INTRODUCTION

Breast cancer is a social health problem due to its high incidence, the mortality rate, therapeutic complexity and the high cost of treatment ^{1 , 2}. Among the therapeutic options, chemotherapy is linked to potential multisystemic adverse effects that result in the lack of adherence to treatment and an increase in morbidity and mortality. This has led to an increased search in recent decades for new substances with anti-tumor potential as alternative treatments, such as natural products derived from marine organisms, which account for more than 60% of the antineoplastic agents currently used ^{3 - 5}.

Various peptides, produced by marine species and their host bacteria, present a wide range of biological activity with antimicrobial, antitumor and antiviral action. Among the mechanisms that involve antineoplastic effects, the blocking of cell division, which mainly affects tubulin, is similar to the effect of alkaloid drugs like vinca and taxanes ⁶. Drugs derived from marine peptides such as trabectedin, cytarabine, vidarabine, and ziconotide have been approved by the European Union and the Food and Drug Administration (FDA) for treating advanced stages of soft tissue sarcoma and ovarian cancer ^{7 - 10}. An estimated 118 natural marine products (NMP) are in pre-clinical trials; 22 NMP are in clinical trials; and 4 NMP are in the pharmaceutical market ¹¹.

Bacteria of the genus Vibrio are known to be indigenous members of the bacterial biota of the seas and estuaries, and constitute 0.1 to 60% of the total heterotrophic bacteria. There are studies that show that the crude extract produced by some species of this genus present antibacterial activity against Staphylococcus aureus, resistant to methicillin, and cytotoxic activity against some cell lines ¹². Other studies indicate that peptides obtained from the genus Vibrio present cytotoxic action against the murine leukemia P388 cell line ¹³. Also, one of the compounds produced by a marine member of Vibrionaceae is in a clinical trial for the treatment of prostate, lung and liver cancer and possibly these metabolites will sensitize the tumor cells to macrophage-mediated cytolysis ¹⁴.

Consequently, the evaluation of the anti-tumor activity of peptides derived from marine Vibrio sp. constitutes an important line of biotechnological and pharmacological research; however, further studies are required to determine the efficacy of their biological activity in solid tumors such as breast cancer. This study aims to evaluate the anti-tumor effect of biopolymers isolated from the marine bacterium Vibrio sp. on N-Methyl-N-nitrosourea (MNU)-induced breast cancer in rats.

MATERIALS AND METHODS

RESULTS

One of the parameters observed to assess disease severity was body weight gain at the end of treatment. The group treated with only the cancer inducer (MNU) presented a mean weight gain of 115 g; while animals treated with MNU + crude extract of biopolymers isolated from Vibrio sp. showed a 141 g mean weight gain, this difference being significantly higher (p = 0.03) to the MNU group and close to the control group (133 g) (Figure 1).

Figure 1 Weight gain at the end of the study in animals treated with marine bacteria Vibrio sp. biopolymers. 

Figure 2 shows the average number of animals that developed tumors and shows that 76.9% of the rats in the control group developed tumors at the end of the experiment. Only 7.7% of the animals in the MNU + crude biopolymer of the marine bacteria Vibrio sp. groups and MNU + tamoxifen developed tumors (p = 0.001). The most frequent locations were axillary, suprainguinal and suprapubic on both sides of the mammary chain. The latency period for tumor occurrence was 7 weeks for the control MNU group and 9 weeks for the MNU + biopolymer group.

Figure 2 Percentage of palpable tumors in animals treated with marine bacteria Vibrio sp. Biopolymers. 

The total number of tumors observed in the control group was 22 with an average of 2.2 tumors per animal, while only one tumor was evident in each of the MNU + crude biopolymer extract of Vibrio sp. groups and MNU + tamoxifen, as shown in Table 1. In addition, it was observed that the average weight of the tumor in the group treated with crude biopolymer extract was greater than that treated with MNU alone, however, for the tamoxifen treated group, the tumor developed, was significantly smaller than the control.

According to the histopathological results, all the removed tumors correspond to breast ductal adenocarcinoma with different patterns: solid, papillary and cystic. Likewise, necrotic foci were observed in 30% of the tumors found in the MNU group, suggesting a greater invasive capacity of the tumor (Table 2).

DISCUSSION

Marine organisms and their metabolites represent an enormous potential of unexplored natural resources and therapeutic products. Bacterial proteins and peptides derived from marine organisms are a promising group of bioactive compounds and possible anti-cancer drugs; these include anti-cancer antibiotics (actinomycin D, bleomycin, doxorubicin, mitomycin C) and bacterial toxins used in cancer treatment, while other substances are in clinical trials or have been tested in in-vitro research ^{20 , 21}.

Using a standard model induced by MNU, it has been possible to reproduce breast cancer in rats; this has allowed us to explore in-vivo the anti-tumor activity of biopolymers extracted from the marine bacterium Vibrio sp. culture. According to the results, the latency period for tumor occurrence in the control group was 7 weeks post-induction; while in the MNU + biopolymer treated group, it was 9 weeks, similar to the tamoxifen treated group, which would demonstrate that the biopolymers would delay the occurrence of MNU-induced tumors. In addition, we note that in the biopolymer-treated groups only 7.7% of the animals developed tumors in contrast to the control group where 77.9% of the animals developed tumors with an average of 2.93 per animal.

Although the protection mechanism is not known, other authors postulate that in solid tumors, biopolymers extracted from marine bacteria would have an immunomodulatory effect, activating the mechanisms against tumor cells. This would produce some tumor regression and, consequently, the latency period for tumor appearance and the life of the animals would be extended ²².

Studies with the Vibrio genus isolated from marine species show that it produces substances with antagonistic activity against other pathogenic bacteria ¹⁶. Conde, et al. ¹² evaluated the cytotoxic and dereplication activity of the fractions extracted from the culture medium of Vibrio diabolicus; the authors showed that the F4 and F6 fractions were cytotoxic against the human cervical epithelial cancer cell line, SiHa, with an IC50 of >100 µg/mL and 80 µg/mL, respectively. On the other hand, the F5 fraction showed higher cytotoxic activity with an IC50 of 28 µg/mL against the same cell line. Furthermore, none of these fractions showed inhibition of the non-tumor cell line (CI50 >100 µg/mL).

Cao, et al. ²³ investigated the molecular mechanisms of the polysaccharide EPS11, of marine bacterial origin (Bacillus sp.), on cytotoxicity in small-cell lung cancer. The authors found that EPS11 significantly affects cell proliferation and blocks adhesion in tumor cells. In addition, the expression of several proteins associated with cell adhesion is negatively regulated and the filiform structures of lung cancer cells are destroyed after treatment with EPS11. This same biopolymer has also been shown to inhibit liver cancer cell growth by blocking cell adhesion and attenuating the formation of filiform structure, and stopping cancer cell metastasis ²⁴.

Studies about the action of biopolymers derived from marine bacteria on breast cancer have only been conducted in in-vitro models of breast carcinoma cell lines. Mahgoub et al. ^{25 )} evaluated the effect of a biopolymer isolated from the marine bacterial strain, Bacillus velezensis, on breast cancer cell lines MCF-7. According to published results, this metabolite increases apoptosis and hinders the proliferation of MCF-7 cells by 5-80 μg/mL compared to the control group. In addition, induced apoptosis is associated with activation of caspase-3 which is dose-dependent and does not have cytotoxicity against normal cells; similar results were reported by Sirpu et al. ^{26 )} with the partially purified crude extract of marine NMK17 Bacillus subtilis (PPCEBS) in the human MCF-7 breast cancer cell line. The findings demonstrated that PPCEBS significantly induces apoptosis in the studied cell line and would result in increased expression of caspase-3 and Bax revealing the possible mechanism of the apoptosis-inducing property. Similarly, PPCEBS also presented antibacterial and antioxidant activities.

These results suggested that the compounds present in PPCEBS of marine bacteria B. subtilis NMK17 would be cytotoxic metabolites that could be candidates for developing a drug specific to breast line tumor cell apoptosis, with minimal toxicity. However, other authors observe that two new cyclic hexapeptides, venturamide A and B obtained from the marine cyanobacterium Oscillatoria sp. have only mild activity when tested against breast cancer cells MCF-7 with a CI50 value of 13.1 and >54 µM, respectively ²⁷. Likewise, when pitiprolamide, a cyclic depsipeptide derived from the marine cyanobacterium Lyngbya majuscule, was isolated, only weak cytotoxic activity against the colorectal carcinoma HCT116 and the breast adenocarcinoma cell lines MCF7 was noted (CI50 33 µM for both) ²⁸.

Besides the anti-tumor effect, it was observed that weight gain was significantly higher for the group treated with crude biopolymer extract derived from Vibrio sp. bacteria in relation to the control group (Figure 2). These results have a positive anti-tumor implication since the weight loss in cancer is due to an increased caloric demand due to the tumors’ presence (with the corresponding competition for nutrients between the patient’s cells and those of the tumor) and malnutrition due to anorexia caused by pro-emaciation cytokines released during the progression of the disease ²⁹.

On the other hand, only one animal in the biopolymer-treated group presented a tumor, the tumors’ weight was higher than the average weight of the control. However, this was only observed in one tumor and in one animal in a ratio of 1/13 in contrast to the control group where there was an average of 2.2 tumors per animal and in a ratio of 10/13; therefore, it could be considered as an isolated event.

Sample size extension and biopolymer dosing are required to obtain a dose-response curve. In addition, the observation time needs to be extended to assess the effect of biopolymers on the natural course of the disease. Although the MNU-induced breast cancer model replicates many pathophysiological features of what is observed in human disease, an in-vivo model with a human tumor cell line infiltration in experimental animals would contribute greatly. Some mechanisms of action of the anti-tumor effect suggested could be related to the activation of tumor cell apoptosis and activation of the immune system, as has been observed in cultures with other bacteria ^{24 , 26}.

The main limitation of this study is that it has not been possible to isolate, identify or perform chemical characterization of the biopolymers obtained and only their crude extract has been evaluated. It would be necessary to perform fractionation and pharmacological tests to identify the fraction with the best antitumor activity. Although it is true that three groups were included (a control group of healthy animals, a group with the disease and another group treated with a standard drug for the treatment of breast cancer), our limitation was not to include an experimental group treated with the biopolymer-free culture medium, and we did not perform acute and chronic toxicity tests on the biopolymers.

Results show that the crude extract of the bacteria Vibrio sp. can constitute a source of biopolymers with anti-tumor activity. Therefore, it is suggested to deepen research about breast cancer, as well as its possible mechanism of action and the active metabolite. The extrapolation of the expected benefits with the application of special isolation techniques of the active principle would allow to be a potential candidate in the prevention and treatment of neoplastic diseases.