New Results

Penicillium purpurogenum exerts antitumor effects and ameliorates inflammations in Erlich mice model

Amanda Mara Teles, Leticia Prince Pereira Pontes, Sulayne Janayna Araujo Guimarães, Ana Luiza de Araújo Butarelli, Gabriel Xavier Silva, Flavia Raquel Fernandes do Nascimento, Geusa Felipa de Barros Bezerra, Carla Junqueira Moragas Tellis, Rui Miguel Gil da Costa, Marcos Antonio Custódio Neto da Silva, Fernando Almeida-Souza, Ana Paula Silva de Azevedo-Santos, Maria do Desterro Soares Brandao Nascimento
doi: https://doi.org/10.1101/2020.07.02.184291

ABSTRACT

Background The bioactive metabolites production contributes to the resistance of fungi towards adverse environmental conditions. Some metabolites often have interesting health-promoting activities. This study addressed the anti-tumoural properties of Penicillium purpurogenum isolated from a polluted lagoon in Northeastern Brazil.

Methods The extract obtained from the polished environment strain P. purpurogenum was fermented, filtered, concentrated and lyophilized, giving rise to the Ethyl Acetate Extracellular Extract (EAE). The metabolites of the extracellular extract of P. purpurogenum were studied using direct infusion mass spectrometry. The solid Ehrlic tumor model was used to evaluate the extract antitumor activity. Female Swiss mice were divided in groups (n=10/group) as follow: Negative control (CTL-) treated with phosphate buffered solution; Positive Control (CTL+) treated with cyclophosphamide (25mg/mL); Extracts treatment at doses 4, 20 and 100mg/Kg; Animals without tumor or treatment (Sham); and animals without tumor treated with intermediate dose (EAE20). All treatments were performed intraperitoneally, daily during 15 days. After, the animals were eutanized and the tumor, lymphoid organs and serum were used for immunological, histological and biochemical parameters evaluation.

Results The extract was rich in meroterpenoids. All doses of the extract significantly reduced tumor size compared to CLT- and were associated with 100% survival. Histologically, the 20 and 100mg/kg doses reduced tumour-associated inflammation and tumour necrosis. The extract also reduced cellular infiltration of lymphoid organs and circulating TNF-α levels when compared with CLT-. The extract did not induce weight loss and renal or hepatic toxic changes.

Conclusions These results indicate that P. purpurogenum from a polluted marine environment produce hybrid natural products of the terpenoid pathway that exhibits immunomodulatory and antitumor properties in vivo. Thus, fungal fermentation is a biotechnological approach for the production of antitumour agents.

BACKGROUND

Fungi are versatile organisms that can be found in several habitats, occupying inhospitable ecological niches in all ecosystems on the planet, with promising therapeutic and biotechnological potential [1]. Several studies show that marine microorganisms are sources of unique natural products including molecules with potential anticancer uses [2,3,4,5].

Penicillium fungi synthesize high amounts of known bioactive secondary metabolites [6,7] including anticarcinogenic drugs and immunosuppressive agents [8,9]. P. purpurogenum species has the ability to synthesize a variety of substances with biotechnological and bioactive potential, but the strains that demonstrate this activity are mutant strains resistant to antibiotics [10,11].

P. purpurogenum MA52 is a strain previously isolated from a polluted marine environment, the Jansen lagoon in the Brazilian Northeastern state of Maranhão [12]. This is a highly pollutes environment receiving domestic effluents from the surrounding city of São Luís [12]. However, the specific characterisitcs of the MA52 strain which allow it to survive in this polluted environment have not yet been described. New drugs are being developed from secondary metabolites in search of less toxic and more effective compounds compared with traditional cancer therapies [13]. Also, some natural products become important nutraceutics with cancer chemopreventive properties [14,15].

Many in vivo models are used for studying breast cancer, the most common cancer in women worldwide [16]. Breast cancer models include genetically-modified animals, xenografted tumours in immune-compromised mice, chemically induced models [17], and syngeneic models like the solid Ehrlich’s tumor, a spontaneous and highly aggressive murine mammary adenocarcinoma [18]. This syngeneic model is immunocompetent and avoids the use of harmful chemicals, making it particularly useful for tumor and chemotherapy studies [19,20]. Considering the search for bioactive compounds capable of being prototypes of new antitumor drugs, the present study aims to describe the in vivo antitumor activity of P. purpurogenum extract compounds obtained from the MA52 strain against solid Ehrlich’s tumor.

METHODS

Fermentation and Preparation of Ethyl Acetate Extracellular Extract (EAE)

P. purpurogenum is a marine fungus found in the coastal region (2°29’56”S 44°17’59”W) of Maranhão, Brazil. The present strain was isolated by the Mycology Laboratory of the Basic and Applied Immunology Center of the Federal University of Maranhão and deposited in the fungi collection of the Federal University of Maranhão under access code MA52. Fungus strain was grown in Potato Dextrose Agar (BDA) at 28 °C for 7 days until complete growth. After that period, superficial circles of mycelium-containing agar were further cultivated in BDA broth for fermentation at 28 °C for 21 days in a rotary shaker at 150 rpm. Afterwards, 300 mL of the fermented broth were macerated for 48 hours with 600 mL of ethanol, to obtain an extracellular extract. Then, aqueous ethanol solution was filtered and concentrated under reduced pressure to remove ethanol and the remaining water was extracted twice with 1:4 (v/v) ethyl acetate, resulting in an organic phase which was concentrated and lyophilized to obtain the ethyl acetate extract used for in vivo testing.

Tandem Mass Spectrometry with Electrospray Ionization (ESI-MS/MS)

Ethyl acetate extracellular extract (EAE) was analyzed by direct infusion (ESI-MS/MS) in a Bruker Ion trap amazon SL mass spectrometer, positive mode (ESI+). EAEE (3mg) was dissolved in methanol certified HPLC grade containing 0.1% formic acid (v/v) using an ultrasonic bath for 20 minutes. The operating conditions were 1 □L/min infusion, 4.0 kV capillary voltage, 100 °C temperature source, and cone voltage of 20-40 V. Mass spectra were recorded and interpreted by Bruker Compass Data Analysis 4.2.

Animals

After approval by the Ethics Committee (CEUA 23115.11239/2017-70), female Swiss mice (n=70), weighing 25-30 g and aged between 3 and 4 months, were provided by the Federal University of Maranhão (UFMA). Animals were kept in room with controlled temperature at 22 ± 3 °C, 50 ± 15% relative humidity, 12 hours light/dark photoperiod, and food and water ad libitum. Animal experiments were conducted according to animal welfare guidelines by the National Council for Control of Animal Experimentation (CONCEA). Every effort has been made to reduce the number of animals used and their discomfort.

Ehrlich solid tumor model

Animals were anesthetized with ketamine/xylazine (120-150 mg/kg). Ehrlich ascitic carcinoma was maintained in mouse via intraperitoneal injections of 2×106cells [21]. For inducing the solid tumor, transplantable neoplastic cells with 7 days of ascitic evolution were aspirated and 200 µL of cell suspension at 2×106 cells/mL was injected into the left posterior foot pad. The cells were found to be more than 99% viable by a Trypan blue exclusion method. The experimental treatment was started 24 hours after the tumor implantation [22].

Treatment Groups

The animals were separated in two groups: with tumor inoculation for antitumor activity and without tumor inoculation used to evaluated the extract toxicity. The tumor inoculation group was subdivided in six subgroups (n=10), the negative control subgroup (CTL-) with tumor induction treated with phosphate buffered solution (PBS); the positive control subgroup (CTL+) treated with cyclophosphamide at dose of 25 mg/kg; and the subgroups treated with extracts at doses of 4 mg/kg (EAE4+Tumor), 20 mg/kg (EAE20+Tumor) and 100 mg/kg (EAE100+Tumor). To determine acute toxicity, two groups without tumor induction were treated with PBS (Sham) or with the extract at medium dose of 20mg/kg (EAE20). All treatments were performed intraperitoneally 24 hours after tumor inoculation during fifteen days, the volume admistrated was 100µL. After treatment, the animals were randomly selected, a part was euthanized (n=5) for biological analysis and another part (n=5) was used for the quality of life/survival test. The euthanasia was performed by the administration of 120 mg/kg ketamine and 150 mg/kg xylazine (2:1 solution) via intraperitoneal [23] and underwent complete necropsy.

Tumor development Assay

The Ehrlich solid model was performed to evaluated antitumoral activity. Paw volume was determined before and after the injection of Ehrlich tumor cells using a digital caliper at 48 h intervals. The volume was calculated by multiplying the thickness, width and length measurements of the paw with tumor presented as mm3. After the treatment performed in fifteen days, the animals of each group were randomly chosen (n=5), euthanised and the paws with a tumor were removed and weighed. The area under the curve was calculated from the kinetics graph obtained from the tumor-inoculated paw development data using the GrafPAd Prism 7 software.

Histopathological analyzes

Immediately after euthanasia, Ehrlich’s tumor tissue (and matched normal tissue from sham groups), liver and kidneys were removed and fixed in 10% neutral buffered formalin. Samples sections were stained with hematoxylin and eosin solution and analyzed by a single researcher with expertise, blinded to the experimental groups. Concerning the tumour samples, the following parameters were analyzed: inflammatory infiltrate distribution (focal, multifocal, diffuse or peripheral); inflammatory infiltrate intensity (scores: absent 0, light 1, moderate 2, intense 3); necrosis (scores: absent 0, focal 1, focally extensive or multifocal 2, diffuse 3); mitotic figures (scores: no mitotic figures 0, occasional mitoses 1, single mitotic figure per 400x field 2, two mitotic figures per 400x field 3); cellular pleomorphism (scores: monomorphic tumour cells 0, minimal intercellular variation 1, variations in nuclear size and shape 2, major variations with bizarre nuclei 3); and tumour invasion (scores: well-defined borders with no obvious invasion 0, well-defined borders with minimal invasion ofadjacent tissues 1, poorly-defined borders with marker invasion 2, unrecognizable borders with multiple tumour foci 3) [24].

Lymphoid organs cellularity

To obtain and quantify cells in the popliteal lymph node and spleen, these solid organs were removed and macerated in 1mL PBS. To obtain bone marrow cells, the femur was removed and perfused with 1 mL of PBS. After, 90 μL of lymph node, spleen and bone marrow cell suspensions were added to 10 μL of violet crystal and the cells were counted in a Neubauer chamber with the aid of a common light optical microscope [25].

Blood samples

Blood samples were obtained by cardiac puncture. The samples were then centrifuged at 5000 rpm for 10 min. Serum were separated and stored in aliquots at - 80°C until needed. Prior to the assay, the samples were thawed at room temperature [23, 24].

Cytokine Quantification

Blood serum was used for quantification of interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6), interferon-γ (IFN-γ), tumor necrosis factor (TNF-α), interleukin 17A (IL-17A) and interleukin-10 (IL-10) by flow cytometry with FACSCalibur equipment (BD Biosciences, San Jose, CA, USA), using the BD ™ Cytometric Bead Array (CBA) cytokine kit Mouse Th1/Th2/Th17 (BD Biosciences, San Jose, CA, USA) following the manufacturer’s recommendations.

Extract toxicity

For the assessment of acute toxicity, we take into account the weight of the animals during the treatment, weighed daily for 15 days. Weight variation was verified before the tumor inoculation until the last day of treatment. We also verified that it is a fungus extract, hepatic parameters in which blood serum was used to perform the biochemical measurement of glutamic-oxalic transaminase (TGO), glutamic-pyruvic transaminase (TGP) through colorimetric analyzes using the Labtest kits, following the manufacturer’s guidelines, and the data was obtained on a visible spectrum plate reader (Lab. Syftemf Multi Skan EX). Histopathological analysis of the liver and kidney was performed. The parameters analyzed in the liver were the presence of mitotic figures, caryatia (more than 10% of hepatocytes with nuclei twice the size of normal hepatocytes) and necrosis present or absent. Hepatitis was classified as mild (hyperplasia of Küpfer cells and/or occasional microabscesses or mild focal periportal leukocytic infiltration) or moderate (multifocal to diffuse leukocytic infiltration in multiple portal spaces or centrilobular veins). Hepatocellular vacuolar degeneration was also classified as mild (restricted to the periportal and/or centrilobular areas) or moderate (extending to the midzonal areas). In the kidney, the presence of tubular degeneration, defined as the swelling of the cells of the outlined proximal or distal tubules, or necrosis of isolated tubular cells or loss of cell vesicles in the tubular lumen (bleeding) was evaluated [26]. The quality of life/survival of the animals was verified for 30 days after treatment, following a quality of survival protocol in which they checked some of the animals that suffered damage, and a pain scale, in which if the animal shows three or more potential signs associated with pain or discomfort they are kept under surveillance and staying for more than 72 hours, being euthanized by excess of anesthetic [27].

Statistical analysis

Results were expressed as mean ± standard error of means (S.E.M. and S.D.). The differences were submitted to analysis of variance (one-way or two-way ANOVA) followed by Newman-Keuls test and by Student’s t-test using GraphPad Prism software, version 7.0. To evaluate the survival curve, the Kaplan-Meier curve was used and the statistical analysis was performed by the Log-Rank test. The significance level for rejection of the null hypothesis was 5% (p <0.05).

RESULTS

P. purpurogenum extracellular extract contains meroterpenoids

The mass spectral data obtained by direct infusion in positive mode (ESI-MS/MS), presented at figure 1, were useful to determine the chemical profile of P. purpurogenum extracellular extract (EAE) and showed the presence of five major compounds (1-5). They were tentatively identified as meroterpenoids, compounds commonly produced by fungi [28], by comparing the mass spectral data with that described in the literature.

Fig 1.
Fig 1. Mass spectra obtained for tentative identification of meroterpenoids 1-5.

Compound 1 (purpurogemutantin), showed experimental pseudo-molecular ion (M + [H]+) at m/z 419,1674 compatible with the molecular formula C24H35O6. Compounds 2 (purpurogemutantin) and/or 3 (macrophorin A) showed the same pseudo-molecular ion (M + [H]+) at m/z 361.1037 corresponding to the molecular formula C22H33O4. These compounds were previously described at P. purpurogenum extracts by Fang et al [29]. 4 (Berkeleyacetal C, molecular formula C24H26O8), another meroterpenoid previously described in mutant P. purpurogenum by Li et al [30] presented pseudo-molecular ion (M + [H]+) at m/z 443,1696 while compound 5 (Rubratoxin B) showed pseudo-molecular ion (M + [H]+) at m/z 519.1758 corresponding to the molecular formula C22H33O4. Rubratoxin B is a known meroterpenoid with anti-cancer activity isolated by P. purpurogenum [31].

P. purpurogenum extract showed activity against Ehrlich’s solid tumor

In tumor growth kinetics, the results showed that throughout the experiment the negative control group presented an upward curve, while those treated with cyclophosphamide and the extract showed a linearity kinetics. The extract significantly reduced tumor growth from the eighth day compared to CLT-, while cyclophosphamide reduced the tumor growth from the tenth day (Figure 2a). The kinetic values of tumor growth have been in accordance with the graph of the area under curve (AUC). The extract at doses 4 mg/kg (422±35.8mm2), 20mg/kg (389.7±49.13 mm2) and 100mg/kg (365.5±40.47mm2) presented an area smaller than the negative control (1240±43.25 mm2) and the chemotherapy (599.8±55.51mm2) (Figure 2b). At the end of the experimental period, the extract at 4mg/kg (0.19±0.02g), 20mg/kg (0.19±0.05g) and 100mg/kg (0.16±0.02g) showed low weight of the tumor when compared to the negative control (0.38±0.07g) and showed similar weight to the positive control (0.15±0.05g) (Figure 2c).

Fig. 2
Fig. 2

Effect of Penicillium purpurogenum extract on the development of Ehrlich’s solid tumor. (a) The kinetic of paw volume inoculated with Ehrlich’s tumor followed by intraperitoneal treatment with phosphate buffer solution (CTL-), cyclophosphamide 25mg/kg (CTL +) and extracts with a concentration of 4 mg/kg (EAE4), 20 mg/kg (EAE20) and 100 mg/kg (EAE10) at 24h intervals, with the animals being euthanized on day 15. (b) Area under the curve (AUC) calculated from the volume growth kinetics. (c) The average weight of the paws with a tumor was determined in the groups after treatment. Valeus expressed as mean ± standard error of means (S.E.M.) deviation and analyzed by the analysis of variance (one-way or two-way ANOVA) with *p<0.05, ** p <0.005, **** p <0.0001 in relation to the negative control (CTL-), #p < 0.05, ## p <0.01 when compared to positive control (CTL +) and Θ p<0.05 difference in tumor growth for the other groups and f shows that, on the eighth day, only the extract inhibited tumor growth.

Histological results

Histopathological analysis showed the presence of tumor masses in foot pad of animals from groups with Ehrlich’s solid tumor. The tumour masses exhibited high cellularity and different growth patterns, central, moderate to very high pleomorphism with bizarre nuclear forms, presence of one or more nucleoli and heterogeneous chromatin patterns. The cytoplasm was eosinophilic, abundant, with poorly defined limits. Occasional multinucleated giant cells (associated with pattern 3 pleomorphism) and up to 2 mitosis figures per 400x field were identified. Groups with tumor induction presented peritumoral, multifocal to coalescent lymphohistiocytic inflammatory infiltrate with variable intensity Areas of multifocal liquefaction necrosis, with accumulation of eosinophilic and amorphous cellular debris associated with neutophilic infiltrate were also observed. Interestingly, the extract showed an inflammatory process induced by the minor tumor in the groups treated with 20 and 100 mg/kg when compared to the negative control. The extract also demonstrated to have smaller areas of tumor necrosis, mitotic activity and invasion when compared to the negative control (Figure 3 and Table 1).

View this table:
Table 1

Ehrlich tumor histopathological scores (mean ± SD) of groups treated with saline (CTL-), cyclophosphamide (CTL+) or Penicillium purpurogenum ethyl acetate extract at 4mg/Kg (EAE4), 20mg/Kg (EAE20) and 100 mg/Kg (EAE100).

Fig. 3
Fig. 3

Tumor and leukocyte infiltration in Ehrlich tumors with and without treatments. Leukocyte infiltrates in Ehrlich tumors. Swiss mice were inoculated in the paw with 2×106 Ehrlich tumor cells and were treated daily with EAE extract intraperitoneally. At the end of the fifteen days of treatment, the animals were euthanized and their feet amputated, weighed and fixed. Histological sections were stained with Hematoxylin-Eosin. In the photos it is possible to see the tumor cells (indicated by letter T) and the inflammatory infiltrate (indicated by letter I) present in the paws of the Sham (A), CLT- (B), CLT + (c) group (100x total magnification). The animals treated with the extract showed a decrease in the infiltrate and necrosis in the EAE doses EAE 4 (D), EAE 20 (E) and EAE 100 (F) (100x total magnification). The Sham group is shown on panel G and animals treated with extract in the EAE 100 dose on panel H (400x total magnification).

P. purpurogenum extract induced immunomodulatory effects

Treatment with EAE at doses of 20 mg/kg and 100 mg/kg presented low number of cells in popliteal lymph nodes (9.6×104±1.0 cells/mL and 4.4×104±0.34 cells/mL, respectively) when compared with negative control (326×104±40.83 cells/mL). The similar results were observed in cyclophosphamide-treated animals (8.0×104±1.88 cells/mL) (Figure 4a). In spleen cells number account, the negative control group (72.7×107±7.06 cells/mL) showed high cellularity when compared to the animals of Sham group (1.6 ×107±0.12 cells/mL), however this difference was not observed in animals inoculated with the tumor and treated with cyclophosphamide (0.7×107±0.02 cells/mL) or with extract doses 20 mg/kg (1.6×107±0.14 cells/mL) and 100 mg/kg (1.2×107±0.04 cells/mL) (Figure 4b). The marrow bone cellularity showed that the cyclophosphamide (12.3×105±3.13 cells/mL) and the extract treatment (61.0×105±8.35 cells/mL, 17.6×105±1.20 cells/mL and 4.0×105±1.08cells/mL, respectively) prevented the high level of cellularity in associated with Ehrlich’s tumor, since the groups without tumor (sham: 4.6×105±0.77 cells/mL and EAE20: 15.44×105±5.04 cells/mL) had fewer cells than negative control group (189.0×105±23.12 cells/mL) (Figure 4c). The results showed that extract’s effect was dose-dependent.

Fig. 4
Fig. 4

Immunomodulatory effects of Penicilium purpurogenum ethyl acetate extract. (a, b e c) The results were expressed as the mean ± standard deviation of the total lymph node cell count, bone marrow, splenocytes obtained from the group without tumor treated with saline solution (Sham), from the groups with solid Ehrlich tumor treated intraperitoneally with extracts at doses respectively 4, 20 and 100mg/kg (EAE4, EAE20 and EAE100), positive and negative control administered cyclophosphamide (CLT +) and e with saline solution (CTL-) respectively. (d) Blood serum was used for quantification of tumor necrosis factor (TNF-α). The data were submitted to statistical analyzes by method the Kruskal-Wallis and Dunn multiple comparison test, with significance of Δ p <0.05, ΔΔ p <0.005, ΔΔΔ p <0.0005, ΔΔΔΔ p <0.0001 in relation to Sham, ϕ p <0.05, ϕϕϕϕ p <0.0001 when compared to EA20 without tumor, #### p <0.0001 when compared to CLT+, * p <0.05, ** p <0.005, *** p <0.0005, **** p <0.0001 when compared to CLT-, ○○ p <0.0001, ○○○ p <0.0001 comparing the extracts with each other.

Cytokine quantification demonstrated that treatment was able to alter only the TNF levels (Supplementary Data 1). The animals with untreated tumours (CTL-) showed an even higher level in TNF-α concentrations (508.8±66.22 pg/mL) when compared with Sham group (156.0±25.14 pg/mL). The cyclophosphamide treatment significantly showed lower TNF-α levels (177.2±27.67 pg/mL) compared to the negative control group. Similarly, the extract at 4, 20 and 100mg/Kg doses also showed TNF-α low concentrations versus the group CLT- (161.7±30.22 pg/mL, 212.4±12.03 pg/mL and 261.4±43.16 pg/mL respectively) in animals with solid tumor. However, the extract treatment in animals without tumor showed high level of TNF-α seric concentration (306.9±17.93 pg/mL) when compared with normal animals (Figure 4d).

Toxicity studies of P. purpurogenum EAE

Daily treatment with P. purpurogenum ethyl acetate extract in the solid Ehrlich tumor maintained the body weight of the tumor-inuculated animals (EAE4: 2.38±0.63 g; EAE20: 2.8±0.76 g and EAE100: 0.96±0.67 g) compared with CTL+ (−1.16±0.46 g) and CTL- (−0.24±0.73 g) (Figure 5a). The data showed that only the group receiving cyclophosphamide had a body weight reduction when compared with Sham group (1.58±0.96 g). There were no significant differences concerning serum AST and ALT levels in any of the groups analyzed, although CTL-animals showed increased average values compared with groups treated with the extract in the presence of the tumor (Table 2). Hepatic and renal hiltological difference was not found between the groups. The survival rate curve showed that saline (79.12%) and cyclophosphamide (85.72%) groups were statistically different, as expected. Importantly, tumor-animals groups treated with the extract showed 100% survival rate in all doses, being significantly different when compared to controls (Figure 5b).

View this table:
Table 2

Effect of Penicilium purpurogenum ethyl acetate extract on ALT and AST serum levels (mean ± SEM) in all experimental groups.

Fig. 5
Fig. 5

Effect of the toxicity of the extracellular extract of Penicillium purpurogenum ethyl acetate extract (EAE). The groups without tumor induction were treated with saline (sham), while groups with induction received extract at doses of 4, 20 and 100 mg/kg (EAE4, EAE20 and EAE100), saline (CTL-) and cyclophosphamide (CTL +), where we can see in the graphs: a) The animals’ body weight in the final treatment. (b) After the treatment of the animals, it remained under observation for thirty days. The data represent mean ± SEM. The difference was statistically analyzed by Kruskal-Wallis and Dunn’s multiple comparison test, with significance of Δ p <0.05, ΔΔp <0.005, ΔΔΔp <0.0005 in relation to Sham, ϕ p <0.05 ϕϕp <0.005, ϕϕϕp <0.0005 when compared to EA20 without tumor, ○ p <0.05 when compared to CLT + and ○○○ p <0.0005 when compared to CLT-.

DISCUSSION

P. purpurogenum isolated from the marine environment is capable of secreting secondary bioactive metabolites with anticancer properties. In the present study, the extracellular extract of the fungus revealed the presence of macrophorin A, purpurogemutanthidine and purpurogemutanthin, corroborating previous findings by Tang et al. [32], Fang et al [29]. and He et al [33]. These compounds showed interesting anti-cancer activity in vitro against cervical cancer, gastric adenocarcinoma and breast cancer cells, while berkeleyacetal C [34,35,36] and rubratoxin B [37,38,39] showed anti-inflammatory activity and anticancer in vitro.

Meroterpenoids, which are formed by the mixed terpenoid-polyketide biosynthetic pathway, are an important class of compounds in the context of the development of new anticancer agents, due to their vast structural diversity and broad spectrum of bioactivities [40]. They are known to have activities against cancer through various mechanisms, such as the blocking of cell survival pathways, activity against oxidative stress, and induction of apoptosis [41]. When checking the presence of compounds with anticancer activity in vitro, we questioned whether the extract isolated from the marine environment and not described in the literature with activity in vivo against Erhlich’s tumor could be active.

The present study is the first to evaluate the in vivo anticancer effects of P. purpurogenum, employing mice inoculated with Ehrlich’s tumor cells. The results showed that extract compounds were efficient to reduce the development and weight of Ehrlich solid tumor.

Ehrlich’s tumor exhibits strong inflammatory phenomena, including edema and inflammatory infiltrates, which are believed to play an essential role in tumor growth in various types of cancer [42, 43, 44, 45, 46]. But the anti-inflammatory effects were reported by meroterpenoids [47] and were observed in this research showing that the extract reduces inflammation, reducing the tumor by an indirect route.

The extract inhibited tumor growth at the same level as the untreated group, since the behavior of the animals and the survival of the group treated with the extract was similar to the sham group, probably acting in tumor cell proliferation, migration, and invasion.

Two possibilities can inhibit tumor growth: the direct effect on the tumor microenvironment caused by macrophorin A, purpurogutididine and purpurogemutanthin identified in the extracts that result in smaller tumor size. In addition to an indirect effect, reducing inflammation which makes the tumor smaller, which may be associated with the presence of Berkeleyacetal C in the extract.

Other mereterpenoides shows inhibition tumor activities. Wang et al [37] studied Guajadial, one of natural dialdehyde meroterpenoids, that was capable to suppress tumor growth in human xenograft mouse models, with probably proliferation inhibition by the block the Ras/MAPK pathway.

In research carried out by Wan et al. [48] they demonstrate that mereterpenoids have anti-inflammatory and antioxidant activity that can allow the reduction of tumor growth. Li et al. [31] found that Berkeleyacetal C causes significant inhibition of the expression of inducible nitric oxide synthase (iNOS) and the production of nitric oxide by macrophages.

Berkeleyacetal C inhibits expression and secretion of major pro-inflammatory factors and chemokines, including tumor necrosis factor-α (TNF-α) interleukin-6 (IL-6), interleukin-1β (IL-1β), macrophage inflammatory protein −1α (PImax) −1α), and the monocyte chemotactic protein-1 (MCP-1) [29, 35].

Imunosupression cells can have pro-tumoral effect. Indeed, higher infiltration by Tregs is observed in tumor tissues, and their depletion augments antitumor immune responses in animal models [44].

There were also systemic effects, with dose-dependent reduction in the cells of the popliteal lymph node, spleen and bone marrow, when compared in the negative control group. This is in line with a marked low levels of TNF-α, which plays an important role in the beginning and applies the activation of adhesion molecules and expression of inflammatory mediators during inflammatory responses [49]. These pro-inflammatory mediators can cause damage to cells and tissues and also activate macrophages in various diseases associated with inflammation [50].

Increased levels of pro-inflammatory cytokines was previously associated with the development of Ehrlich’s tumor [51]. Our data also agree with those of Calixto-Campos et al [52], Aldubayan et al [53] and Harun et al [54], who studied the protective role of fungal extracts against inflammatory events induced by LPS in vitro and found that all the extracts inhibited the TNF-α expression. Taken together, these results show that the extracellular extract of P. purpurogenum has a potent anticancer activity in vivo against Ehrlich’s breast adenocarcinoma and this effect can be mediated, at least in part, by imnomodulatory mechanisms.

Another important set of observations from the present study concerns the safety of the extract. Remarkably, P. purpurogenum’s extracellular extract was able to improve mouse survival at all doses, compared with the negative control and even with cyclophosphamide, providing 100% survival at the end of the study. The extract preserved body weight of the animals, preventing the wasting syndrome that is often associated with cancer and intensified by chemotherapy [55,56,57,58,59], as previously reviewed [60,61].

These data suggest that P. purpurogenum extract may have potential for clinical use in combination therapies, to prevent the loss of body weight in cancer patients. Considering that fungal extracts often display hepatic toxicity, we evaluated ALT and AST serum levels and hepatic histology. Results did not demonstrate acute hepatic toxicity; instead, the extract not showed hepatic histological lesions associated with Ehrlich’s tumour and presented low serum levels of hepatic transaminases. In line with these observations, no changes were observed in renal histology. These results support the hypothesis that the extract has a favourable toxicological profile, although these observations should be confirmed and complemented by additional studies.

Thus, the results show that the extract inhibited tumor growth when compared to the negative control, at the same level as a standard therapy (cyclophosphamide), even when applied at the lowest dose.

Interestingly, the activity of the extract occurred before the cyclophosphamide, suggesting an intense anti-tumour activity in vivo. In addition, these findings correlate with morphological changes at the histological level, showed that P. purpurogenum presented low mitotic activity and invasive behaviour of tumour cells. The extract’s antitumour activity was associated with marked immunomodulatory effects.

Conclusions

Overall, the present results indicate that the P. purpurogenum extract extract has potent in vivo anticancer activity against Ehrlich’s solid tumour, suggesting the presence of immunomodulatory mechanisms. Chemical components present in the extract may serve as lead compunds for the development of new compounds with antitumor effect and immune response modulators. The extract showed being well-tolerated and improved animal survival compared with cyclophosphamide, suggesting a favourable toxicity profile and potential applications in combination therapies. Further preclinical studies are required to clarify the potential uses of P. purpurogenum and to understand the production of bioactive metabolites in fungi for biotechnological applications.

Ethics approval and consent to participate

Animal studies were approved by ethical and research committee of Universidade Federal do Maranhão. This article does not describe any studies with human participants performed by any of the authors.

Consent for publication

All the authors have consented for publication

Availability of data and materials

The datasets supporting the conclusions of this article are included within the article and its additional files.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported financially by Universidade Federal do Maranhão, Brazil. Amanda Mara Teles has received this research grant by the Foundation for the Support of Research and Scientific and Technological Development of Maranhão (FAPEMA).

Authors’ contributions

AMT, MDSBN and APSAS designed the concept and experiments under study. AMT and GFBB preparation of the fungus extract. APSAS and FRFN Implementation and standardization of the Erlich model. AMT, LPPS, SJAG, ALAB, GXS and APSAS performed the experiments. RMGC and GXS slide reading in the histological test. CJMT, AMT and FAS carried out the chemical characterization of the extract. FAS performed the quantification cytokines by FACS. AMT and APSAS Data curation; Formal analysis; Statistical analysis; Treatment of results in vivo and preparation of figures; Writing - original draft; proofreading and editing. MACNS: Writing - original draft; proofreading and editing. MDSBN Acquisition of resources; Project management; Supervision.

Acknowledgements

We would like to thank to Fundação Oswaldo Cruz for providing the “in vitro” experimental procedures.

We would like to thank to Fundação de Amparo à Pesquisa e ao Desenvolvimento Científico e Tecnológico do Estado do Maranhão for financial support.

List of abbreviations

ALT
alanine aminotransferase
AST
aspartate aminotransferase
AUC
Area under the curve
CTL
negative control
CTL +
positive control
EAE
ethyl acetate extract
IFN-γ
Interferon gamma
iNOS
inducible nitric oxide synthase
IL-1β
interleukin-1β
IL-2
interleukin-2
IL-4
interleukin-4
IL-6
interleukin-6
IL-10
interleukin-10
IL- 17a
interleukin-17a
MCP-1
monocyte chemotactic protein-1
PImax-1α
macrophage inflammatory protein −1α
SEM
standard error of means
TNF-α
alpha Tumor Necrosis Factor

References

  1. 1.↵
    Teixeira TR, Santos GSD, Armstrong L, Colepicolo P, Debonsi, HM. Antitumor Potential of Seaweed Derived-Endophytic Fungi. Antibiotics (Basel). 2019 Oct 31;8(4).
  2. 2.↵
    Pejin B, Jovanović KK, Mojović M, Savić AG. New and highly potent antitumor natural products from marine-derived fungi: Covering the period from 2003 to 2012. Curr. Top. Med. Chem. 2013;13:2745–2766.
    OpenUrl
  3. 3.↵
    Rateb ME, Ebel R. Secondary metabolites of fungi from marine habitats. Nat. Prod. Rep. 2011;28:290–344.
    OpenUrlCrossRefPubMed
  4. 4.↵
    Bhatnagar I, Kim SK. Immense essence of excellence: Marine microbial bioactive compounds. Mar. Drugs. 2010;8:2673–2701.
    OpenUrlCrossRefPubMedWeb of Science
  5. 5.↵
    Newman DJ, Cragg GM. Marine-sourced anti-cancer and cancer pain control agents in clinical and late preclinical development. Mar. Drugs. 2014;12:255–278.
    OpenUrl
  6. 6.↵
    Cafêu MC, Silva GH, Teles HL, Bolzani VS, Araújo AR, Young MCM, Pfenning LH. Substâncias antifúngicas de Xylaria sp., um fungo endofítico isolado de Palicourea marcgravii (Rubiaceae). Quim Nov. 2005; 28 (6): 991–5.
    OpenUrl
  7. 7.↵
    Frisvad JC, Smedsgaard J, Larsen T, Samson RA. Mycotoxins, drugs and other extrolites produced by species in Penicillium subgenus Penicillium. Stud. Mycol. 2004; 49: 201–41.
    OpenUrlCrossRef
  8. 8.↵
    Baker CJO, Shaban-Nejad A, Su X, Haarslev V, Butler G. Semantic Web Infrastructure for Fungal Enzyme Biotechnologists. Journal of Web Semantics First Look. 2006; 4(3): 168–180..
    OpenUrl
  9. 9.↵
    Samson RA. Food and indoor fungi. CBS-KNAW Fungal Biodiversity Centre, 2010.
  10. 10.↵
    Chai YJ, Cui CB, Li CW, Wu CJ, Tian CK, Hua W. Activation of the dormant secondary metabolite production by introducing gentamicin-resistance in a marine-derived P. purpurogenum G59. Mar. Drugs. 2012;10:559–582
    OpenUrlCrossRefPubMed
  11. 11.↵
    Wu CJ, Yi L, Cui CB, Li CW, Wang N, Han X. Activation of the silent secondary metabolite production by introducing neomycin-resistance in a marine-derived Penicillium purpurogenum G59. Mar Drugs. 2015;13(4):2465–2487. Published 2015 Apr 22.
    OpenUrl
  12. 12.↵
    Cutrim MVJ, Ferreira FS, dos Santos AKD, Cavalcanti LF, de Oliveira Araújo B, Gomes de Azevedo-Cutrim AC, Lima Oliveira AL. Trophic state of an urban coastal lagoon (northern Brazil), seasonal variation of the phytoplankton community and environmental variables. Estuar Coast Shelf S. 2019; 217: 98–109
    OpenUrl
  13. 13.↵
    Santos JMO, Moreira-Pais A, Neto T, Peixoto da Silva S, Oliveira PA, Ferreira R, Mendes J, Bastos MMSM, Lopes C, Casaca F, Silva S, Sweeney C, Medeiros R, Gil da Costa RM. Dimethylaminoparthenolide reduces the incidence of dysplasia and ameliorates a wasting syndrome in HPV16-transgenic mice. Drug Development Research. 2019; 80:824–830.
    OpenUrl
  14. 14.↵
    Medeiros-Fonseca B, Mestre VF, Colaço B, Pires MJ, Martins T, Gil da Costa RM, Neuparth MJ, Medeiros R, Moutinho MSS, Dias MI, Barros L, Bastos MMSM, Félix L, Venâncio C, Ferreira ICFR, Antunes LM, Oliveira PA. Laurus Nobilis (laurel) aqueous leaf extract’s toxicological and anti-tumor activities in HPV16-transgenic mice. Food and Function. 2018;9:4419–4428.
    OpenUrl
  15. 15.↵
    Santos S, Ferreira T, Almeida J, Pires MJ, Colaço A, Lemos S, Gil da Costa RM, Medeiros R, Bastos MMSM, Neuparth MJ, Abreu H, Pereira R, Pacheco M, Gaivão I, Rosa E, Oliveira PA. Dietary supplementation with the red seaweed Porphyra umbilicalis protects against DNA damage and pre-malignant dysplastic skin lesions in HPV-transgenic mice. Marine Drugs; 2019; 17:615
    OpenUrl
  16. 16.↵
    Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 coutries. CA Cancer J Clin. 2018; 68 (6): 394–424.
    OpenUrlCrossRefPubMed
  17. 17.↵
    Faustino-Rocha AI, Silva A, Gabriel J, Gil da Costa R, Gama A, Ferreira R, Oliveira PA, Ginja M. Ultrasonographic, thermographic and histologic evaluation of MNU-induced mammary tumors in female Sprague-Dawley rats. Biomed Pharmacother. 2013; 67: 771–776.
    OpenUrl
  18. 18.↵
    Faustino-Rocha AI, Colaço B, Oliveira PA. Experimental mammarycarcinogenesis - Rat models. Alvarado A1, Life Sci. 2017;173:116–134.
    OpenUrl
  19. 19.↵
    Segura JA, Barbero LG, Márquez J. Ehrlich ascites tumour unbalances splenic cell populations and reduces responsiveness of T cells to Staphylococcus aureus enterotoxin B stimulation. Immunol Lett. 2000;74(2):111–115
    OpenUrlCrossRefPubMed
  20. 20.↵
    Ali AD, Badr El-Din KN, Abou-El-Magd FR. Antioxidant and hepatoprotective activities of grape seeds and skin against Ehrlich solid tumor induced oxidative stress in mice. Egyptian Journal of Basic and Applied Sciences. 2015;2(2):98–109.
    OpenUrl
  21. 21.↵
    Dagli MLZ, Guerra JL, Saldiva PHN. An experimental study on the lymphatic dissemination of the solid Ehrlich tumor in mice. Braz. J. Vet. Res. An. Sci. 1992; 29 (1): 97–103.
    OpenUrl
  22. 22.↵
    Fortes TS, Fialho SEM, Reis AS, Assunção AKM, Azevedo APS, Barroqueiro ESB, Guerra RNM, Nascimento FRF. Desenvolvimento do tumor de Ehrlich em camundongos após tratamento in vitro com mesocarpo de babaçu Mart. Revista de Ciências da Saúde. 2009; 11:101–5.
    OpenUrl
  23. 23.↵
    Machado JL, Assunção AK, da Silva MC, Reis ASR, Costa GC, Arruda DS, et al. Brazilian Green Propolis: Anti-Inflammatory Property by an Immunomodulatory Activity. Evid Based Complement Alternat Med. 2012: 2012:157652.
    OpenUrl
  24. 24.↵
    Nascimento FR, Cruz GV, Pereira PV, Maciel MC, Silva LA, Azevedo APS, et al. Ascitic and solid Ehrlich tumor inhibition by Chenopodium ambrosioides L. treatment. Life Sci; 2006; 78(22):2650–3
    OpenUrlPubMed
  25. 25.↵
    Fialho SEM, Maciel MCG, Silva ACB, Reis AA, Assuncao AKM, Fortes TS, et al. Immune cells recruitment and activation by Tityus serrulatus scorpion venom. Toxicon (Oxford). 2011; 1:1–6
    OpenUrl
  26. 26.↵
    Bancroft JD, Cook, HC. Manual of Histological Techniques and Their Diagnostic Application. Edinburgh, London, New York, Tokyo: Churchill Livingstone; 1994.
  27. 27.↵
    Conselho Nacional de Controle de Experimentação Animal. Normativa do CONCEA para produção, manutenção ou utilização de animais em atividades de ensino ou pesquisa científica, 2015.
  28. 28.↵
    Geris R, Simpson TJ. Meroterpenoids produced by fungi. Nat Prod Rep. The Royal Society of Chemistry; 2009;26:1063–94.
    OpenUrl
  29. 29.↵
    Fang SM, Cui CB, Li CW, Wu CJ, Zhang ZJ, Li L. et al. Purpurogemutantin and purpurogemutantidin, new drimenyl cyclohexenone derivatives produced by a mutant obtained by diethyl sulfate mutagenesis of a marine-derived Penicillium purpurogenum G59. Marine Drugs. 2012; 10 (6): 1266–1287.
    OpenUrl
  30. 30.↵
    Natori S, Sakaki S, Kurata H, Udagawa SI, Ichinoe M. Production of rubratoxin B by Penicillium purpurogenum Stoll. Appl Microbiol. American Society for Microbiology (ASM). 1970;19:613–7.
    OpenUrl
  31. 31.↵
    Li SS, Li J, Sun J, Guo R, Yu LZ, Zhao YF, et al. Berkeleyacetal C, a meroterpenoid isolated from the fungus Penicillium purpurogenum MHZ 111, exerts anti-inflammatory effects via inhibiting NF-κB, ERK1/2 and IRF3 signaling pathways. Eur J Pharmacol. 2017;814: 283–93
    OpenUrl
  32. 32.↵
    Tang MC, Cui X, He X, Ding Z, Zhu T, Tang Y, Li D. Late-stage terpene cyclization by an integral membrane cyclase in the biosynthesis of isoprenoid epoxycyclohexenone natural products. Organic Letters. 2017;19(19): 5376–79.
    OpenUrl
  33. 33.↵
    He WJ, Zhou XJ, Qin XC, Mai YX, Lin XP, Liao SR, et al. Quinone/hydroquinone meroterpenoids with antitubercular and cytotoxic activities produced by the sponge-derived fungus Gliomastix sp. ZSDS1-F7. Nat Prod Res. 2017; 31(5): 604–9.
    OpenUrl
  34. 34.↵
    Stierle DB, Stierle AA, Patacini B. The berkeleyacetals, three meroterpenes from a deep water acid mine waste Penicillium. J Nat Prod. 2007;70(11):1820–3.
    OpenUrlPubMed
  35. 35.↵
    Etoh T, Kin YP, Tanaka H, Hayashi M. Anti-inflammatory effect of berkeleyacetal C through the inhibition of interleukin-1 receptor-associated kinase-4 activity. Eur J Pharmacol. 2013; 698 (1-3): 435–443.
    OpenUrl
  36. 36.↵
    Nagashima H, Goto T. Calcium channel blockers verapamil and diltiazem impaired rubratoxin B-caused toxicity in HL60 cells. Toxicol Lett. 2000; 118(1-2): 47–51.
    OpenUrlPubMed
  37. 37.↵
    Wang T, Zhang Y, Wang Y, Pei Y. Anti-tumor effects of Rubratoxin B on cell toxicity, inhibition of cell proliferation, cytotoxic activity and matrix metalloproteinase-2,9. Toxicology in Vitro. 2007; 21(4): 646–650.
    OpenUrlPubMed
  38. 38.↵
    Nagashima H. Rubratoxin-B-induced secretion of chemokine ligands of cysteine– cysteine motif chemokine receptor 5 (CCR5) and its dependence on heat shock protein 90 in HL60 cells. Environm Toxicol Pharmacol. 2015; 40(3): 997–1000.
    OpenUrl
  39. 39.↵
    Qin XJ, Yu Q, Yan H, Khan A, Feng MY, Li PP, et al. Meroterpenoids with Antitumor Activities from Guava (Psidium guajava). J Agric Food Chem. 2017; 65(24):4993–4999.
    OpenUrl
  40. 40.↵
    Sharma SH, Thulasingam S, Nagarajan S. Terpenoids as anti-colon cancer agents - A comprehensive review on its mechanistic perspectives. Eur J Pharmacol. 2017; 795:169–178.
    OpenUrl
  41. 41.↵
    Qin FY, Yan YM, Tu ZC, Cheng YX. Meroterpenoid dimers from Ganoderma cochlear and their cytotoxic and COX-2 inhibitory activities. Fitoterapia. 2018;129:167–172.
    OpenUrl
  42. 42.
    Silva IP, Brissow E, Kellner Filho LC, Senabio J, Siqueira KA, Vandresen Filho S, et al. Bioactive compounds of Aspergillus terreus—F7, an endophytic fungus from Hyptis suaveolens (L.) Poit. World J Microbiol Biotechnol. 2017;33(3):62.
    OpenUrl
  43. 43.
    Santos OJ, Sauaia Filho EN, Nascimento FRF, Silva Júnior FC, Fialho EDS, Santos RHP, et al. Use of raw Euphorbia tirucalli extract for inhibition of ascitic Ehrlich tumor. Rev Col Bras Cir. 2016; 43(1):18–21
    OpenUrl
  44. 44.↵
    Bianchi-Frias D, Damodarasamy M, Hernandez SA, Gil da Costa RM, Vakar-Lopez F, Coleman I, Reed MJ, Nelson PS. The aged microenvironment influences the tumorigenic potential of malignant prostate epithelial cells. Molecular Cancer Research. 2019; 17: 321–331.
    OpenUrlAbstract/FREE Full Text
  45. 45.
    da Costa RMG, Bastos MMSM, Medeiros R, Oliveira PA. The NFkB signalling pathway in papillomavirus-induced lesions: friend or foe? Anticancer Res. 2016; 36:2073–83.
    OpenUrlAbstract/FREE Full Text
  46. 46.
    Gupta I, Burney I, Al-Moundhri MS, Tamimi Y. Molecular genetics complexity impeding research progress in breast and ovarian cancers. Mol Clin Onc. 2017;7(1):3–14.
    OpenUrl
  47. 47.↵
    Zbakh H, Zubía E, de Los Reyes C, Calderón-Montaño JM, López-Lázaro M, Motilva V. Meroterpenoids from the Brown Alga Cystoseira usneoides as Potential Anti-Inflammatory and Lung Anticancer Agents. Mar Drugs. 2020;18(4):207.
    OpenUrl
  48. 48.↵
    Wan H, Li J, Zhang K, Zou X, Ge L, Zhu F, et al. A new meroterpenoid functions as an anti-tumor agent in hepatoma cells by downregulating mTOR activation and inhibiting EMT. Sci Rep. 2018;8(1):13152.
    OpenUrl
  49. 49.↵
    McCoy MK, Ruhn KA, Blesch A, Tansey MG. TNF: A Key Neuroinflammatory Mediator of Neurotoxicity and Neurodegeneration in Models of Parkinson’s Disease. Adv Exp Med Biol. 2011; 691: 539–40.
    OpenUrlCrossRefPubMed
  50. 50.↵
    Yoon WJ, Ham YM, Kim SS, Yoo BS, Moon JY, Baik JS, Lee NH, Hyun CG. Supressão de citocinas pró-inflamatórias, iNOS e expressão de COX-2 por algas marrons Sargassum micracanthum em macrófagos RAW264.7. Eurásia J. Biosci. 2009; 3: 130–143.
    OpenUrl
  51. 51.↵
    Fronza AS, Sabel R, Carrenho LZB, Pollo L, Bratti LOS, Monteiro FBF, et al. Antitumoral activity of sesquiterpene lactone diacethylpiptocarphol in mice. J Ethnopharmacol. 2017;198:262–267.
    OpenUrl
  52. 52.↵
    Calixto-Campos C, Corrêa MP, Carvalho TT, Zarpelon AC, Hohmann MSN, Rossaneis AC, et al. Quercetin reduces Ehrlich tumor-induced cancer pain in mice. Anal Cell Pathol, 2015;2015:285708.
    OpenUrl
  53. 53.↵
    Aldubayan MA, Elgharabawy RM, Ahmed AS, Tousson E. Antineoplastic Activity and Curative Role of Avenanthramides against the Growth of Ehrlich Solid Tumors in Mice. Oxid Med Cell Longev. 2019; 1–12.
  54. 54.↵
    Harun A, Vidyadaran S, Meng Lim S, Cole ALJ, Ramasamy K. Malaysian endophytic fungal extracts-induced anti-inflammation in Lipopolysaccharide-activated BV-2 microglia is associated with attenuation of NO production and, IL-6 and TNF-α expression. BMC Compl Alt Med. 2015;15(1):166.
    OpenUrl
  55. 55.
    Murphy RA, Mourtzakis M, Chu QSC, Baracos VE, Reiman T, Mazurak VC. Nutritional intervention with fish oil provides a benefit over standard of care for weight and skeletal muscle mass in patients with nonsmall cell lung cancer receiving chemotherapy. Cancer. 2011;117(8): 1775–1782.
    OpenUrlCrossRefPubMedWeb of Science
  56. 56.
    Lordick F, Hacker U. Gewichtsverlust aus onkologischer Sicht. DMW-Deutsche Medizinische Wochenschrift. 2016;141(4):247–252.
    OpenUrl
  57. 57.
    Lau SKM, Iyengar P. Implications of weight loss for cancer patients receiving radiotherapy. Curr Opin Support Pa. 2017; 11(4):261–5.
    OpenUrl
  58. 58.
    Iyengar NM, Gucalp A, Dannenberg AJ, Hudis CA. Obesity and Cancer Mechanisms: Tumor Microenvironment and Inflammation. J Clin Oncol. 2016;34(35):4270–76.
    OpenUrl
  59. 59.
    Hess LM, Barakat R, Tian C, Ozols RF, Alberts DS. Weight change during chemotherapy as a potential prognostic factor for stage III epithelial ovarian carcinoma: a Gynecologic Oncology Group study. Gynecol Oncol. 2007;107(2):260–5.
    OpenUrlCrossRefPubMed
  60. 60.↵
    Moreira-Pais A, Ferreira R, da Costa RMG. Platinum-induced muscle wasting in cancer chemotherapy: mechanisms and potential targets for therapeutic intervention. Life Sciences. 2018; 208: 1–9.
    OpenUrl
  61. 61.↵
    Peixoto da Silva S, Santos JMO, Costa e Silva MP, Gil da Costa RM, Medeiros R. Cancer cachexia and its pathophysiology: links with sarcopenia, anorexia and asthenia. Journal of Cachexia, Sarcopenia and Muscle. 2020.
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Posted July 02, 2020.
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Penicillium purpurogenum exerts antitumor effects and ameliorates inflammations in Erlich mice model
Amanda Mara Teles, Leticia Prince Pereira Pontes, Sulayne Janayna Araujo Guimarães, Ana Luiza de Araújo Butarelli, Gabriel Xavier Silva, Flavia Raquel Fernandes do Nascimento, Geusa Felipa de Barros Bezerra, Carla Junqueira Moragas Tellis, Rui Miguel Gil da Costa, Marcos Antonio Custódio Neto da Silva, Fernando Almeida-Souza, Ana Paula Silva de Azevedo-Santos, Maria do Desterro Soares Brandao Nascimento
bioRxiv 2020.07.02.184291; doi: https://doi.org/10.1101/2020.07.02.184291
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