance level of p< 0.05. Int.J.Mol.Sci.2024,25,74914of20 Figure1.TotalproteinconcentrationsdeterminedbyBradfordassay(A)andtotalcarbohydrate concentrationsdeterminedbyanthronereaction(B)fromthelarvalhemolymphofselectedinsect species—T.molitor(TM)andZ.morio(ZM).Valuesarereportedasthemeanoftriplicatemeasurementsperformedonhemolymphobtainedfrom100larvaeofeachinsectspecies,respectively,with theerrorbarsrepresentingthestandarddeviationofthemeasurements.Asterisks(*)abovethebars denotestatisticallysignificantdifferencesinthemeasuredvaluesofthebiochemicalparametersbetweenthetwodifferentinsectspecies,determinedbyone-wayANOVAfollowedbyTukey’spost hoctestforasignificancelevelofp<0.05. Whenitcomestotheanalysisofhemolymphantioxidativestatus,theresultsofferric reducingantioxidantpower(FRAP)andlipidperoxidation(MDA)assayshaveshown thatthehemolymphofZ.moriohasahigherantioxidativecapacity,aswellasleveloflipid peroxidation,comparedtoT.molitorhemolymph.UsingtheantioxidativepowerofvitaminCasthestandard,larvalhemolymphfromZ.moriowasshowntohavemorethan 30%higherantioxidativecapacitythanthehemolymphfromT.molitor(Figure2A).When itcomestotheamountofmalondialdehyde(MDA)produced,asameasureoflipidperoxidationlevels,hemolymphfromZ.moriowasdemonstratedtocontaindoublethe amountofMDAcomparedtoitsT.molitorcounterpart(Figure2B). Figure2.AntioxidativecapacityofT.molitor(TM)andZ.morio(ZM)larvalhemolymphmeasuredbythe antioxidativepowerofmgofvitaminCpermgoftotalprotein,determinedbyFRAPassay(A),aswell aslipidperoxidationlevelsmeasuredinnmolofproducedMDApermgofprotein,determinedbyMDA assay(B).Valuesarereportedasthemeanoftriplicatemeasurementsperformedonhemolymphobtainedfrom100larvaeofeachinsectspecies,respectively,withtheerrorbarsrepresentingthestandard deviationofthemeasurements.Asterisks(*)abovethebarsdenotestatisticallysignificantdifferencesin themeasuredvaluesofthebiochemicalparametersbetweenthetwodifferentinsectspecies,determined byone-wayANOVAfollowedbyTukey’sposthoctestforsignificancelevelofp<0.05. 2.2.CytotoxicityAssays ThedatainFigure3showthatthehighestconcentrationofT.molitorhemolymph proteins(2000µg/mL)decreasedcellviabilitybelowthe80%viabilitythresholdalready 24haftertreatment.AsignificantcytotoxiceffectonU-87cellsalsooccurs48haftertreatmentwith1000µg/mLofT.molitorhemolymphtotalproteins,withanearly70%reductionincellviabilitycomparedtoafter24h.Cellviabilitywasfurthernegativelyaffected Figure 2. Antioxidative capacity of T. molitor (TM) and Z. morio (ZM) larval hemolymph measured by the antioxidative power of mg of vitamin C per mg of total protein, determined by FRAP assay (A), as well as lipid peroxidation levels measured in nmol of produced MDA per mg of protein, determined by MDA assay (B). Values are reported as the mean of triplicate measurements performed on hemolymph obtained from 100 larvae of each insect species, respectively, with the error bars representing the standard deviation of the measurements. Asterisks (*) above the bars denote statistically significant differences in the measured values of the biochemical parameters between the two different insect species, determined by one-way ANOVA followed by Tukey’s post hoc test for significance level of p< 0.05. 2.2. Cytotoxicity Assays The data in Figure 3show that the highest concentration of T. molitor hemolymph proteins (2000 µ g/mL) decreased cell viability below the 80% viability threshold already 24 h after treatment. A significant cytotoxic effect on U-87 cells also occurs 48 h after treatment with 1000 µ g/mL of T. molitor hemolymph total proteins, with a nearly 70% reduction in cell viability compared to after 24 h. Cell viability was further negatively affected 72 h after treatment. Also, it was observed that hemolymph protein concentrations ranging from 125 µ g/mL to 500 µ g/mL exerted a slight negative effect on U-87 cell viability 48 h after treatment, whereby 72 h after treatment, a recovery of cell viability to the values recorded 24 h after treatment was noticed. Overall, hemolymph protein concentrations below 1000 µ g/mL had no negative effect on U-87 cell viability at any time point. Additionally, according to the results of the two-way analysis of variance (two-way ANOVA),
Int. J. Mol. Sci. 2024,25, 7491 5 of 20 protein concentration and incubation time, as well as the interaction of these two factors, contributed to the decrease in U-87 cell viability. Int.J.Mol.Sci.2024,25,74915of20 72haftertreatment.Also,itwasobservedthathemolymphproteinconcentrationsrangingfrom125µg/mLto500µg/mLexertedaslightnegativeeffectonU-87cellviability48 haftertreatment,whereby72haftertreatment,arecoveryofcellviabilitytothevalues recorded24haftertreatmentwasnoticed.Overall,hemolymphproteinconcentrations below1000µg/mLhadnonegativeeffectonU-87cellviabilityatanytimepoint.Additionally,accordingtotheresultsofthetwo-wayanalysisofvariance(two-wayANOVA), proteinconcentrationandincubationtime,aswellastheinteractionofthesetwofactors, contributedtothedecreaseinU-87cellviability. Figure3.U-87cellviability24h,48h,and72haftertreatmentwithdifferentT.molitorhemolymph totalproteinconcentrations,determinedbyMTTassay.Errorbarsrepresentthestandarddeviation ofthreeindependentexperiments,withfourbiologicalreplicateseach,foreverymeasurement.The bluedashedlinerepresentsthecontrolgrouptakenas100%cellviability.Thereddottedlinerepresentstheviabilitycutoffof80%,belowwhichitisconsideredthatthetreatmentsarecytotoxic. Asterisks(*)abovethebarsdenotestatisticallysignificantdifferencesinmeasuredcellviabilitybetweenthecorrespondingbarslinkedwithlines,determinedbyone-wayANOVAfollowedby Tukey’sposthoctestforsignificancelevelofp<0.05.Interactionbetweenproteinconcentration(C) andincubationtime(T)forthewholeexperimentwasanalyzedbytwo-wayANOVAfollowedby Tukey’sposthoctestforsignificancelevelofp<0.05(topleft). ThedatainFigure4showtheeffectofdifferentT.molitorhemolymphproteinson theviabilityofMRC-5cells24,48,and72haftertreatment.Itcanbeseenthataslight cytotoxiceffectfirstoccurs48haftertreatmentwithatotalproteinconcentrationof1000 µg/mL.Interestingly,here,thetreatmentwith2000µg/mLofhemolymphproteinsdid nothaveanegativeeffectonMRC-5cellviabilityafter24h,incontrasttothesametreatmentinthesetupwithU-87cells.ThisconcentrationonlystartedaffectingMRC-5cells48 haftertreatment,comparedto24hforU-87cells.Additionally,ageneraltrendofagradualdecreaseinMRC-5cellviabilityovertimewasobservedafteralltreatments.Thatbeingsaid,500µg/mLandbelowhemolymphproteinconcentrationsdidnotbringcellviabilitybelow80%even72haftertreatment.AswiththeU-87cellline,theresultsofthe two-wayANOVAshowedthatproteinconcentrationandincubationtime,aswellasthe Figure 3. U-87 cell viability 24 h, 48 h, and 72 h after treatment with different T. molitor hemolymph total protein concentrations, determined by MTT assay. Error bars represent the standard deviation of three independent experiments, with four biological replicates each, for every measurement. The blue dashed line represents the control group taken as 100% cell viability. The red dotted line represents the viability cutoff of 80%, below which it is considered that the treatments are cytotoxic. Asterisks (*) above the bars denote statistically significant differences in measured cell viability between the corresponding bars linked with lines, determined by one-way ANOVA followed by Tukey’s post hoc test for significance level of p< 0.05. Interaction between protein concentration (C) and incubation time (T) for the whole experiment was analyzed by two-way ANOVA followed by Tukey’s post hoc test for significance level of p< 0.05 (top left). The data in Figure 4show the effect of different T. molitor hemolymph proteins on the viability of MRC-5 cells 24, 48, and 72 h after treatment. It can be seen that a slight cytotoxic effect first occurs 48 h after treatment with a total protein concentration of 1000 µ g/mL. Interestingly, here, the treatment with 2000 µ g/mL of hemolymph proteins did not have a negative effect on MRC-5 cell viability after 24 h, in contrast to the same treatment in the setup with U-87 cells. This concentration only started affecting MRC-5 cells 48 h after treatment, compared to 24 h for U-87 cells. Additionally, a general trend of a gradual decrease in MRC-5 cell viability over time was observed after all treatments. That being said, 500 µ g/mL and below hemolymph protein concentrations did not bring cell viability below 80% even 72 h after treatment. As with the U-87 cell line, the results of the two-way ANOVA showed that protein concentration and incubation time, as well as the interaction of these two factors, had a significant effect on the decrease in MRC-5 cell viability after treatment with higher concentrations of T. molitor hemolymph proteins, as evidenced by the pvalues equaling 0, i.e., <0.05, for both analyzed factors and their interactions.
Int. J. Mol. Sci. 2024,25, 7491 6 of 20 Int.J.Mol.Sci.2024,25,74916of20 interactionofthesetwofactors,hadasignificanteffectonthedecreaseinMRC-5cellviabilityaftertreatmentwithhigherconcentrationsofT.molitorhemolymphproteins,asevidencedbythepvaluesequaling0,i.e.,<0.05,forbothanalyzedfactorsandtheirinteractions. Figure4.MRC-5cellviability24h,48h,and72haftertreatmentwithdifferentT.molitorhemolymphtotalproteinconcentrations,determinedbyMTTassay.Errorbarsrepresentthestandard deviationofthreeindependentexperiments,withfourbiologicalreplicateseach,foreverymeasurement.Thebluedashedlinerepresentsthecontrolgrouptakenas100%cellviability.Thered dottedlinerepresentstheviabilitycutoffof80%,belowwhichitisconsideredthatthetreatments arecytotoxic.Asterisks(*)abovethebarsdenotestatisticallysignificantdifferencesinmeasuredcell viabilitybetweenthecorrespondingbarslinkedwithlines,determinedbyone-wayANOVAfollowedbyTukey’sposthoctestforsignificancelevelofp<0.05.Interactionbetweenproteinconcentration(C)andincubationtime(T)forthewholeexperimentwasanalyzedbytwo-wayANOVA followedbyTukey’sposthoctestforsignificancelevelofp<0.05(topleft). WhenitcomestotheeffectofZ.moriohemolymphproteinsontheviabilityoftheU-87 cellline,thedatainFigure5showthatasignificantcytotoxiceffectfirstoccurs48haftertreatmentwithatotalproteinconcentrationofatleast250µg/mL,wherecellviabilitywasreduced byapproximately40%comparedtothevaluesafter24h.However,concentrationsof500 µg/mLandhigherinducemuchstrongercytotoxiceffects,withareductionincellviabilityof around60%onaverageafter48h.Interestingly,incontrasttotheT.molitorsamples,theproteinconcentrationof2000µg/mLonlystartednegativelyaffectingU-87cellviability48hafter treatment.Also,itwasobservedthathemolymphproteinconcentrationsofZ.morioranging from15.625µg/mLto62.5µg/mLexertedaslightnegativeeffectonU-87cellviability48h aftertreatment.AlthoughlessprofoundcomparedtothedataobtainedfromT.molitorsamples(Figure3),asimilartrendincellviabilityrecovery72haftertreatmentwasobserved.That beingsaid,itcanbeseenthattreatmentwithhemolymphproteinconcentrationsuptoand including125µg/mLdidnotnegativelyaffectU-87cellviabilityatanytimepoint.AswithT. molitor,theresultsoftwo-wayANOVAanalysesshowedthatwhereverasignificantdecrease inU-87cellviabilitydidoccur,bothproteinconcentrationandincubationtime,aswellasthe interactionbetweenthesetwofactors,weresignificantcontributorstothiseffect. Figure 4. MRC-5 cell viability 24 h, 48 h, and 72 h after treatment with different T. molitor hemolymph total protein concentrations, determined by MTT assay. Error bars represent the standard deviation of three independent experiments, with four biological replicates each, for every measurement. The blue dashed line represents the control group taken as 100% cell viability. The red dotted line represents the viability cutoff of 80%, below which it is considered that the treatments are cytotoxic. Asterisks (*) above the bars denote statistically significant differences in measured cell viability between the corresponding bars linked with lines, determined by one-way ANOVA followed by Tukey’s post hoc test for significance level of p< 0.05. Interaction between protein concentration (C) and incubation time (T) for the whole experiment was analyzed by two-way ANOVA followed by Tukey’s post hoc test for significance level of p< 0.05 (top left). When it comes to the effect of Z. morio hemolymph proteins on the viability of the U-87 cell line, the data in Figure 5show that a significant cytotoxic effect first occurs 48 h after treatment with a total protein concentration of at least 250 µ g/mL, where cell viability was reduced by approximately 40% compared to the values after 24 h. However, concentrations of 500 µ g/mL and higher induce much stronger cytotoxic effects, with a reduction in cell viability of around 60% on average after 48 h. Interestingly, in contrast to the T. molitor samples, the protein concentration of 2000 µ g/mL only started negatively affecting U-87 cell viability 48 h after treatment. Also, it was observed that hemolymph protein concentrations of Z. morio ranging from 15.625 µ g/mL to 62.5 µ g/mL exerted a slight negative effect on U-87 cell viability 48 h after treatment. Although less profound compared to the data obtained from T. molitor samples (Figure 3), a similar trend in cell viability recovery 72 h after treatment was observed. That being said, it can be seen that treatment with hemolymph protein concentrations up to and including 125 µ g/mL did not negatively affect U-87 cell viability at any time point. As with T. molitor, the results of twoway ANOVA analyses showed that wherever a significant decrease in U-87 cell viability did occur, both protein concentration and incubation time, as well as the interaction between these two factors, were significant contributors to this effect.
Int. J. Mol. Sci. 2024,25, 7491 7 of 20 Int.J.Mol.Sci.2024,25,74917of20 Figure5.U-87cellviability24h,48h,and72haftertreatmentwithdifferentZ.moriohemolymph totalproteinconcentrations,determinedbyMTTassay.Errorbarsrepresentthestandarddeviation ofthreeindependentexperiments,withfourbiologicalreplicateseach,foreverymeasurement.The bluedashedlinerepresentsthecontrolgrouptakenas100%cellviability.Thereddottedlinerepresentstheviabilitycutoffof80%,belowwhichitisconsideredthatthetreatmentsarecytotoxic. Asterisks(*)abovethebarsdenotestatisticallysignificantdifferencesinmeasuredcellviabilitybetweenthecorrespondingbarslinkedwithlines,determinedbyone-wayANOVAfollowedby Tukey’sposthoctestforsignificancelevelofp<0.05.Interactionbetweenproteinconcentration(C) andincubationtime(T)forthewholeexperimentwasanalyzedbytwo-wayANOVAfollowedby Tukey’sposthoctestforsignificancelevelofp<0.05(topleft). ThedatainFigure6showthatasignificantcytotoxiceffectonMRC-5cellsfirstoccurs 48haftertreatmentwithatleast500µg/mLoftotalproteinsfromthehemolymphofZ.morio, witharound60%reductionincellviabilitycomparedtovaluesafter24h.Similareffectswere inducedbythehighestconcentrations48haftertreatment.Also,itwasobservedthathemolymphtotalproteinconcentrationsrangingfrom7.8125µg/mLto125µg/mLexertedaslight negativeeffectonMRC-5cellviability48haftertreatment,albeittheviabilitywasstillabove the80%cutoff.AlthoughlessprofoundcomparedtotheresultsshowninFigure4,thesame trendincellviabilityrecovery72haftertreatmentispresent.Ingeneral,onlyproteinconcentrationsof250µg/mLandlowerhadnosignificantantiproliferativeeffectonMRC-5cells,as opposedtoT.molitorhemolymphproteinswherenosucheffectwasshownevenatconcentrationsof500µg/mL.Theresultsofthetwo-wayANOVAhaveshownthesametrendasin previouscases,whereproteinconcentrationandincubationtime,aswellastheinteractionof thesetwofactors,hadasignificanteffectonthemeasureddecreaseinMRC-5cellviability aftertreatmentwithhighhemolymphproteinconcentrations. Figure 5. U-87 cell viability 24 h, 48 h, and 72 h after treatment with different Z. morio hemolymph total protein concentrations, determined by MTT assay. Error bars represent the standard deviation of three independent experiments, with four biological replicates each, for every measurement. The blue dashed line represents the control group taken as 100% cell viability. The red dotted line represents the viability cutoff of 80%, below which it is considered that the treatments are cytotoxic. Asterisks (*) above the bars denote statistically significant differences in measured cell viability between the corresponding bars linked with lines, determined by one-way ANOVA followed by Tukey’s post hoc test for significance level of p< 0.05. Interaction between protein concentration (C) and incubation time (T) for the whole experiment was analyzed by two-way ANOVA followed by Tukey’s post hoc test for significance level of p< 0.05 (top left). The data in Figure 6show that a significant cytotoxic effect on MRC-5 cells first occurs 48 h after treatment with at least 500 µ g/mL of total proteins from the hemolymph of Z. morio, with around 60% reduction in cell viability compared to values after 24 h. Similar effects were induced by the highest concentrations 48 h after treatment. Also, it was observed that hemolymph total protein concentrations ranging from 7.8125 µ g/mL to 125 µ g/mL exerted a slight negative effect on MRC-5 cell viability 48 h after treatment, albeit the viability was still above the 80% cutoff. Although less profound compared to the results shown in Figure 4, the same trend in cell viability recovery 72 h after treatment is present. In general, only protein concentrations of 250 µ g/mL and lower had no significant antiproliferative effect on MRC-5 cells, as opposed to T. molitor hemolymph proteins where no such effect was shown even at concentrations of 500 µ g/mL. The results of the two-way ANOVA have shown the same trend as in previous cases, where protein concentration and incubation time, as well as the interaction of these two factors, had a significant effect on the measured decrease in MRC-5 cell viability after treatment with high hemolymph protein concentrations.
Int. J. Mol. Sci. 2024,25, 7491 8 of 20 Int.J.Mol.Sci.2024,25,74918of20 Figure6.MRC-5cellviability24h,48h,and72haftertreatmentwithdifferentZ.moriohemolymph totalproteinconcentrations,determinedbyMTTassay.Errorbarsrepresentthestandarddeviation ofthreeindependentexperiments,withfourbiologicalreplicateseach,foreverymeasurement.The bluedashedlinerepresentsthecontrolgrouptakenas100%cellviability.Thereddottedlinerepresentstheviabilitycutoffof80%,belowwhichitisconsideredthatthetreatmentsarecytotoxic. Asterisks(*)abovethebarsdenotestatisticallysignificantdifferencesinmeasuredcellviabilitybetweenthecorrespondingbarslinkedwithlines,determinedbyone-wayANOVAfollowedby Tukey’sposthoctestforsignificancelevelofp<0.05.Interactionbetweenproteinconcentration(C) andincubationtime(T)forthewholeexperimentwasanalyzedbytwo-wayANOVAfollowedby Tukey’sposthoctestforsignificancelevelofp<0.05(topleft). Next,thetwoinsectspecieswerecomparedinregardtotheafter-treatmenteffectsthat thedifferentproteinconcentrationshavehadontheviabilityofU-87(Figure7A–C)andMRC5cells(Figure7D–E).ObservingtheresultsforU-87cells(Figure7A–C),itisnoticeablethata pronouncedcytotoxiceffectwasachievedalreadyafter24hwithaconcentrationof2000 µg/mLT.molitorhemolymphproteins,whilealltheothertreatmentsdidnothavesuchan effectatthesametimepoint(Figure7A).AtthisconcentrationofT.molitorhemolymphproteins,theviabilityofU-87cellswasreducedbyover30%comparedtothetreatmentwiththe sameconcentrationofZ.moriohemolymphproteins.However,astimeprogressed,i.e.,at48 haftertreatment,itwasobservedthathigherconcentrationsofZ.moriohemolymphproteins (250µg/mLandhigher)exhibitedmoresignificantcytotoxicityonU-87cellscomparedtothe sameconcentrationsofT.molitorhemolymphproteins(Figure7B,C).Thisdifferenceincytotoxiceffectwasespeciallyobservedatahemolymphproteinconcentrationof500µg/mL, wheretheviabilityofU-87cellswasreducedbyapproximately50%more48haftertreatment (Figure7B)andapproximately75%more72haftertreatmentwithZ.moriohemolymph(Figure7C),incomparisontotreatmentwithT.molitorhemolymph.Theexceptionistheprotein concentrationof2000µg/mL,where72haftertreatment,ahighercytotoxiceffectwasstill shownbyT.molitorhemolymphproteinscomparedtothoseoriginatingfromZ.morio.Additionally,theresultsofthetwo-wayANOVAshowedthatfactorssuchasproteinconcentration andinsectspecies,aswellastheinteractionofthesetwofactors,hadasignificanteffectonthe measuredU-87cellviabilityafterallincubationtimepoints. Figure 6. MRC-5 cell viability 24 h, 48 h, and 72 h after treatment with different Z. morio hemolymph total protein concentrations, determined by MTT assay. Error bars represent the standard deviation of three independent experiments, with four biological replicates each, for every measurement. The blue dashed line represents the control group taken as 100% cell viability. The red dotted line represents the viability cutoff of 80%, below which it is considered that the treatments are cytotoxic. Asterisks (*) above the bars denote statistically significant differences in measured cell viability between the corresponding bars linked with lines, determined by one-way ANOVA followed by Tukey’s post hoc test for significance level of p< 0.05. Interaction between protein concentration (C) and incubation time (T) for the whole experiment was analyzed by two-way ANOVA followed by Tukey’s post hoc test for significance level of p< 0.05 (top left). Next, the two insect species were compared in regard to the after-treatment effects that the different protein concentrations have had on the viability of U-87 (Figure 7A–C) and MRC-5 cells (Figure 7D,E). Observing the results for U-87 cells (Figure 7A–C), it is noticeable that a pronounced cytotoxic effect was achieved already after 24 h with a concentration of 2000 µ g/mL T. molitor hemolymph proteins, while all the other treatments did not have such an effect at the same time point (Figure 7A). At this concentration of T. molitor hemolymph proteins, the viability of U-87 cells was reduced by over 30% compared to the treatment with the same concentration of Z. morio hemolymph proteins. However, as time progressed, i.e., at 48 h after treatment, it was observed that higher concentrations of Z. morio hemolymph proteins (250 µ g/mL and higher) exhibited more significant cytotoxicity on U-87 cells compared to the same concentrations of T. molitor hemolymph proteins (Figure 7B,C). This difference in cytotoxic effect was especially observed at a hemolymph protein concentration of 500 µ g/mL, where the viability of U-87 cells was reduced by approximately 50% more 48 h after treatment (Figure 7B) and approximately 75% more 72 h after treatment with Z. morio hemolymph (Figure 7C), in comparison to treatment with T. molitor hemolymph. The exception is the protein concentration of 2000 µ g/mL, where 72 h after treatment, a higher cytotoxic effect was still shown by T. molitor hemolymph proteins compared to those originating from Z. morio. Additionally, the results of the two-way ANOVA showed that factors such as protein concentration and insect species, as well as the interaction of these two factors, had a significant effect on the measured U-87 cell viability after all incubation time points.
Int. J. Mol. Sci. 2024,25, 7491 9 of 20 Int.J.Mol.Sci.2024,25,74919of20 Figure7.ComparisonsoftheeffectsofdifferentT.molitor(TM)andZ.morio(ZM)hemolymphtotal proteinconcentrationsonU-87(24h(A),48h(B),and72h(C)aftertreatment)andMRC-5(24h(D), 48h(E),and72h(F)aftertreatment)cellviability,determinedbyMTTassay.Errorbarsrepresentthe standarddeviationofthreeindependentexperiments,withfourbiologicalreplicateseach,forevery measurement.Thebluedashedlinerepresentsthecontrolgrouptakenas100%cellviability.The reddottedlinerepresentstheviabilitycutoffof80%,belowwhichitisconsideredthatthetreatments arecytotoxic.Asterisks(*)abovethebarsdenotestatisticallysignificantdifferencesinmeasuredcell viabilitybetweenthecorrespondingbarslinkedwithlines,determinedbyone-wayANOVAfollowed Figure 7. Comparisons of the effects of different T. molitor (TM) and Z. morio (ZM) hemolymph total protein concentrations on U-87 (24 h (A), 48 h (B), and 72 h (C) after treatment) and MRC-5 (24 h (D), 48 h (E), and 72 h (F) after treatment) cell viability, determined by MTT assay. Error bars represent the standard deviation of three independent experiments, with four biological replicates each, for every measurement. The blue dashed line represents the control group taken as 100% cell viability. The red dotted line represents the viability cutoff of 80%, below which it is considered that the treatments are cytotoxic. Asterisks (*) above the bars denote statistically significant differences in measured cell viability between the corresponding bars linked with lines, determined by one-way ANOVA followed by Tukey’s post hoc test for significance level of p< 0.05. Interaction between protein concentration (C) and insect species (I) for the whole experiment was analyzed by two-way ANOVA followed by Tukey’s post hoc test for significance level of p< 0.05 (top left).
Int. J. Mol. Sci. 2024,25, 7491 16 of 20 4.4.2. Treatment of U-87 and MRC-5 Cells with Different Total Hemolymph Protein Concentrations In accordance with the total hemolymph protein concentrations determined with the Bradford assay, the following dilutions of T. molitor and Z. morio total hemolymph proteins for cell treatments were made with the appropriate cell culture medium [ µ g/mL]: 15.625, 31.25, 62.5, 125, 250, 500, 1000, 2000, 4000. Following cell incubation, 100 µ L of cell culture medium was carefully removed from each well, after which 100 µ L of each treatment was added in four replicates. Thus, the final concentrations of T. molitor and Z. morio total hemolymph proteins used as treatments for cell toxicity assays were as follows [ µ g/mL]: 7.8125, 15.625, 31.25, 62.5, 125, 250, 500, 1000, 2000. For control, 100 µ L of appropriate cell culture medium was added per well. The plates were incubated for 24 h, 48 h, and 72 h at 37 ◦C and 5% CO2in a humified CO2incubator. 4.4.3. MTT Assay After 24 h, 48 h, and 72 h of incubation, an MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) was performed to measure cellular metabolic activity, which is an indicator of cell viability, proliferation, and cytotoxic effects. The culture medium was carefully removed from treated and control wells and replaced with 100 µ L/well of MTT (5 mg MTT/10 mL serum-free appropriate medium). The plates were incubated for 3 h at 37 ◦ C and 5% CO 2 in a humified CO 2 incubator. After incubation, the solution was carefully removed from the wells and replaced with 100 µ L/well of solvent (42 µ L 35% HCl/10 mL isopropanol, i.e., 0.04 M HCl in isopropanol). The plates were incubated for 10 min at room temperature in the dark. After that, absorbance was measured at wavelengths of 540 nm (A 540 ) and 690 nm (A 690 ) using a Multiskan GO spectrophotometer and SkanIt version 5.0 software. This experiment was replicated independently three times for each incubation time point—24 h, 48 h, and 72 h. The obtained A 690 values for each well were subtracted from the A540 values: Asample = A540 −A690, after which the average value of control sample absorbance (A control ) was determined, and the percentage of cell viability (% cell viability) was calculated according to the following formula: % cell viability = Asample ×100/Acontrol 4.5. Statistical Data Analysis—One-Way and Two-Way ANOVA The results of the biochemical analyses and cytotoxicity assays were statistically analyzed using the Statistica version 14.0 software (StatSoft, Inc., Tulsa, OK, USA). First, normal distribution and equal group variance of the data were confirmed with the Brown– Forsythe test. Next, the statistical significance of the differences was tested with a oneway analysis of variance (one-way ANOVA) followed by Tukey’s post hoc analysis for a significance level of p< 0.05. Additionally, the combined effects of hemolymph protein concentration and incubation time, as well as hemolymph protein concentration and insect species at different time points, on cell viability, were tested using two-way analysis of variance (two-way ANOVA) followed by Tukey’s post hoc analysis for a significance level of p< 0.05. The results are presented as bar charts, and graphs were created using GraphPad Prism version 8.0.2 software (Dotmatics, Boston, MA, USA). Statistically significant results are highlighted with asterisks (explanation given in individual graph descriptions). 5. Conclusions In this study, it was shown that the T. molitor larval hemolymph is rich in proteins and total carbohydrates, and it also exhibits a lower level of lipid peroxidation and antioxidant capacity than larval Z. morio hemolymph. Additionally, T. molitor larval hemolymph exerts a weaker cytotoxic effect on both cancer and normometabolic cell lines compared to hemolymph from Z. morio larvae. Specifically, the cytotoxic effect was observed only after treatment with the highest concentrations of hemolymph proteins, and it was generally
Int. J. Mol. Sci. 2024,25, 7491 17 of 20 more potent on cancer cells. Considering that the hemolymph of T. molitor larvae was found to be richer in carbohydrates compared to Z. morio larval hemolymph, this could explain its milder cytotoxic effects. The higher carbohydrate content could translate into more metabolic fuel for cell proliferation after the treatments. These findings align well with the status of T. molitor as an edible species and an alternative source of proteins to be used in human and animal consumption. Regarding the hemolymph of Z. morio, it was demonstrated to contain high concentrations of total proteins, a high level of lipid peroxidation, and therefore high antioxidative capacity, and a low level of total carbohydrates in comparison to T. molitor larval hemolymph. These characteristics are potentially linked to its more potent cytotoxic activity against both cancer and normometabolic cells compared to T. molitor hemolymph. However, considering that Z. morio hemolymph exhibited greater cytotoxicity to cancer cells, it is hypothesized to contain anticancer agents warranting further investigation. Concerning the exploration of Z. morio hemolymph’s potential in a food-related context, its documented high antioxidant capacity suggests the presence of antioxidant molecules that could potentially enhance food quality, substituting synthetic ones commonly utilized. Therefore, further research into the hemolymph of both insect species from this study, as well as specific constituents of the hemolymph, is necessary to fully explore their potential for application in various fields of the food, feed, and pharmaceutical industries. Indeed, following the results of this study, the next step would be to ascertain the specific biomolecular composition of the hemolymph from the larvae of these insects, which would help identify key bioagents responsible for the cytotoxic effects recorded here. Additionally, while different degrees of hemolymph cytotoxic effects on both analyzed cell lines were shown, further analyses must be undertaken to better describe them and show whether these effects are due to some inherent anticancer properties of these hemolymph extracts, or due to some other antiproliferative mechanisms. Author Contributions: Conceptualization, T.K., Ž.D.P. and I.G.; methodology, Ž.D.P. and I.G.; validation, T.K., M.A. and Ž.D.P.; formal analysis, T.K., M.A. and V.T.; investigation, T.K. and M.A.; resources, M.P.; data curation, T.K. and M.A.; writing—original draft preparation, T.K.; writing—review and editing, M.A., Ž.D.P., I.G., M.P. and V.T.; visualization, T.K. and V.T.; supervision, M.A., Ž.D.P. and I.G.; project administration, Ž.D.P. and I.G.; funding acquisition, Ž.D.P., I.G., M.A. and M.P. All authors have read and agreed to the published version of the manuscript. Funding: The authors gratefully acknowledge the financial support of the European Union’s Horizon 2020 Research and Innovation Programme (ANTARES Project under Grant No. 739570 for the BioSense Institute and IPANEMA Project under the Marie Skłodowska-Curie Grant No. 872662 for the BioSense Institute and the Faculty of Sciences), the Ministry of Science, Technological Development and Innovation of the Republic of Serbia (Grant No. 451-03-66/2024-03/200358 for the BioSense Institute; Grants No. 451-03-66/2024-03/200125 and 451-03-65/2024-03/200125 for the Faculty of Sciences; Grant No. 451-03-65/2024-03/200117 for the Faculty of Agriculture), and the Provincial Secretariat for Higher Education and Scientific Research of the Autonomous Province of Vojvodina, Republic of Serbia (Grant No. 001119852 2024 09418 003 000 000 001 04 003 for the Faculty of Sciences and the BioSense Institute). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available from the corresponding authors upon reasonable request. Acknowledgments: The authors would like to thank Sofija Beki´c from the Faculty of Sciences, University of Novi Sad for providing them with a license number (GH26OON8YB) for the BioRender Scientific Image and Illustration Software that was used to prepare the graphical abstract (https://www.biorender.com/, accessed on 11 April 2024). Conflicts of Interest: The authors declare no conflicts of interest.
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