Marine-Derived Penicillium purpurogenum Reduces Tumor Size and Ameliorates Inflammation in an Erlich Mice Model

Abstract

Background: This study addresses the antitumoral properties of Penicillium purpurogenum isolated from a polluted lagoon in Northeastern Brazil.

Methods: Ethyl Acetate Extracellular Extract (EAE) was used. The metabolites were studied using direct infusion mass spectrometry. The solid Ehrlich tumor model was used for antitumor activity. Female Swiss mice were divided into groups (n = 10/group) as follows: The negative control (CTL-), treated with a phosphate buffered solution; the positive control (CTL+), treated with cyclophosphamide (25 mg/kg); extract treatments at doses of 4, 20, and 100 mg/kg; animals without tumors or treatments (Sham); and animals without tumors treated with an intermediate dose (EAE20). All treatments were performed intraperitoneally, daily, for 15 days. Subsequently, the animals were euthanized, and the tumor, lymphoid organs, and serum were used for immunological, histological, and biochemical parameter evaluations.

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

Conclusions: These results indicate that P. purpurogenum exhibits immunomodulatory and antitumor properties in vivo. Thus, fungal fermentation is a valid biotechnological approach to the production of antitumor agents.

Keywords: Ehlich’s tumor; P. purpurogenum; antitumor; inflammation; meroterpenoids.

Figure 1

HPLC-DAD-UV chromatogram (300 nm) obtained from the Penicillium purpurogenum extract showing 12 peaks of which the UV spectral data (λ max) are characteristic of meroterpenoid compounds.

Figure 2

Mass spectra obtained for the tentative identification of meroterpenoids 1–5.

Figure 3

Effect of the Penicillium purpurogenum extract on the development of Ehrlich’s solid tumor. (a) The kinetic of the paw volume inoculated with Ehrlich’s tumor followed by intraperitoneal treatment with phosphate buffer solution (CTL−), cyclophosphamide 25 mg/kg (CTL+), and extracts with a concentration of 4 mg/kg (EAE4), 20 mg/kg (EAE20), and 100 mg/kg (EAE10) at 24 h intervals, with the animals being euthanized on day 15. (b) The 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. Values are expressed as the mean ± standard error of means (SEM) deviation and analyzed by analysis of variance (one-way or two-way ANOVA) with * p < 0.05, ** p < 0.005, and **** p < 0.0001 relative to the negative control (CTL−); # p < 0.05, ## p < 0.01 when compared to the positive control (CTL+); and Θ p < 0.05 shows a difference in tumor growth for the other groups; φ shows that, on the eighth day, only the extract inhibited tumor growth.

Figure 4

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 treated daily with EAE extract intraperitoneally. At the end of the fifteen days of treatment, the animals were euthanized, and their feet were amputated, weighed, and fixed. Histological sections were stained with Hematoxylin–Eosin. In the photos, it is possible to see the tumor cells (indicated by the letter T) and the inflammatory infiltrate (indicated by letter I) present in the paws of the Sham (A), CLT− (B), and CLT+ (C) groups (100× total magnification). The animals treated with the extract showed a decrease in the infiltrate and necrosis are EAE doses of EAE 4 (D), EAE 20 (E), and EAE 100 (F) (100× total magnification). The Sham group is shown in panel G, and the animals treated using the extract with a dose of EAE 100 are shown in panel H (400× total magnification).

Figure 5

Immunomodulatory effects of Penicillium purpurogenum ethyl acetate extract. (a–d) The results are expressed as the mean ± standard deviation of the total lymph node cell count; bone marrow and splenocytes obtained from the group without tumors treated with saline solution (Sham) from the groups with solid Ehrlich tumors treated intraperitoneally with extracts at doses of 4, 20, and 100 mg/kg, respectively (EAE4, EAE20, and EAE100); positive and negative control-administered cyclophosphamide (CLT+) and saline solution (CTL−), respectively. Blood serum was used to quantify the tumor necrosis factor (TNF-α). The data were submitted to statistical analyses via Kruskal–Wallis and Dunn multiple comparison tests, with a significance of ∆ p < 0.05, ∆∆ p < 0.005, ∆∆∆ p < 0.0005, and ∆∆∆∆ p < 0.0001 in relation to the 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, and **** p < 0.0001 when compared to CLT−; and οο p < 0.0001 οοο p < 0.0001 when comparing the extracts with each other.

Figure 6

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 the groups with induction received the extract at doses of 4, 20, and 100 mg/kg (EAE4, EAE20, and EAE100), as well as saline (CTL−) and cyclophosphamide (CTL+). The graphs show (a) the animals’ body weights in the final treatment. (b) After the treatment of the animals, the animals remained under observation for thirty days. The data represent the mean ± SEM. The difference was statistically analyzed by a Kruskal–Wallis and Dunn’s multiple comparison test, with significance of ∆ p < 0.05, ∆∆ p < 0.005, and ∆∆∆ p < 0.0005 in relation to the Sham, ϕ p < 0.05 ϕϕ p < 0.005, and ϕϕϕ p < 0.0005 when compared to EA20 without a tumor, ο p < 0.05 when compared to CLT+, and οοο p < 0.0005 when compared to CLT−, * p < 0.05, ** p < 0.005, and #### p < 0.0001.

Figure 7

Dose–response curve of Penicillium purpurogenum ethyl acetate extract (EAE) for MCF7 cell viability. Data represent the means ± SD of the viable MCF7 cell percentage in relation to the untreated cells after 24 and 48 h of treatment with EAE (A) and doxorubicin (B).