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... 9/5/2023 - Open Access Bitter melon extract suppresses metastatic breast cancer cells (MCF-7 cells) growth possibly by hindering glucose uptake Abhinav Kakuturu1*, Heeyun Choi1*, Leah G Noe1, Brianna N Scherer1, Bikram Sharma2, Bilon Khambu3, Bhupal P Bhetwal1 1Division of Biomedical Sciences, Marian University College of Osteopathic Medicine, Marian University - Indiana, Indianapolis, Indiana, United States 2Department of Biology, Ball State University, Muncie, Indiana, United States 3Department of Pathology and Laboratory Medicine, School of Medicine , Tulane University, New Orleans, Louisiana, United States To whom correspondence should be addressed: bpbhetwal@marian.edu *These authors contributed equally. Abstract Breast cancer is one of the most commonly diagnosed cancers among women, however the complete cure for metastatic breast cancer is lacking due to poor prognosis. There has been an increasing trend of dietary modifications including consumption of natural food for the prevention of cancer. One of the popular natural foods is bitter melon. Bitter melon grows in tropical and subtropical areas. Some of the beneficial effects of bitter melon towards disease including cancer have been reported at the whole body/organismal level. However, specific cellular mechanisms by which bitter melon exerts beneficial effects in breast cancer are lacking. In this study, we used a human metastatic breast cancer cell line, MCF-7 cell, to study if bitter melon alters glucose clearance from the culture medium. We co-cultured MCF-7 cells with bitter melon extract in the presence and absence of supplemented insulin and subsequently measured MCF-7 cells viability. In this study, we report a noble finding that bitter melon extract exerts cytotoxic effects on MCF-7 cells possibly via inhibition of glucose uptake. Our findings show that insulin rescues MCF-7 cells from the effects of bitter melon extract. 9/5/2023 - Open Access Figure 1. Insulin rescues human metastatic breast cancer cells (MCF-7 cells) from cytotoxic effects of bitter melon extract (BME): (A) MCF-7 cell viability determined by using Trypan blue assay. Different doses of bitter melon extract (BME) was used [0.5% to 10% (v/v)]. As mentioned in the description section, the bitter melon extract was obtained by grinding whole bitter melon (with seeds in the bitter melon). Cumulative data of average % viable cells (SD) with N=3. Average % significantly different from the no BME control cells (**P < 0.01, ***P < 0.001), one-way ANOVA. (B) Microscopic images of MCF-7 cells (cultured in low glucose DMEM medium) after washing with 1X PBS. (a) control cells with no BME added to the cell culture. (b), (c), (d), (e), and (f) are the MCF-7 cells co-cultured with different doses of BME(v/v %). Note that (f) contains some cell debris from dead cells that were not completely washed during PBS wash. These are not live cells. (g) Cells cocultured with insulin (50 ng/mL). (h) Cells co-cultured with 2% BME and insulin (50ng/mL). (C) Glucose concentration remaining in MCF-7 cell culture media. The cells were cultured in high glucose (450mg/dL) DMEM medium. Glucose remaining in the medium was measured by a glucometer after the 48 hours co-culturing with different doses of BME. The green bar represents cells co-cultured with BME (2%) and insulin (50ng/mL). Cumulative data of average amount of glucose in mg/dL (SD) with N=5. Average mg/dL significantly different (*P < 0.01), one-way ANOVA. (D) Cell viability of MCF-7 cells cultured in high glucose (450mg/dL) DMEM medium . Cell viability was determined by using MTT assay. Cells were co-cultured with 2% BME, insulin (200 ng/mL), or both 2% BME & insulin (50 or 100 or 200 ng/mL). Cumulative data of average % cell viability (SD) with N=3. Average % significantly different (*P < 0.01), one-way ANOVA. NS; Non significant. (E) Cell viability of MCF-7 cells cultured in low glucose (100mg/dL) DMEM medium. Cell viability was determined by using MTT assay. Cells were co-cultured with 2% BME, insulin (200 ng/mL), or both 2% BME & insulin (50 or 100 or 200 ng/mL). Cumulative data of average % cell viability (SD) with N=3., DMEM; Dulbeccos Modified Eagles Medium. 9/5/2023 - Open Access Description According to recent statistics, breast cancer has been one of the most commonly diagnosed cancers among women in the United States (Siegel, Miller, & Jemal, 2019). Numerous risk factors such as alcohol use, obesity, family history, hormonal therapy, diet, and age, etc. have been associated with breast cancer development (Akram, Iqbal, Daniyal, & Khan, 2017; Wood, Cuke, & Bedrosian, 2019). Even though several treatment plans have been developed, complete cure for metastatic breast cancer is lacking due to poor prognosis (Peart, 2017). Although various types of breast cancer cells have been used in research laboratories, MCF-7 cells are perhaps one of the most well studied cell lines (Jiang et al., 2016). The name of MCF-7 is derived from the Michigan Cancer Foundation, which were isolated from a 69-year-old woman with metastasis (Comsa, Cimpean, & Raica, 2015). MCF-7 cells are estrogen receptor (ER)-positive and progesterone receptor (PR)-positive cells with metastatic potential. The metastasis is developed by the secretion of vascular endothelial growth factor (VEGF-A) by MCF-7 cells and the VEGF induces migration and invasion of the cancer cells (Comsa et al., 2015). Using this cell line, researchers have found effective pharmacological targets to treat breast cancer. While there are some treatment options for breast cancer patients, strategies to prevent the disease altogether is the most effective way to deal with it, and one of the ways to prevent cancer is by regulating diet (De Cicco et al., 2019). Throughout the world, people have looked for and consumed natural diets due to health benefits they provide. A popular natural food is bitter melon. Bitter melon grows in tropical and subtropical areas such as Asia, mostly in India and Southeast Asia, Africa, the Caribbean, and California, Florida, and Texas in the United States (Pahlavani et al., 2019; Perez, Jayaprakasha, Crosby, & Patil, 2019; Raina, Kumar, & Agarwal, 2016). There are multiple ways to consume bitter melon: bitter melon juice or tea, salad, and stir-fry, and it has been used traditionally as folk medicine as well (Raina et al., 2016). For this reason, many researchers have paid attention to its health benefits, and they have investigated the effect of bitter melon on various cell functions using in vitro and in vivo study models. Researchers have found that BME inhibits the expression of cell cycle regulatory proteins such as cyclin B1 and D1, blocks G2-M transition of the cell cycle, and thus inducing apoptosis in cancer cells (Cao et al., 2015; Ray, Raychoudhuri, Steele, & Nerurkar, 2010). While anti-proliferative, proapoptotic, and autophagic effects of bitter melon were found in previous studies, these studies did not use the entire fruit of bitter melon when studying the anti-cancer effects of bitter melon on MCF-7 cells. Instead, deseeded bitter melon extract (BME) or any isolated compounds from bitter melon was used (Bai et al., 2016; Cao et al., 2015; Grossmann et al., 2009; Muhammad, Steele, Isbell, Philips, & Ray, 2017; Ray et al., 2010; Weng et al., 2013). In reality, people do not just eat isolated compounds of bitter melon or deseeded fruit; rather, they usually consume the entire fruit of bitter melon. Therefore, it is important to determine the effects of whole bitter melon extract on MCF-7 cells. We investigated the role of bitter melon extract from the entire fruit including seeds in this study. Furthermore, whether the effects of bitter melon on cellular functions is dose dependent has not been well established. We hypothesized that the juice extracted from whole bitter melon fruit (with seeds in the bitter melon) will exert cytotoxic effects on MCF-7 cells, similar to the effects observed from deseeded bitter melon or isolated compounds of bitter melon. To test this hypothesis, we co-cultured MCF-7 cells with increasing doses of BME followed by viability assays and microscopic imaging of cells. MCF-7 cells viability was decreased by BME in a dose dependent manner (Figure 1A). Microscopic imaging of cell culture revealed dose-dependent decline in the number of cells remaining in the culture dish (Figure 1B). The conditioned media from these cultures were separated and glucose concentration was measured in them. The cultures treated with the highest dose of BME that resulted in the least viability of MCF-7 cells (Figure 1A) had the highest concentration of remaining glucose in the conditioned media (Figure 1C). Reciprocally, the cultures treated with the lowest dose of BME that resulted in relatively the highest cell viability (Figure 1A) had the lowest concentration of remaining glucose in the medium (Figure 1C). These observations suggest that BME exerts cytotoxic effects on cells in a dose dependent manner. Furthermore, stronger cytotoxic effects on MCF-7 cells may lead to less number of remaining cells, which in turn likely results into less glucose clearance from the culture medium. Our findings in cancer cells is interesting compared to the effects of BME in glucose clearance by the healthy tissues (skeletal muscles and adipose tissues) reported by others (Ma, Yu, Xiao, & Wang, 2017; Nkambo, Anyama, & Onegi, 2013) (Shih, Lin, Lin, & Wu, 2009) (Roffey, Atwal, Johns, & Kubow, 2007). Both the in vitro and in vivo studies have reported that BME enhances glucose clearance from the extracellular fluid (culture medium or blood) by the healthy skeletal muscle cells or adipose cells. Thus, BME was reported to lower the blood glucose levels by increasing GLUT-4 transporter mediated downstream signaling pathways resulting in enhanced glucose clearance from blood. In the major insulin sensitive healthy tissues, insulin stimulates glucose uptake by enhancing mobilization of endocytosed GLUT-4 transporters to the cell membrane, which then facilitates glucose transport into the cells, a vital biochemical process for the metabolism & viability of mammalian cells. Thus, in our study, we wanted to test if enhancing glucose uptake by the MCF-7 cells would increase their viability under basal or stressed conditions. To the best of our knowledge, effects of altering glucose uptake by the MCF-7 cells on these cells viability have not been investigated. We used insulin to enhance glucose uptake by these cells and test if insulin supplementation will rescue BMEs cytotoxic effects in MCF-7 cell cultures. We 9/5/2023 - Open Access hypothesized that insulin supplementation will rescue BME induced toxicity on MCF-7 cells. To test this hypothesis, we cocultured MCF-7 cells with 2% BME & three different doses of insulin (50, 100, and 200 ng/mL). From the BME dose response experiments, 5% and 10% BME exerted such a strong cytotoxic effects that we observed almost zero viability of the MCF-7 cells in culture (Figure 1B). Thus, we decided to use a middle dose of BME (2%) for our rescue experiments. The higher dose of insulin alone (200 ng/mL), with no BME added, neither inhibited nor activated the cells cultured in high glucose medium (Figure 1D). However, we found that the insulin supplementation rescued the BME-induced cytotoxicity (Figure 1D) most likely by increasing glucose uptake by the cells. Among the three doses of insulin (50, 100, and 200 ng/mL), 50 ng/mL insulin rescued the cell toxicity close to normal viability. To our surprise, compared to 50 ng/mL insulin, higher doses of insulin (100, and 200 ng/mL) did not show a cumulative increase in the rescue effect (Figure 1D). Interestingly, at doses higher than 50 ng/mL, insulin appeared to show decreasing effects (Figure 1D). We do not know why higher doses of insulin become less effective in rescuing the cells from the BMEs cytotoxic effects. One possibility could be that maximal glucose uptake is achieved by the 50 ng/mL insulin. Furthermore, higher doses of insulin may exert suppressive effects on cells-a phenomenon named as insulin toxicity (Kolb, Kempf, Rohling, & Martin, 2020). It appears that when cells are exposed to higher doses of insulin, downregulation of insulin signaling occurs, which is not primarily due to less insulin receptor expression on the cell surface but due to impaired insulin signal transduction as a result of receptor dysfunction (Bertacca et al., 2005; Catalano et al., 2014). Bertacca and colleagues have reported that higher dose of insulin downregulates GLUT4 receptor expression on the cell surface (Bertacca et al., 2005). Downregulation of GLUT4 receptor is likely to add stress to the cells as they will not be efficient to take up glucose from the medium. Thus, the cells co-cultured with high insulin dose in a low glucose medium (100 mg/dL glucose compared to high glucose medium with 450 mg/dL glucose) are likely to experience double stress due to downregulation of GLUT4 receptors and low glucose environment. To further evaluate this possibility, we repeated the rescue experiments in low glucose DMEM medium in which glucose concentration is approximately five-fold lower. In the low glucose medium, concentration gradient of glucose between the culture medium and cell cytoplasm decreases. Thus, low glucose medium is likely to result into less glucose influx into the cells, which in turn can induce stress and thereby decrease viability in cells. We found that in contrast to the cells cultured in high glucose DMEM medium, cells cultured in low glucose DMEM medium lost rescue sensitivity to 200 ng/mL insulin. Moreover, higher dose of insulin alone (200 ng/mL), with no BME added, neither inhibited nor activated the cells cultured in low glucose medium (Figure 1E), similar to what we found in high glucose medium culture (Figure 1D). We do not know why 200 ng/mL insulin alone is neither beneficial nor harmful to cells. One of the limitations of this study is that we do not know if the BME (obtained from whole fruit with seeds in it) inhibits glucose uptake by the MCF-7 cells or glucose clearance from the medium is suppressed as a secondary effect of BMEs cytotoxicity on MCF-7 cells. Cancer cells express different subtypes of GLUT transporters (GLUT1, GLUT2, GLUT3, GLUT4 etc.) (Ancey, Contat, & Meylan, 2018). Previous studies have emphasized that ubiquitous GLUT-1 transporter plays major roles in glucose uptake in cancer cells (Shin & Koo, 2021). However, whether relative contributions of GLUT-1 and GLUT-4 transporters are different in MCF-7 cells under healthy versus stressful situations is not known. Our findings suggest that insulin dependent glucose uptake is critical to overcome stressors such as BME induced cytotoxicity. Our findings also suggest that the rescue effect of insulin is dependent on the glucose concentration in the media, particularly at higher doses of insulin (Figure 1D and Figure 1E). Studies have found overexpression of IGF-1 receptor in metastatic breast cancer cells compared to the normal cells (Bartucci, Morelli, Mauro, Ando, & Surmacz, 2001; Cullen et al., 1990; Surmacz, Guvakova, Nolan, Nicosia, & Sciacca, 1998). Furthermore, insulin and IGF-1 ligands can potentially cross stimulate their receptors (Griffeth, Bianda, & Nef, 2014). Therefore, enhanced glucose uptake could also be secondary to the signaling pathways associated with IGF-1 receptor stimulation and not necessarily via the well-established insulin-GLUT-4 pathway. Several of these possibilities could be investigated in the future. Methods Bitter Melon Extract (BME) Preparation Fresh bitter melons were purchased from an Asian grocery store. The whole bitter melon (with seeds in them) were washed and cut into small pieces to fit in the juice extractor (Black & Decker; Product # JE2400BD). The pieces of the entire fruits of bitter melons, seeds included, were used. The juice extract was centrifuged nine times for 15 minutes each at 5000 rpm, and then filter sterilized. BME aliquots were stored at - 80C. Cell culture & BME dose response Equal number of MCF-7 cells (1.0 x 105 viable cells/cm; Thermo Fisher Scientific, catalog # HTB-22) were plated in the 75 cm2 tissue culture treated culture flasks (Thermo Fisher Scientific, catalog # 156499) containing 30 mL medium (Millipore Sigma, DMEM-high glucose, catalog # D6429). The medium was supplemented with 10% Fetal Bovine Serum (Millipore 9/5/2023 - Open Access Sigma, catalog # F2442). Any other appropriate treatments such as BME, insulin etc were added to the medium just before adding the cell inoculum to the complete growth medium. Cells were incubated in an incubator (37C, 5% CO2, 0% O2) for 48 hours. After 48 hours, cultures were washed with 1X phosphate buffered saline (Millipore Sigma, catalog # TMS-012) followed by capturing of pictures using a microscope at the 40x magnification (Nikon, product # 25726). The glucose concentration remaining in the culture medium was also measured. Cell viability was also measured using Trypan Blue Exclusion assay. Glucose measurement After culturing cells for 48 hours, culture media were removed and centrifuged at 2000 rpm for 3 minutes, and then the media were transferred to sterile centrifuge tubes to measure the glucose levels from each tube using a glucometer (BioReactor Sciences, Model # GM100). Trypan blue exclusion assay After MCF-7 cells were cultured with the treatments for the required amount of time, the supernatant was collected from each culture flask. After supernatant was collected from each culture flask, the cells were washed with 1X PBS three times, and they were trypsinized from the bottom of the culture flasks with Trypsin-EDTA (0.25%), phenol red (catalog # 25200056). Trypsinized cells were combined with cells from the supernatant. The cells were resuspended in 1 mL DMEM (Millipore Sigma, DMEM-high glucose, catalog # D6429) and mixed softly with pipette. 20 L cells from the cell suspension were added to a cryovial. Equal parts of 0.4% trypan blue solution (VWR, CAS # 72-57-1) were added to the cryovial containing the cells to obtain a 1 to 1 dilution, and 9 L of the solution was pipetted from the cryovial and loaded into the hemocytometer chamber (Fisher Scientific, catalog # 02-671-55A). Then, cells were counted using a microscope at total magnification of 100X. Both total number of cells and blue-stained cells were counted from the four chamber of the hemocytometer, and number of bluestained cells were subtracted from the total number of cells to give the number of viable cells. After that, the average was taken from the number of viable cells, and then it was multiplied by 10,000 which represented the number of cells per mL and by a dilution factor, thus giving the number of viable cells per mL. MTT; 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay The cells were seeded at a concentration of 0.8 105 cells/mL into a 96-well plate. The cells were incubated (37 C, 5% CO2, 0% O2) with the medium alone or with the appropriate supplements (different doses of BME, different doses of insulin or both insulin and 2% BME) in the medium. After culturing cells for 48 hours, medium was aspirated from each well and 100 L fresh medium containing DMEM and 10% FBS was added to the wells. Then 10 L of MTT reagent (Millipore Sigma, catalog # 11465007001) was added to the wells. The contents of wells in the 96-well plate were mixed using a shaker followed by incubation of the plate (37 C, 5% CO2, 0% O2) for 4 hours. After 4 hours, 100 L of detergent (0.01N HCl with 10% sodium dodecyl sulfate) was added to each well and the plate was incubated in dark at room temperature for four hours. After the incubation, the plate was read using spectrophotometer plate reader (Molecular Devices; Filter Max F3 Multi-Mode Microplate Reader) and a reading software (SoftMax Pro 6.2.1), and absorbance for each well was recorded at 570nm. The cells viability was estimated by measuring absorbance at 570 nm. The cell viability percentage was calculated based on the absorbance ratio between culture wells with different treatments and the untreated control well multiplied by 100 (percentage of control, %). Acknowledgements: We would like to thank the members of the Bhetwal laboratory for their support. We thank Dr. Colleen Doci for providing cell line and her constructive feedbacks. References Akram M, Iqbal M, Daniyal M, Khan AU. 2017. Awareness and current knowledge of breast cancer. Biol Res 50(1): 33. PubMed ID: 28969709 Ancey PB, Contat C, Meylan E. 2018. Glucose transporters in cancer - from tumor cells to the tumor microenvironment. FEBS J 285(16): 2926-2943. 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Cucurbitane Triterpenoid from Momordica charantia Induces Apoptosis and Autophagy in Breast Cancer Cells, in Part, through Peroxisome Proliferator-Activated Receptor Activation. Evid Based Complement Alternat Med 2013: 935675. PubMed ID: 23843889 Wood ME, Cuke M, Bedrosian I. 2019. Prevention Therapy for Breast Cancer: How Can We Do Better? Ann Surg Oncol 26(7): 1970-1972. PubMed ID: 30798445 Funding: This study was supported by the Faculty Research Development grants-Marian University College of Osteopathic Medicine to Dr. Bhupal P. Bhetwal. Author Contributions: Abhinav Kakuturu: data curation, formal analysis, investigation, methodology, writing - original draft. Heeyun Choi: data curation, formal analysis, investigation, methodology, writing - original draft. Leah G Noe: data curation, methodology. Brianna N Scherer: data curation, methodology. Bikram Sharma: formal analysis, writing - review editing. Bilon Khambu: formal analysis, writing - review editing. Bhupal P Bhetwal: conceptualization, investigation, funding acquisition, project administration, supervision, writing - original draft, writing - review editing. Reviewed By: Benjamin Spears History: Received August 17, 2023 Revision Received September 1, 2023 Accepted September 5, 2023 Published Online September 5, 2023 Indexed September 19, 2023 Copyright: 2023 by the authors. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International (CC BY 4.0) License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Citation: Kakuturu, A; Choi, H; Noe, LG; Scherer, BN; Sharma, B; Khambu, B; Bhetwal, BP (2023). Bitter melon extract suppresses metastatic breast cancer cells (MCF-7 cells) growth possibly by hindering glucose uptake. microPublication Biology. 10.17912/micropub.biology.000961  ...

... 4/5/2023 - Open Access The impact of C. elegans ceramide glucosyltransferase enzymes on the beneficial effects of B. subtilis lifespan Chelsey L Arvin 1, Zachary Sibila 2, Regina Lamendella 3, Jason Chan 2, Trisha Staab 2 1Marian University College of Osteopathic Medicine, Indianapolis, Indiana, USA 2Marian University College of Arts and Sciences, Indianapolis, Indiana, USA 3Biology, Juniata College, Huntingdon, Pennsylvania, USA To whom correspondence should be addressed: tstaab@marian.edu Abstract Ceramide glucosyltransferase (CGT) adds sugar moieties to ceramide, forming glucosylceramides that play roles in immune signaling, stress response, and host-bacterial interactions. Here, we examined whether mutations in cgt block the beneficial effects of Bacillus subtilis on C. elegans lifespan. We found that loss of cgt-1 or cgt-3 reduces lifespan compared to wildtype worms, but did not prevent the lifespan-extending phenotype of B. subtilis. However, cgt-1(ok1045) and cgt-3(tm504) did play a minor role in blocking stress resistance of 5-day old worms treated with B. subtilis. Further studying CGTs may elucidate potential roles of glucosylceramides in host-bacterial interaction. 4/5/2023 - Open Access Figure 1. Loss of CGT does not impair the beneficial effects of B. subtilis: A) Survival curves of wild-type (N2) and cgt mutants (cgt-1(tm1027), cgt-1(ok1045), cgt-2(tm1192), cgt-3(tm504)) fed on E. coli (OP50; yellow) or B. subtilis (3A1T; blue). Worms were tracked for lifespan starting at L4 stage (day 0) and scored every 2 days. Bagged or missing worms were censored (indicated by a crossline). Significant differences were found between survival of animals treated with E. coli vs B. subtilis. The bottom right panel in (A) shows the survival curves of all animals fed E. coli (OP50) on the same graph. B) Table showing sample size, mean, standard error deviation, & median values for the lifespans of worms grown on the control bacteria (E. coli) and experimental bacteria (B. subtilis). For B, * indicates significant difference compared to the respective animal on B. subtilis and # indicates significant difference compared to N2 on E. coli. C) Acute stress response survival curves of wild-type (N2) and cgt mutants treated with 100mM paraquat. Experiments were performed on 1-day, 5-day, and 10-day old animals. Worms were grown to respective ages on either E.coli (OP50; yellow) or B. subtilis (3A1T; blue) bacterial lawns prior to the stress test. For all, survival curves were analyzed using Kaplan-Meier estimates, and pairwise comparisons were performed using a log-rank test. Description At the membrane surface of intestinal cells, there is a rich complement of glucosylceramides. Ceramides are a type of sphingolipid that play a role in lipid microdomains, stress response, and cell death (Rohrhofer et al. 2021). The enzyme ceramide glucosyltransferase (CGT) catalyzes the addition of sugar moieties onto ceramide in the lipid bilayer. However, it is not known how bacteria-host interactions are affected by glucosylceramide metabolism. Could the presence of different cgt enzymes affect the impact of the beneficial effects of bacteria on the host physiology? There are three genes (cgt-1, cgt-2, and cgt-3) that are thought to have CGT enzymatic activity in C. elegans. Previous studies suggest that cgt-1 and cgt-3 have a greater number of amino acids relating to functional enzymatic activity; thus, mutations in cgt-1 and cgt-3 may have more of a negative impact on animal physiology than cgt-2 (Marza et al. 2009). Specifically, cgt-1;cgt-3 double mutants have larval phenotypes, cgt-1 and cgt-3 are highly expressed in the worm intestine, and they are known to serve developmental roles; furthermore, loss of all cgts (cgt-1, cgt-2, and cgt-3) are lethal (Marza et al. 2009). Re-expression of cgt enzymes in the intestine can rescue larval phenotypes of cgt-1;cgt-3 double mutants, suggesting CGTs have important intestinal functions (Marza et al. 2009). CGTs have also been shown to help establish intestinal cell polarity during development (Zhang et al. 2011). More recently, there has been observation of CGTs acting on autophagolysosomes to recruit clathrin and mediate lysosome recycling (Wang et al. 2021). The commensal bacteria Bacillus subtilis has been demonstrated to increase lifespan and promote survival to the oxidative stressor juglone and thermotolerance of the nematode Caenorhabditis elegans (Donato et al. 2017; Smolentseva et al. 2017). Interestingly, these effects were dependent on the biofilm forming nature of B. subtilis. Biofilms are protective structures composed of extracellular matrix proteins and signaling molecules, providing a place for bacteria to grow and survive (Vlamakis et al. 2013). It also acts as a point of contact between bacteria and the host intestinal membrane. However, less is known about whether glucosylceramides mediate the beneficial effects of B. subtilis. Recent studies show that glycosylated ceramides are targets of Bacillus thuringiensis binding, leading to C. elegans infection (Griffitts et al. 2005). Furthermore, CGT inhibition can weaken the colon cell barrier to the Bacteroides fragilis toxin (Patterson et al. 2020). Given the effect of B. subtilis on stress response and lifespan, along with the roles of cgt enzymes in the intestine, we aimed to examine whether the protective effects of B. subtilis require cgt enzymes (cgt-1, cgt-2, and cgt-3). To do this, we compared lifespan and stress response of wild-type and mutant animals when grown on either the B. subtilis wild-type isolate (3AIT) or the common lab bacteria E. coli (OP50). First, we found that B. subtilis increased the survival of wild-type animals approximately 10% (Figure 1A), which is similar to the 15% increased survival demonstrated in other studies (Donato et al. 2017). However, B. subtilis also increased the survival of all cgt mutant animals examined. When comparing wild-type animals to cgt mutants on OP50, we found that mutations in cgt-1 and cgt-3 reduced lifespan compared to wild-type (Figure 1A,B). Similarly, Wang et al. (2021) found that cgt-3 RNAi reduced lifespan. Whereas loss of particular ceramide glucosyltransferase genes can reduce lifespan, it does not block the beneficial effects of the commensal bacteria B. subtilis. However, it is not clear how specific cgt manipulations may affect total levels of glucosylceramides. Indeed, one study showed that only double-knockout mutants (cgt-3;cgt-1) demonstrated observable phenotypes (Marza et al. 2009); but, another showed pharmacological inhibition of glucosylceramides, particularly glucosylceramide transferase 2, actually increase lifespan (Cutler et al. 2014). Furthermore, the enzymes may have location- specific cell functions that have not been explored. Previously, it was found that wild-type B. subtilis can promote tolerance to heavy metal, osmotic, oxidative, pathogenic, and temperature stress (Donato et al. 2017; Smolentseva et al. 2017). To examine the effect of mutations in cgt enzymes on adult stress response, we performed an oxidative stress assay by examining acute survival to the oxidative stressor paraquat (PQ) in 1, 5, and 10 day old animals. We found that wild-type N2 worms fed B. subtilis performed worse in response to 100mM PQ at 4/5/2023 - Open Access 1-day old than N2 worms fed OP50 (Figure 1C). This was also observed in cgt-1(tm1027) mutants. However, 5-day-old N2 worms fed B. subtilis improved acute survival to PQ compared to those who fed OP50 (p=0.007; Figure 1C); however, this effect was not observed at 10-days of age. The improved response to PQ in 5-day old wild-type animals was not observed in the presence of cgt mutations. In summary, we show that loss of individual CGTs impact does not block the lifespan extending effects of the bacteria B. subtilis. Prior research demonstrates that single CGT knockouts may play a minor role in C. elegans response to stress (Marza et al. 2009). However, others have found that cgt-3 RNAi alone can reduce survival to the oxidative stressor TBHP (Wang et al. 2021). Thus, further experiments deleting or knocking down multiple CGTs may provide further insight into the roles of glucosylceramides in host-bacterial interactions. It was interesting that there were some differences between 1 day and 5 day responses of wild-type animals fed OP50 vs B. subtilis to PQ (from a slightly detrimental effect to a slightly beneficial effect), which may suggest that colonization of B. subtilis is needed to impact hosts. Although speculative, this finding supports the model that biofilm formation in B. subtilis is necessary to promote host lifespan and physiology (Donato et al. 2017; Smolentseva et al. 2017). Nevertheless, our data suggests that CGTs may play minor roles in the beneficial effects of the commensal bacteria B. subtilis, unlike findings from pathogenic bacteria. Future work examining how sphingolipid enzymes affect intestinal membranes may inform our understanding of how bacteria, including those that form biofilms, impact host physiology. Methods Strains. Wild-type N2 animals were obtained from the Caenorhabditis Genetics Center (CGC), which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). Mutants for cgt-1(tm1027), cgt-2(tm1192), cgt-3(tm504) were obtained from the Mitani lab at the National BioResource Project and cgt-1(ok1045) was obtained from the Caenorhabditis Genetics Center (CGC). Strains were not backcrossed into lab strains of N2. All tm strains are thought to eliminate CGT function or enzymatic activity (Marza et al. 2009). ok1045 is a large ~1800 deletion spanning 7 exons (Wormbase). All worms were maintained on Nematode Growth Medium (Stiernagle 2006) and maintained at 20 on respective bacteria. Gene Name Allele Sequence Name Strain Source cgt-1 tm1027 T06C12.10 JPC21 NBRC cgt-1 ok1045 T06C12.10 JPC22 CGC cgt-2 tm1192 F20B4.6 JPC23 NBRC cgt-3 tm504 F59G1.1 JPC24 NBRC N2 CGC wildtype Bacterial cultures. E. coli was obtained from the CGC and B. subtilis from the Bacillus Genetic Stock Center (BGSC). To make E. coli cultures, a single isolate of E. coli OP50 was inoculated in LB broth and incubated at 37. When the OD was around 0.5 (approximately 24 hours), the solution was stored at 4C. For B. subtilis cultures, a single isolate of B. subtilis (3AIT) was inoculated nutrient broth and incubated at 32 with 125 RPM shaking. When the OD was around 0.5 (approximately 48 hours), the solution was stored at 4C. Both OP50 and 3AIT bacteria were seeded on NGM or NGM plates supplemented with 50M FUdR. Lifespan Assay. L4 animals were placed on NGM plates supplemented with 50 M FUdR. Lifespan measurements were made every two days and bagged/missing worms were censored on corresponding days. The plates were kept at 20 . Animals were transferred to new 50 M FUdR NGM plates with their respective bacteria every four days. Statistical tests for lifespan were analyzed using Kaplan-Meier survival estimates and log-rank tests, with Bonferroni correction, in the R (version 4.0) statistical package survival (version 3.5-0) and survminer (version 0.4.9). Acute Paraquat Assay. N2 and cgt mutants at L4 stage were placed onto 50 M FUdR NGM plates seeded with either E. coli (OP50) or B. subtilis (3AIT) and kept at 20. On days 1 (24 hours after L4), 5 and 10, around 60 animals of each group were transferred into 15 L of M9 buffer in a 96 well plate (10 animals per well in six replicates). Fifteen microliters of 200 mM paraquat were then added for a final test concentration of 100 mM paraquat in each well. Animals in each well were checked 4/5/2023 - Open Access for survival every 30 minutes, for five hours. Survival was determined by movement to gentle prodding. Statistical tests for survival to PQ were analyzed using Kaplan-Meier estimates and log-rank tests, with Bonferroni correction, in the R (version 4.0) statistical package survival (version 3.5-0) and survminer (version 0.4.9). Reagents Product Product Number Source paraquat 856177 Sigma FUdR F0503 Sigma References Cutler RG, Thompson KW, Camandola S, Mack KT, Mattson MP. 2014. Sphingolipid metabolism regulates development and lifespan in Caenorhabditis elegans. Mechanisms of Ageing and Development 143-144: 9-18. PubMed ID: 25437839 C. elegans Deletion Mutant Consortium. 2012. Large-scale screening for targeted knockouts in the Caenorhabditis elegans genome. G3 (Bethesda) 2: 1415-25. PubMed ID: 23173093 Donato V, Ayala FR, Cogliati S, Bauman C, Costa JG, Leini C, Grau R. 2017. Bacillus subtilis biofilm extends Caenorhabditis elegans longevity through downregulation of the insulin-like signalling pathway. Nat Commun 8: 14332. 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PubMed ID: 21926990 Funding: This work was supported by R15AG063103 and R15AG052933 grants. Author Contributions: Chelsey L Arvin : conceptualization, data curation, formal analysis, investigation, methodology, project administration, resources, validation, visualization, writing - original draft, writing - review editing. Zachary Sibila : conceptualization, data curation, formal analysis, investigation, methodology, project administration, resources, validation, visualization, writing - original draft, writing - review editing. Regina Lamendella : conceptualization. Jason Chan : conceptualization, data curation, formal analysis, funding acquisition, investigation, methodology, project administration, resources, software, supervision, validation, visualization, writing - original draft, writing - review editing. Trisha Staab : conceptualization, project administration, resources, writing - review editing, supervision. Reviewed By: Anonymous, Michelle Mondoux 4/5/2023 - Open Access History: Received January 27, 2023 Revision Received March 28, 2023 Accepted April 3, 2023 Published Online April 5, 2023 Indexed April 19, 2023 Copyright: 2023 by the authors. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International (CC BY 4.0) License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Citation: Arvin , CL; Sibila , Z; Lamendella , R; Chan , J; Staab , T (2023). The impact of C. elegans ceramide glucosyltransferase enzymes on the beneficial effects of B. subtilis lifespan. microPublication Biology. 10.17912/micropub.biology.000758  ...