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In vitro cytotoxic effect of a chitin-like polysaccharide produced by Mortierella alpina on adrenocortical carcinoma cells H295R, and its use as mitotane adjuvant
Luis Daniel Goyzueta Mamani 1 . Julio Cesar de Carvalho 1 D . Sandro J. R. Bonatto2 . Valcineide A. O. Tanobe 3 . Carlos Ricardo Soccol1
Received: 9 September 2020 / Accepted: 11 March 2021 / Editor: Tetsuji Okamoto C The Society for In Vitro Biology 2021
Abstract
This study presents an in vitro evaluation of the antitumor potential of a chitin-like exopolysaccharide (EPS, produced by Mortierella alpina) on Adrenocortical carcinoma cells (ACC) compared to mitotane, a commercial drug commonly used in ACC treatment, and known for its side effects. Techniques of cellular viability determination such as MTT and fluorescence were used to measure the cytotoxic effects of the EPS and mitotane in tumoral cells (H295R) and non-tumoral cells (VERO), observing high cytotoxicity of mitotane and a 10% superior pro-apoptotic effect of the EPS compared to mitotane (p <0.05). The cytotoxic effect of the EPS was similar to the effect of 50 uM mitotane on tumoral cells (p < 0.05). A decrement of the lysosomal volume was also noted in tumoral cells treated with the EPS. To enhance the antitumor effect, a combination of mitotane at a lower dosage and the EPS (as adjuvant) was also tested, showing a slight improvement of the cytotoxicity effect on tumoral cells. Therefore, the results indicate a cytotoxic effect of the EPS produced by Mortierella alpina on adrenocortical carcinoma, and a possible application in biomedical formulations or additional treatments.
Keywords Exopolysaccharide · Mortierella alpina · Anti-tumor effect · Mitotane · Adrenocortical carcinoma
Introduction
Mortierella alpina is a GRAS (Generally Recognized As Safe) filamentous fungi, industrially used to produce arachi- donic acid. There is scarce information or research on the potential use of other metabolites of this microorganism, such as polysaccharides.
Currently, polysaccharides have been used in cancer trials since they can trigger a nonspecific reaction against tumor cells. Various polysaccharides have shown efficiency in treating different types of cancer, such as lung, breast, gastric,
☒ Júlio Cesar de Carvalho jccarvalho@ufpr.br
1 Department of Bioprocess Engineering and Biotechnology, Federal University of Paraná, P.O. box 19011, Curitiba, Paraná 81531-990, Brazil
2 Instituto de Pesquisa Pelé Pequeno Príncipe, Curitiba, Brazil
3 Department of Chemistry, Centro Universitario de Ciencias Exactas e Ingenierías-CUCEI. C.P.44430, Guadalajara University, Guadalajara, Jalisco, Mexico
and colon (Khan et al. 2019). Their potential as adjuvants of conventional drugs (used as chemotherapeutics) has also been demonstrated (Vannucci et al. 2013). Polysaccharides, i.e., glucans, have shown a reduction of metastasis processes (Taki et al. 1995) and induction of apoptosis in human pros- tate cancer and proliferation suppression of colon and breast cancer cells (Yamamoto et al. 1981; Zhang et al. 2005).
According to Zhang et al. 2007, one mechanism of action of polysaccharides is the direct activity on the tumor-possibly arresting the cell life cycle and generat- ing an apoptosis cell death (Song et al. 2011); this could explain the anti-proliferative process in cancer cells (Zaidman et al. 2005).
In this study, we tested the effect of a chitin-like exopolysaccharide from M. alpina on adrenocortical carcino- ma (ACC), which is a uncommon type of malignant tumor (Dackiw et al. 2001), with an incidence of 1.7-2 cases per million people per year and is rare in children (Else et al. 2014). However, in Southern Brazil, ACC is more frequent in children, reaching a rate of 3.4 to 4.2 per million children versus an estimated worldwide incidence of 0.3 per 1 million children younger than 15 years (Custódio et al. 2013; Grossman et al. 2013).
The ACC pathogenesis and prognosis are still controversial (Wajchenberg et al. 2000). In most cases, this type of cancer can be diagnosed due to increased steroid hormone secretion by the tumor (cortisol). In any case, to diagnose the malignan- cy stage, a histopathologic study must be performed (Flack and Chrousos 1996; Vassilopoulou-Sellin and Schultz 2001; Allolio and Fassnacht 2006).
The ACC can be removed at early stages, but its local recurrence is frequent, triggering a metastatic process (Ng and Libertino 2003). At advanced stages, when surgery is not suitable, the treatment with drugs is mandatory, and mitotane is the treatment of choice, combined with some other cytotoxic drugs such as etoposide, doxorubicin, cisplatin, and streptozotocin (Kasperlik-Zalułska et al. 1995; Berruti et al. 2005). Unfortunately, mitotane can present some side effects of gastrointestinal and neurological events (Terzolo et al. 2007). The survival rate is around 16 to 38% after 5 yr of diagnosis (Wajchenberg et al. 2000).
According to Steiner 1954, this cancer represents 0.2% of deaths from cancers in the USA. Allolio and Fassnacht 2006, affirm that women between the fourth and fifth decade are more vulnerable.
The current study aimed to evaluate the potential antitumor effect of the EPS produced by M. alpina, in vitro, on H295R cells, comparing its bioactivity to mitotane, the most common chemotherapeutic drug known for ACC treatment. In a second approach, we evaluated the EPS application as mitotane adjuvant.
Material and methods
EPS production and purification The EPS was identified as a fungal chitin-like exopolysaccharide (Goyzueta et al. 2020). The fixed culture medium composition for EPS production was in (g.L-1): glucose 46.01, urea 7.48, KH2PO4 2.3, KNO3 1.0, MgSO4.7H2O 0.3, and in (mg.L-1): CaCl2-2H2O 0.62, FeCl3-6H2O 1.5, ZnSO4-7H2O 1.0, CuSO4-5H2O 0.1, and MnCl2-4H2O 1.0. Fermentation was carried out at 25℃ for 5 days at 120 rpm.
After the fermentation process, the biomass was removed by filtration, and EPS was precipitated after ethanol addition (3:1, v/v). The mixture was kept at 5℃ overnight to improve the precipitation process. The EPS was recovered by centrifu- gation, resuspended in water, and dialyzed (cut-off 20 kDa) against distilled water (48 h) and ultrapure water (24 h). The suspension of the EPS was then lyophilized.
Tumor cell lines and culture conditions The adrenocortical carcinoma H295R (ATCC CRL_2128) cell line (purchased from the American Type Culture Collection-ATCC bank, Manassas, VA) and the non-tumoral VERO (BCRJ_0245) cell line from kidney purchased from the cell bank of Rio de
Janeiro-Brazil (APABCAM-Associação Técnico Científica Paul Erlich) were cultivated in Dulbecco’s modified Eagle’s medium F12 (DMEM F12) (Sigma-Aldrich®, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS) (Gibco®, Gaithersburg, MD), and an antibiotic solution of 10 U.mL-1 of streptomycin and 20 U.mL-1 of penicillin (Invitrogen®, Carlsbad, CA). Cell lines used in this research had been au- thenticated by Short Tandem Repetition analysis at DTAPEP on 11 November 2020 (Supplementary Material, STR Profile Report, Figure 1S) and DNA Barcode at Labio INMETRO on 29 August 2019 (Supplementary Material, STR Profile Report, Figure 2S). Cell lines used in this research were my- coplasma-free, as evidenced in the authentication in the sup- plementary material section.
The confluent cells were collected using 0.25% trypsin (Invitrogen®), and viability was evaluated using the trypan blue exclusion test. The concentration of cells used was 1 x 10° cells.mL-1, pipetted in a 96-well-flat-bottomed plate. The incubation process was carried out for 24 h at 37℃ in a CO2 (5%) humidified incubator.
Effect on cell viability of EPS The evaluation of the EPS effect on the cell viability was carried out at the Research Institute Pelé Pequeno Príncipe, Curitiba, Paraná, Brazil (IPPP).
Viability was evaluated using the 3-(4,5-dimethylthiazol-2- yr)-2,5- diphenyltetrazolium bromide (MTT) assay. One hun- dred microliters of cell suspension was plated into each well and incubated for 24 h at 37℃ with 5% CO2. After the incu- bation process, the culture medium was removed, and 180 LL of fresh culture medium and 20 µL of the treatment substances were added.
After 24 h of incubation, 20 uL of the MTT solution to a final concentration of 0.5 mg.mL-1 was added to each well and incubated for 3 h at 37℃ and CO2 5%. Then, after careful removal of the supernatant, 100 µL of DMSO (dimethyl sulf- oxide) (Panreac®, Barcelona, Spain) was added and gently agitated for 5 min to dissolve the formazan crystals. The read- ings were performed at 595 nm on an ELISA microplate read- er (Biotek®, Winooski, VT). The viability of the untreated cell lines group was considered as 100%. All assays were performed in five replicates. The viability was calculated ac- cording to the following equation: %cell viability = (100. A595e)/A595b, Where A595e is the mean value of the treatment samples, and A 595b is the mean value of the blanks.
The treatments were: EPS at 1.5 mg.mL-1 solubilized in ultrapure water by ultrasonic treatment at 20% amplitude (12 W.cm-3) for 10 min in an ice-water bath (Wang et al. 2010), Mitotane at concentrations of 20, 31.3, 50, and 62.5 uM, and a mixture of EPS and Mitotane at 1.5 mg.mL-1 and 31.3 µM, respectively. Cell control, vehicle control, and positive control (Mitotane at 1 mM) were also included for comparison. The IC 50 concentration of EPS (1.5 mg.mL-1) determined in our previous work was chosen for this research.
Fluorescence viability assay (live/dead fluorescence imaging) H295R and VERO cells were plated at 8 × 104 cells/well in a black-walled 96-wells plate with an optically clear-bottom surface, cells were incubated for 24 h for adhesion, and then the medium was aspirated and replaced with DMEM supplemented with 10% FBS, and then, the treatments were added. After 24 h, the medium was aspirated and stained for 30 min in the dark with Hoechst 33342 (5 µg.mL-1), ethidium bromide (0.6 uM), and calcein (0.3 uM) in phosphate- buffered saline (PBS) (pH 7.4). Fluorescence images from 4 fields within each well were taken on the IN Cell Analyzer 1000 (GE Healthcare, Chalfont St. Giles, UK) to give the number of dead cells (ethidium bromide, red) and the total number of cells (Hoechst-stained, blue) in each well as a func- tion of the treatments. The number of dead cells was subtracted from the total number to obtain the number of live cells in each well, represented as a percentage of the total number (% viability). Assays were performed in five repli- cates. The treatments were: EPS at 1.5 mg.mL-1 solubilized as mentioned before, Mitotane at concentrations of 20, 31.3, 50, and 62.5 µM and finally, a mixture of EPS Mitotane at 1.5 mg.L-1 and 31.3 uM, respectively. Cell control, vehicle con- trol, and positive control (Mitotane at 1 mM) were also includ- ed in the comparison.
Apoptosis flow cytometry analysis For apoptosis analysis, H295R cells were plated at a density of 1 × 106 cells per well into 6-wells microplates. After 24 h of incubation, the treat- ments were added and incubated for another 24 h. Control wells, vehicle controls, and positive controls were also includ- ed for comparison. After treatment and incubation, the cells were trypsinized and stained with Annexin V according to the manufacturer’s instruction (Annexin V Apoptosis Detection Kit I, BD Bioscience®, San Jose, CA). In this assay, Annexin V was used in conjunction with 7-Aminoactinomycin D (7- AAD) to discriminate apoptotic cells, dead cells, and viable cells, and collected in 300 uL of a solution of human serum albumin 5% in PBS and measured with a FACS Canto II (BD Biosciences®, San Jose, CA) cytometer. The results were analyzed using the software BD FACSDiva™ (BD Bioscience®). The flow cytometer was calibrated with mag- netic beads (BD™M CS&T Beads, BD Biosciences®) before analysis according to the fabricant’s instructions.
The 7-AAD is a membrane-impermeant dye that is gener- ally excluded from viable cells, and ANNEXIN V is a specific protein that can bind to phosphatidylserine, which appears at the outer leaflet of the plasma membrane during apoptosis. This event is related to the loss of activity of the aminophospholipid translocase, causing a flip-flop of phos- pholipids (Bratton et al. 1997).
The treatments evaluated were: EPS at 1.5 mg.mL-1 solu- bilized as mentioned before, Mitotane at concentrations of 31.3 and 62.5 uM, and finally, a mixture of EPS and
Mitotane at 1.5 mg.mL-1 and 31.3 uM, respectively. Cell control and vehicle control were also included in the comparison.
Cell metabolism The evaluation of lysosomal volume followed the methodology proposed by Pipe et al. 1995 modified by Bonatto et al. 2004, in which a density of 1 x 106 H295R cells per well into 96-wells microplates was placed. After 24 h incubation, 20 uL of treatments were added, and finally, after 24 h, 20 uL of 2% neutral red (Sigma-Aldrich®) was added and incubated for 30 min. After discarding the supernatant, neutral red was solubi- lized by adding 100 uL of extraction solution (0.1 mL of 10% acetic acid plus 40% ethanol solution). The absor- bance was read at 595 nm, and lysosomal volume was expressed as absorbance (per 1 × 10° cells.mL-1).
Superoxide anion production was estimated by the nitro blue tetrazolium reduction assay (NBT-Sigma). According to Sim Choi et al. 2006, this assay is specific for determining superoxide anion radicals (Liu et al. 2009). NBT can be re- duced to a monoformazan cation (MF+). MF+ can be further reduced into diformazan (DF) when sufficient reducing sources are available. MF and DF precipitates are water- insoluble (Sim Choi et al. 2006).
After treatment incubation for 24 h, the culture medium was removed and replaced with 100 µL of DEMEM F12 and 100 µL of NBT (nitroblue tetrazolium, 0.25%) (Amresco®, Solon, OH) and Zymosan solution. After 30 min incubation at 37°C, the supernatant was discarded, and the cells were fixed by adding 100 uL of methanol (50%) for 10 min. Next, the supernatant was discarded, and the plate was dried. Finally, 120 uL of KOH (2M) and 140 µL of dimethyl sulfoxide were added to the wells. After 30 min, the reduction of NBT resulted in blue formazan formation. The absorbance was read at 595 nm, and the results were expressed as absorbance (per 1 × 10° cells.mL-1).
Hydrogen peroxide production was based on the horserad- ish peroxidase-dependent conversion of phenol red into a col- ored compound by H2O2 (Pick and Mizel 1981). H295R cells (100 µL) were incubated in the presence of glucose (5 mM), phenol red solution (0.56 mM), and horseradish peroxidase (Sigma-Aldrich®) (8.5 U.mL-1) and Zymosan (Sigma- Aldrich®) in the dark for 30 min at 37℃. Finally, 10 uL of 1 M NaOH (Fluka Chemika®, Buchs, Switzerland) was added to the wells to stop the reaction. The absorbance was read at 620 nm, and the results were expressed as absorbance (per 1 × 10° cells.mL-1).
The treatments tested were EPS at 1.5 mg.mL-1 solubilized as mentioned before, Mitotane at concentrations of 20, 31.3, 50, and 62.5 uM and finally a mixture of EPS and Mitotane at 1.5 mg.L-1 and 31.3 uM, respectively. Cell control, vehicle control, and positive control (Mitotane at 1 mM) were also included in the comparison.
Statistical analysis Results (means ± SEM) from at least three independent experiments with three replicates were expressed as the fold change from a control value. Post-hoc comparisons were performed with the ANOVA-Newman-Keuls test in GraphPad Prism 6 software. p < 0.05 was considered to indi- cate a statistically significant difference.
Results and discussions
Effect of the EPS on cell viability Mitotane is a potent chemo- therapeutic drug used for ACC treatment (Oddie et al. 2018). The adrenocortical tumoral cell line (H295R) and non-tumoral cell line (VERO) were incubated under different treatments and con- centrations of Mitotane, EPS, and a mixture of these two. A mixture of Mitotane and EPS was proposed to evaluate the po- tential use of EPS as an adjuvant of mitotane. The EPS concen- tration is the IC50 value, chosen from the screening performed in our previous work (IC50 value)(Goyzueta et al. 2020).
The serum concentration window of ACC patients treated with mitotane can vary from 14 (43.75 uM) to a maximum of 20 µg.mL-1 (62.5 [M). In this study, we considered 62.5 uM, the upper limit, as the window concentration to corroborate our findings with those of Lehmann et al. 2013.
The window concentration of mitotane (62.5 [M) showed a significant decrease of 2.7-fold in VERO cell viability (p < 0.05), while the EPS and the mixture of mitotane and EPS decreased viability by 1.6 and 1.7-fold, respectively (Fig. 1). In H295R cells, lower concentrations of mitotane (20-50 LM) did not affect the viability, while at the window concentration, a 10-fold viability decrease was observed. EPS at 1.5
mg.mL-1 and the mixture of mitotane and EPS (31.3 µM + 1.5 mg.mL-1) decreased the viability by 2.5-fold (Fig. 1).
The mitotane concentrations influenced VERO cells cyto- toxicity; the effect was negative and exponential.
Although mitotane showed cytotoxicity of 84.2% on the H295R cell population at the window concentration, it is cru- cial to consider that mitotane reduced the VERO cell popula- tion to 34.5% (p < 0.05) as well, demonstrating its negative effect and harmfulness on non-tumoral cells.
On the other hand, when EPS and EPS as an adjuvant of mitotane were evaluated, a living population up to 58% (p < 0.05) of VERO cells was observed, which indicates a lower cytotoxicity effect when compared to mitotane treatments.
In this first screening, the MTT method was used to eval- uate the viability of cells by their metabolism, but this method does not give an accurate proportion of living and dead cells. Therefore, a test of the living/dead dye exclusion assay was carried out to investigate the EPS effects further.
Fluorescence viability assay (live/dead fluorescence imaging)
This method was used to analyze live/dead populations by dye exclusion, using Hoechst 33342 to dye viable cells, and ethidium bromide, which dyes specifically nuclei of cells with lost plasma membrane integrity.
The use of mitotane at different concentrations showed cytotoxicity in VERO cells diminishing the viability com- pared to the control (p < 0.05) by 1.35-fold at 20, 31.3 uM, and by 1.6- and 2.9-fold at 50 and 62.5 uM, respectively. Only 34.5% of cells survived at the window concentration (Fig. 2). EPS and the mixture of EPS and mitotane decreased the cell viability in 1.7- and - 1.9-fold compared to the control.
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Figure 2 shows a viability reduction of H295R cells when high concentrations of 50 and 62.5 uM were used, in which a maximum decrement of 6.3-fold of viability was noted. The use of EPS (1.5 mg.mL-1) and a mixture of mitotane and EPS showed a decrement of 2.1-fold of total viability. In this assay, a significant cytotoxicity effect of mitotane on VERO cells at the window concentration was observed.
The viability of VERO and H295R was confirmed by both tests. In a parallel assay, an evaluation of the cellular morphol- ogy change was carried out for 48 h (Fig. 3) in an IN-Cell Analyzer 1000 (GE Health Sciences, Chalfont St. Giles, United Kingdom), showing lysis of the plasmatic membrane when mitotane was used. Debris was observed at 48 h of treatment, especially at 62.5 uM of mitotane.
The results of viability in this work agree with different authors’ results, supporting the evidence of high toxicity of mitotane on H295R cells at different concentrations (Fassnacht et al. 2012; Lehmann et al. 2013; Kroiss et al. 2016).
Apoptosis flow cytometry analysis In cancer treatment, the ideal treatments are based on immunotherapy, enhancing the immune system response or stimulating apoptosis (Riley et al. 2019). To evaluate the effect of mitotane, EPS, and their com- bination on cellular death, an evaluation of apoptosis by cy- tometry analysis was performed. Only cells marked with ANNEXIN V were considered apoptotic cells positive, and cells marked by ANNEXIN V and 7-AAD were considered dead cells. Some authors have considered cells marked by ANNEXIN V and 7-ADD as apoptotic cells positive (Poli et al. 2013a; Germano et al. 2014).
The results of the treatments were 2% (mitotane), 13.5% (EPS), and 17% (EPS plus mitotane) of apoptotic cells com- pared to the control at p < 0.05. The results were expressed as events percentage and were distributed, as shown in Fig. 4.
No increase in apoptosis was observed when higher EPS concentrations were tested (data not shown).
According to some researchers, the apoptotic effect of mitotane is unclear, and the determination of cellular death by necrosis or apoptosis must be studied (Wilhelm et al. 1998; Högel et al. 2011; Lehmann et al. 2013). The necrosis hypothesis could explain the high quantity of cellular debris observed at 62.5 uM mitotane treatment at 24 h and 48 h (data not shown).
Cell metabolism The evaluation of neutral red dye retention by lysosomes is a relatively new method to indicate the function- ality of lysosomes.
The lysosomal volume of H295R cells did not change when treated with mitotane concentrations below 50 uM at 24 h. The window concentration (62.5 uM) caused a considerable adverse alteration in the volume, 22% less than the control (Fig. 5). Most of the cell population, at the positive control concentration, could have been dead by the end of the experiment due to mitotane cytotoxicity. The mitotane might induce necrosis due to mitochondrial oxidative injury, as reported by Lehmann et al. 2013 and Poli et al. 2013b, and lysosomal damage, inducing cellu- lar death triggering an apoptotic process (Rizk-Rabin et al. 2008).
According to Krecic and Swanson 1999, the action of mitotane in the depletion of the protein hnRNP (protein
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implicated in mRNA metabolism and tumorigenesis process) could lead to tumorigenesis increasing levels of cathepsin D, a lysosomal protease involved in cellular death (Roberg 2001).
The use of EPS and EPS plus mitotane showed a lysosomal volume decrement of 22 and 27% (p < 0.05), respectively. This behavior might explain the apoptotic cells observed in the cytometry assay previously described.
The production of the superoxide anion (a reactive oxygen species, ROS) was not significant when mitotane was used at concentrations below 50 uM. At the window concentration (62.5 uM), a decrement of 16% of production was noted, presumably due to mitotane cytotoxicity. The same behavior was observed in the positive control (Fig. 6).
When EPS and EPS combined with mitotane as an adju- vant were tested, ROS production of 25 and 33% were
observed, respectively. The production could respond to an oxidative burst stimulation caused by the antioxidant effect of the EPS (as determined in our previous work), which can be formed in the mitochondria or via activation of oxidases (Rajamohan et al. 2012; McDowell et al. 2015).
Generation of ROS is a survival alternative mechanism of tumoral cells making up the tumor microenvironment and re- sistant to apoptosis (Weinberg et al. 2019). However, it can also lead to death when high quantities are produced (Calder 2012). One of the objectives of the action mechanism of chemothera- peutics is the induction of intracellular ROS synthesis (McDowell et al. 2015). In this study, the increase in ROS production might be related to the pro-apoptosis effect of EPS.
The increase in the production of hydrogen peroxide was not significant among the treatments when compared to the
IN VITRO CYTOTOXIC EFFECT OF A CHITIN-LIKE POLYSACCHARIDE PRODUCED BY MORTIERELLA ALPINA
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control, as shown in Fig. 7. A significant production decre- ment was noted at mitotane concentrations up to 50 uM, but this might also be due to the cytotoxic effect on H295R cells. An apoptotic process induced by the increase in hydrogen peroxide level was not observed in this study.
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Conclusions
In this study, for the first time, the anti-tumor potential of a chitin-like exopolysaccharide obtained from Mortierella alpina was evaluated in vitro. Viability evaluation
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demonstrated the high cytotoxicity effect of Mitotane on tu- moral (H295R) and non-tumoral cells (VERO), suggesting a more necrotic than a pro-apoptotic effect, specifically at the concentration of treatment as a chemotherapeutic drug. On the other hand, the EPS showed an antitumor action by a 3.3-fold increase in the pro-apoptotic effect on tumoral cells and low damage on non-tumoral cells. A slight enhancement of the pro-apoptotic effect was observed when the EPS was used as mitotane adjuvant (4%), and cell death was mainly due to pro-apoptotic induction instead of necrosis.
In summary, this work may contribute to further studies on the finding of polysaccharides against ACC and used them to develop bioproducts for treatments.
Supplementary Information The online version contains supplementary material available at https://doi.org/10.1007/s11626-021-00560-y.
Acknowledgements The authors thank the Pelé Pequeno Principe Research institution. J.C.C. and C.R.S. are Research Members of CNPq. LDGM acknowledges a Ph.D. scholarship from the PROEX project.
Authors’ contributions Conceptualization, methodology, investigation, formal analysis: Luis Daniel Goyzueta M. Formal analysis: Valcineide O.A Tanobe. Conceptualization, methodology, supervision, formal anal- ysis: Sandro J.R. Bonatto. Conceptualization, formal analysis, project administration: Júlio Cesar de Carvalho. Resources, writing-review and editing: Carlos Ricardo Soccol. All authors read and approved the final manuscript.
Funding The funding agency CAPES supported this research-the Brazilian Coordination for Improvement of Higher Education Personnel-PROEX program.
Declarations There is no animal testing of the compound made in this study, and therefore no animal ethical clearance is required for this study.
Conflict of interest The authors declare that there are no conflicts of interest.
References
Allolio B, Fassnacht M (2006) Adrenocortical carcinoma: clinical update. J Clin Endocrinol Metab 91:2027-2037
Berruti A, Terzolo M, Sperone P, Pia A, Della Casa S, Gross DJ, Carnaghi C, Casali P, Porpiglia F, Mantero F, Reimondo G, Angeli A, Dogliotti L (2005) Etoposide, doxorubicin and cisplatin plus mitotane in the treatment of advanced adrenocortical carcino- ma: a large prospective phase II trial. Endocr Relat Cancer 12:657- 666
Bonatto SJR, Folador A, Aikawa J, Yamazaki RK, Pizatto N, Oliveira HHP, Vecchi R, Curi R, Calder PC, Fernandes LC (2004) Lifelong exposure to dietary fish oil alters macrophage responses in Walker 256 tumor-bearing rats. Cell Immunol 231:56-62
Bratton DL, Fadok VA, Richter DA, Kailey JM, Guthrie LA, Henson PM (1997) Appearance of phosphatidylserine on apoptotic cells requires calcium-mediated nonspecific flip-flop and is enhanced by loss of the aminophospholipid translocase. J Biol Chem 272:26159-26165 Calder PC (2012) Mechanisms of Action of (n-3) Fatty Acids, 2. J Nutr 142:592S-599S
Custódio G, Parise GA, Kiesel Filho N, Komechen H, Sabbaga CC, Rosati R, Grisa L, Parise IZS, Pianovski MAD, Fiori CMCM, Ledesma JA, Barbosa JRS, Figueiredo FRO, Sade ER, Ibañez H, Arram SBI, Stinghen ST, Mengarelli LR, Figueiredo MMO, Carvalho DC, Avilla SGA, Woiski TD, Poncio LC, Lima GFR, Pontarolo R, Lalli E, Zhou Y, Zambetti GP, Ribeiro RC, Figueiredo BC (2013) Impact of neonatal screening and surveillance for the TP53 R337H mutation on early detection of childhood adre- nocortical tumors. J Clin Oncol 31:2619-2626
Dackiw APB, Lee JE, Gagel RF, Evans DB (2001) Adrenal cortical carcinoma. World J Surg 25:914-926
Else T, Kim AC, Sabolch A, Raymond VM, Kandathil A, Caoili EM, Jolly S, Miller BS, Giordano TJ, Hammer GD (2014) Adrenocortical Carcinoma. Endocr Rev 35:282-326. https://doi. org/10.1210/er.2013-1029
Fassnacht M, Terzolo M, Allolio B, Baudin E, Haak H, Berruti A, Welin S, Schade-Brittinger C, Lacroix A, Jarzab B, Sorbye H, Torpy DJ, Stepan V, Schteingart DE, Arlt W, Kroiss M, Leboulleux S, Sperone P, Sundin A, Hermsen I, Hahner S, Willenberg HS, Tabarin A, Quinkler M, de la Fouchardière C, Schlumberger M, Mantero F, Weismann D, Beuschlein F, Gelderblom H, Wilmink H, Sender M, Edgerly M, Kenn W, Fojo T, Müller HH, Skogseid B (2012) Combination chemotherapy in advanced adrenocortical carcinoma. N Engl J Med 366:2189-2197. https://doi.org/10.1056/ NEJMoa1200966
Flack MR, Chrousos GP (1996) Neoplasms of the adrenal cortex. Cancer Med 1:1567
Germano A, Rapa I, Volante M, Lo Buono N, Carturan S, Berruti A, Terzolo M, Papotti M (2014) Cytotoxic activity of gemcitabine, alone or in combination with mitotane, in adrenocortical carcinoma cell lines. Mol Cell Endocrinol 382:1-7
Goyzueta MLD, Noseda MD, Bonatto SJR et al (2020) Production, char- acterization, and biological activity of a chitin-like EPS produced by Mortierella alpina under submerged fermentation. Carbohydr Polym 247:116716. https://doi.org/10.1016/j.carbpol.2020.116716
5
Grossman AB, Jameson JL, De Groot LJ (2013) Endocrinology adult and pediatric: the adrenal gland e-book. Elsevier Health Sciences
Högel H, Rantanen K, Jokilehto T, Grenman R, Jaakkola PM (2011) Prolyl hydroxylase PHD3 enhances the hypoxic survival and G1 to S transition of carcinoma cells. PLoS One 6:e27112
Kasperlik-Zalułska AA, Migdalska BM, Zgliczyński S, Makowska AM (1995) Adrenocortical carcinoma. A clinical study and treatment results of 52 patients. Cancer 75:2587-2591
Khan T, Date A, Chawda H, Patel K (2019) Polysaccharides as potential anticancer agents-a review of their progress. Carbohydr Polym 210:412-428. https://doi.org/10.1016/j.carbpol.2019.01.064
Krecic AM, Swanson MS (1999) hnRNP complexes: composition, struc- ture, and function. Curr Opin Cell Biol 11:363-371
Kroiss M, Plonné D, Kendl S, Schirmer D, Ronchi CL, Schirbel A, Zink M, Lapa C, Klinker H, Fassnacht M, Heinz W, Sbiera S (2016) Association of mitotane with chylomicrons and serum lipoproteins: practical implications for treatment of adrenocortical carcinoma. Eur J Endocrinol 174:343-353
Lehmann TP, Wrzesiński T, Jagodziński PP (2013) The effect of mitotane on viability, steroidogenesis and gene expression in NCI- H295R adrenocortical cells. Mol Med Rep 7:893-900
Liu R, Fu S, Zhan H, Lucia LA (2009) General spectroscopic protocol to obtain the concentration of the superoxide anion radical. Ind Eng Chem Res 48:9331-9334. https://doi.org/10.1021/ie9007826
McDowell RE, Amsler MO, Li Q et al (2015) The immediate wound- induced oxidative burst of Saccharina latissima depends on light via photosynthetic electron transport. J Phycol 51:431-441
Ng L, Libertino JM (2003) Adrenocortical carcinoma: diagnosis, evalu- ation and treatment. J Urol 169:5-11
Oddie PD, Albert BB, Hofman PL, Jefferies C, Laughton S, Carter PJ (2018) Mitotane in the treatment of childhood adrenocortical carci- noma: a potent endocrine disruptor. Endocrinol Diabetes Metab Case Rep 2018
Pick E, Mizel D (1981) Rapid microassays for the measurement of su- peroxide and hydrogen peroxide production by macrophages in cul- ture using an automatic enzyme immunoassay reader. J Immunol Methods 46:211-226
Pipe RK, Coles JA, Farley SR (1995) Assays for measuring immune response in the mussel Mytilus edulis. Tech Fish Immunol 4:93-100
Poli G, Guasti D, Rapizzi E, Fucci R, Canu L, Bandinelli A, Cini N, Bani D, Mannelli M, Luconi M (2013a) Morphofunctional effects of mitotane on mitochondria in human adrenocortical cancer cells. Endocr Relat Cancer 20:537-550
Poli JS, Dallé P, Senter L, et al (2013b) Fatty acid methyl esters produced by oleaginous yeast Yarrowia lipolytica QU21 : an alternative for vegetable oils. 203-208
Rajamohan SB, Raghuraman G, Prabhakar NR, Kumar GK (2012) NADPH oxidase-derived H2O2 contributes to angiotensin II- induced aldosterone synthesis in human and rat adrenal cortical cells. Antioxid Redox Signal 17:445-459
Riley RS, June CH, Langer R, Mitchell MJ (2019) Delivery technologies for cancer immunotherapy. Nat Rev Drug Discov 18:175-196
Rizk-Rabin M, Assie G, Rene-Corail F, Perlemoine K, Hamzaoui H, Tissier F, Lieberherr M, Bertagna X, Bertherat J, Bouizar Z (2008) Differential expression of parathyroid hormone-related protein in adrenocortical tumors: autocrine/paracrine effects on the growth and signaling pathways in H295R cells. Cancer Epidemiol Prev Biomarkers 17:2275-2285
Roberg K (2001) Relocalization of cathepsin D and cytochrome c early in apoptosis revealed by immunoelectron microscopy. Lab Investig 81:149-158
Sim Choi H, Woo Kim J, Cha Y, Kim C (2006) A quantitative nitroblue tetrazolium assay for determining intracellular superoxide anion production in phagocytic cells. J Immunoass Immunochem 27:31- 44
Song K-S, Li G, Kim J-S, Jing K, Kim TD, Kim JP, Seo SB, Yoo JK, Park HD, Hwang BD, Lim K, Yoon WH (2011) Protein-bound polysaccharide from Phellinus linteus inhibits tumor growth, inva- sion, and angiogenesis and alters Wnt/ß-catenin in SW480 human colon cancer cells. BMC Cancer 11:307
Steiner PE (1954) Cancer: Race and geography: some etiological, envi- ronmental, ethnological, epidemiological, and statistical aspects in Caucasoids, Mongoloids, Negroids, and Mexicans. Williams & Wilkins Company, Philadelphia
Taki H, Ohishi K, Okano A et al (1995) Antimetastatic effect of lentinan on liver metastasis of colon carcinoma (colon26): possible role of activated kupffer cells. Int J Immunother 11:29-38
Terzolo M, Angeli A, Fassnacht M, Daffara F, Tauchmanova L, Conton PA, Rossetto R, Buci L, Sperone P, Grossrubatscher E, Reimondo G, Bollito E, Papotti M, Saeger W, Hahner S, Koschker AC, Arvat E, Ambrosi B, Loli P, Lombardi G, Mannelli M, Bruzzi P, Mantero F, Allolio B, Dogliotti L, Berruti A (2007) Adjuvant mitotane treat- ment for adrenocortical carcinoma. N Engl J Med 356:2372-2380 Vannucci L, Krizan J, Sima P et al (2013) Immunostimulatory properties and antitumor activities of glucans. Int J Oncol 43:357-364
Vassilopoulou-Sellin R, Schultz PN (2001) Adrenocortical carcinoma: clinical outcome at the end of the 20th century. Cancer Interdiscip Int J Am Cancer Soc 92:1113-1121
Wajchenberg BL, Albergaria Pereira MA, Medonca BB, Latronico AC, Carneiro PC, Ferreira Alves VA, Zerbini MCN, Liberman B, Gomes GC, Kirschner MA (2000) Adrenocortical carcinoma: clin- ical and laboratory observations. Cancer 88:711-736
Wang ZM, Cheung YC, Leung PH, Wu JY (2010) Ultrasonic treatment for improved solution properties of a high-molecular weight exopolysaccharide produced by a medicinal fungus. Bioresour Technol 101:5517-5522. https://doi.org/10.1016/j.biortech.2010. 01.134
Weinberg F, Ramnath N, Nagrath D (2019) Reactive Oxygen Species in the Tumor Microenvironment: An Overview. Cancers (Basel) 11: 1191. https://doi.org/10.3390/cancers11081191
Wilhelm S, Wagner H, Häcker G (1998) Activation of caspase-3-like enzymes in non-apoptotic T cells. Eur J Immunol 28:891-900
Yamamoto T, Yamashita T, Tsubura E (1981) Inhibition of pulmonary metastasis of Lewis lung carcinoma by a glucan, Schizophyllan. Invasion Metastasis 1:71-84
Zaidman B-Z, Yassin M, Mahajna J, Wasser SP (2005) Medicinal mush- room modulators of molecular targets as cancer therapeutics. Appl Microbiol Biotechnol 67:453-468
Zhang M, Chen H, Huang J, Li Z, Zhu C, Zhang S (2005) Effect of lycium barbarum polysaccharide on human hepatoma QGY7703 cells: in- hibition of proliferation and induction of apoptosis. Life Sci 76: 2115-2124
Zhang M, Cui SW, Cheung PCK, Wang Q (2007) Antitumor polysac- charides from mushrooms: a review on their isolation process, struc- tural characteristics and antitumor activity. Trends Food Sci Technol 18:4-19