In vitro antitumor activity
The current research was planned to detect the potential immunomodulation and antitumor efficacy of the extracts of P. pavonia and J. rubens inhabiting the Egyptian water. Our overall data has succeeded to present the extracts derived from P. pavonia and J. rubens as novel immune regulators and a promising anticancer agent against Ehrlich ascites breast carcinoma (EAC). Cancer is the world's most serious health concern, and the best treatment is cancer chemotherapy, which is cytotoxic to normal cells as well. Seaweeds are new, inexpensive, healthy, and efficient anticancer agents with minimal side effects on normal cells (Alves et al., 2018; Okolie et al., 2018; Zandi et al., 2010). Seaweed-derived extracts, such as P. pavonia and J. rubens, have been described as immunomodulatory and anticancer drugs, immunological regulators stimulating immune cells, enhancing immune function, and acting as adjuvants in cancer immunotherapy (Costa, et al, 2010; Liang et al., 2014; Xue et al., 2012; Yende et al., 2014;).
In a dose-dependent manner, treatment of EAC cells with P. pavonia and J. rubens extracts resulted in significant reductions in the number and survival of EAC tumor cells in an in vitro investigation. Regardless of differences in the chemical composition of the P. pavonia and J. rubens extracts; both induced direct antitumor effects, indicating that the biological products of genus pavonia and rubens have potent antitumor effects. Despite the antitumor effects of these two agents were not the same as those of cisplatin® (reference drug), our data had shown the promising antineoplastic effects of P. pavonia and J. rubens extracts especially at high doses (1000 µg/ml) in vitro. It is well known that cancer cells possess a high proliferative rate and low apoptotic rate -opposite to normal cells and the antitumor potential of conventional chemotherapeutic drugs such as Cisplatin® mostly mediated at least in part, by increasing apoptosis, and decreasing DNA proliferation of rapidly dividing cells (Sinicrope et al., 1996; Wang et al., 2005).
We found that P. pavonia and J. rubens extracts caused an elevation in EAC cell apoptosis Accompanied by a decrease in cell proliferation post i.p. treatment of EAC tumor cells for 24 h in vitro. In vitro studies also showed that the J. rubens extract had a higher apoptotic impact than T. atomaria, P. pavonia, and C. elongate extracts in enhancing the rate of EAC apoptosis, with IC50 values of 475.1 µg/ml, 580 µg/ml, 613 µg/ml, and 630.9 g/ml, respectively. Previously, it was reported that polysaccharides crude extracts of P. pavonia, J. rubes, and Corallina officinalis significantly inhibited the human breast MCF7 and colon cancer CoCa2, liver cancer cell line HePG2 cell lines proliferation in vitro when compared to the reference drug doxorubicin (Gheda et al., 2018; Matloub et al., 2015). The biological and antitumor activity detected in the tested seaweed extracts may be associated with the presence of oxygenated sterols and diterpenes (Awad et al., 2008), even though these should be further studied. The differences in the antitumor potentials between P. pavonia extracts and J. rubens extracts may be due to specific variations, environmental habitats, the used concentrations, or other factors that need further studies.
Antitumor effects of different doses of seaweeds extracts on the host tumor growth in vivo
In vitro investigations of growth suppression of EAC tumor cells are consistent and strongly suggest that P. pavonia and J. rubens extracts may have anticancer characteristics. Next, we tended to evaluate whether the direct immunomodulation and antitumor efficacy of the P. pavonia and J. rubens extracts can also be observed in vivo. In comparison to naïve EAC mice and Cisplatin®-treated EAC mice, i.p. treatment of EAC-bearing mice with P. pavonia extract at low, high, and prophylactic doses (1.3 g/mouse, 2.5 g/mouse, and 2.5 g/mouse, correspondingly) or J. rubens extract at low, high, and prophylactic doses (1.2 µg/mouse, 2.3 µg/mouse and 2.3 µg/mouse, respectively) have resulted in a considerable reduction in the number of ascetic EAC tumor cells. Previously, Ulva rigdia extracts significantly decrease the tumor's developmental size, J. rubens extract at 250 mg/kg and 500 mg/kg arrest the cell proliferation and tumor cell growth, and Eucheuma cottonii extract suppressed tumor growth and reduce mammary gland tumour development in rats (Ahmed et al., 2011; Mohini et al., 2014; Shamsabadi et al., 2013).
Changes in the level of leucocytes profile
In addition to the direct antitumor effect exerted by both extracts, we hypothesized that both extracts may exert an immunomodulatory effect that could indirectly potentiate their antitumor efficacy in vivo. We investigated the systemic immunomodulatory potentials of tumor inoculation and the ameliorative effect of P. pavonia and J. rubens extracts. The i.p. inoculation of EAC tumor cells induced significant leukocytosis. Furthermore, an elevated leukocyte count linked to the onset of cancer has been found as one of the predictors of an increased risk of venous thromboembolism (VTE) in cancer patients. Thrombosis is the second greatest cause of cancer-related mortality. (Khorana and colleagues, 2008). Interestingly, we explored that treatment of EAC mice with either Cisplatin® or P. pavonia extract or J. rubens extract significantly increased the leukocytosis induced by tumor inoculation. Moreover, both P. pavonia and J. rubens extracts displayed a more powerful efficacy than Cisplatin® in the inhibition of leukocytosis. Accordingly, this revealed that P. pavonia and J. rubens extracts may be potent tools for not only cancer treatment but also, for inhibition of thrombosis. Hence, they will help better the prognosis for cancer patients.
We hypothesized that i.p inoculation of EAC mice with P. pavonia and J. rubens extracts did not increase the total number of WBCs but elevated their flow to the tumor site. The migratory capacity of leukocytes is crucial to their role as defensive cells implicating their role in the progression and spread of tumors. Different leukocyte populations, including neutrophils, dendritic cells, macrophages, eosinophils, mast cells, and lymphocytes, are known to have a role in tumor-associated inflammation (Balkwill and Mantovani, 2001). Our results revealed that i.p. inoculation with P. pavonia and J. rubens extracts managed to decrease neutrophilia induced by tumor inoculation. Neutrophilia is one of the haematological findings related to poor prognosis in human metastatic melanoma, pancreatic carcinoma, and renal carcinoma (Fogar et al., 2006; Schmidt et al., 2005).
Remarkably, treatment of EAC mice with P. pavonia and J. rubens extracts at low, high, and prophylactic doses induced a slight increase in the lymphocytes relative number compared to naïve EAC mice and Cisplatin®-treated EAC mice. Unlike neutrophils, the increased lymphocytic count is considered a good prognostic candidate in diverse cancers (Lissoni et al., 2006).
Monocyte subsets are immune cells that have recently been identified as key regulators of cancer growth and development. Phagocytosis, tumouricidal mediator secretion, angiogenesis, extracellular matrix remodeling, lymphocyte recruitment, and tumor-associated macrophages differentiation and dendritic cells are all functions performed by different monocyte subsets that associate to the antitumoral immunity (Olingy et al., 2019). Intraperitoneal treatment of EAC mice with P. pavonia at high or prophylactic doses and J. rubens extracts at low, high or prophylactic doses has successfully decreased the monocytes relative number compared to naïve EAC mice and Cisplatin®-treated mice. Meanwhile, a low dose of P. pavonia extract seemed to increase the relative number of monocytes compared to naïve EAC mice. Such data could be consistent with the associated neutropenia induced by EAC tumor cells treatment. As, withdrawal of neutrophils numbers is accompanied by their transformation into other myeloid cells including MDSCs (Sagiv et al., 2015).
Flow cytometry analysis
Algal extracts have been widely used in cancer immunotherapy due to their properties as an initiator and modulator of immune responses, and their vaccinations appear to be safe and enhance immunological responses in cancer treatment as well as long-term clinical remissions, but there are some issues that need to be addressed before they can be used clinically. According to flow cytometric analysis of phenotypic expression of immune lymphocyte cells subset surface markers, treatment of mice with P. pavonia and J. rubens extracts at low, high, and prophylactic doses showed putative immune-modulatory effects on the expression and proliferative response of spleen lymphocyte subpopulations; CD4 + T, CD8 + T, and NK cells.
P. pavonia and J. rubens administration increased the percentage of lymphocyte subpopulations; CD4+ T cells, CD8+ T cells, and CD335 cells and lymphocyte proliferative responses that were stimulatory at all tested doses, and there was a dose-dependent increase in the proliferative activity of CD4 + T-cells, CD8 + T cells, and NK cells in EAC-bearing mice comparing to naïve EAC mice and Cisplatin®-tread EAC mice.
More specifically, i.p. inoculation of EAC mice with P. pavonia and J. rubens extracts induced an immune response favoring tumor cell death, tumor antigen cross-presentation in vivo, and the production of cytokines favoring homeostatic proliferation and/or ablation of immunosuppression mechanisms (Baxevanis et al., 2009). The change in the ratio of CD4+T-cell, CD8+T-cell, may be attributed to the migration of immune cells populations to the developed tumor, leading to a significant reduction of the same population in the spleen.
Natural killer ( NK ) cells are an innate immune system subpopulation of lymphocytes that play a key role in the host defense against tumor growth and infectious pathogens (Shen et al., 2008). NK cells can detect and kill a broad variety of abnormal cells, like tumor cells, without damaging healthy cells (Caligiuri, 2008). The same findings as polysaccharide from Laminaria digitate activated immune systems, B and helper T lymphocytes were evaluated in the literature (Hoffman et al., 1995). Later, the fucoidan isolated from Sargassum sp and Fucus vesiculosus stimulates natural killer cell activity in vivo in noncancerous mice (Ale et al., 2011). Data from in vivo studies observed inhibitory activity of cancer, which could be attributed in part to increased innate and specific immunity (Lowenthal and Fitton, 2015). Fucoidan from Undaria pinnatifida sporophyll increased survival in P-388 tumor-bearing mice, which was linked to the enhancement of natural killer (NK) lymphocyte activity and increased interferon-gamma production by T cells (Maruyama et al., 2003). Gracilaria lemaneiformis Sulfated polysaccharides prevented tumor growth; increased CD8+ T cells, splenocyte proliferation, macrophage phagocytosis (Fan et al. 2012; Gesheva et al., 2014).
Our results show that one of the antitumor mechanisms of the extract of P. pavonia and J. rubens may be to mediate an increase in T-lymphocytes subsets of helper (CD4+ T-cell), cytotoxic (CD8+ T-cell), and NK cells in the spleen that resulted in the production of immunostimulation of the immune system triggering the antitumor response development.
Assessment of apoptosis by flow cytometry
In the current study, we aimed to analyze the potentiation of both extracts on the apoptosis rate and proliferative rate of EAC tumor cells by Annexin V-FITC Apoptosis assay using flow cytometry, which targets the loss of integrity of the plasma membrane (Vermes et al., 1995; Wlodkowic et al., 2009). It is well known that cancerous cells have a high proliferative degree and low apoptotic rate -contradictory to normal cells. The antitumor efficacy of conventional chemotherapeutic agent such as Cisplatin® is generally mediated at least in part, by elevating apoptosis rate, and reducing DNA proliferation of rapidly dividing cells (Sinicrope et al., 1996; Wang et al., 2005).
Interestingly, P. pavonia and J. rubens extracts at all tested doses increased the percentages of apoptosis in a dose-dependent manner as high doses increase the percentages of apoptosis compared to low ones. Besides, analysis of the cell cycle revealed that the P. pavonia extract was of a more potent effect than J. rubens extract in increasing apoptosis of EAC cells in vivo. As it successfully increased EAC apoptosis even more than the therapeutic dose of Cisplatin®. It is recommended a strongly related activity of P. pavonia and J. rubens extracts to develop an antitumor response in a tumor-bearing host.
Our data revealed that i.p. inoculation of EAC mice with P. pavonia and J. rubens extracts at low, high, and prophylactic doses resulted in an elevation in EAC cell apoptosis accompanied by a decrease in cell proliferation. Additionally, in vivo data indicated that the P. pavonia extract was of a more apoptotic effect than J. rubens extracts in increasing the rate of EAC apoptosis. The apoptosis mode of action may varyr among different anticancer bioactive compounds, and the main anticancer mechanism of the P. pavonia and J. rubens extracts is the apoptosis induction (Jose, 2015). Several other anticancer studies of sulfated polysaccharides from P. pavonia and Jania rubens (Gheda et al., 2018; Jose et al., 2018 ) were published, with identical findings for both qualitative and quantitative apoptosis evaluation.
Liver and kidney functions
Finally, serum liver transaminases (ALT and AST) and albumin are tested to rule out hepatocellular injury and hepatotoxicity as a side effect of therapy. Their increases in serum are a cellular outflow marker and a loss of functional integrity of hepatocyte cell membranes (Ghouri et al., 2010; Rajesh and Latha, 2004). We also investigate the serum kidney creatinine and urea, the most widely used biomarkers for determining the amount of nephrocellular damage, to rule out nephrocellular damage and nephrotoxicity caused by chemical stressors (Orinya et al., 2016; Singh et al., 2011).
Our data revealed that i.p. inoculation of J. rubens extracts at low, high or prophylactic doses, but not P. pavonia extract doses, has successfully decreased the level of serum AST, ALT, and albumin and showed a significant effect in restoring their levels to be very close to the naïve mice comparing to naïve EAC mice and Cisplatin®-treated EAC mice, indicating restoring liver function and integrity and promoting the antioxidant hepatoprotective role of J. rubens extracts (Bodansky, 1973). A previous study reported that treatment of experimental animals with extract of J. rubens, Sargassum subrepandum, and Ulva lactuca maintained liver enzymes in the range of the normal levels compared to the naïve tumor group. Therefore, methanol extract of J. rubens may be promising hepatoprotective agents that could synergistically inhibit the process of hepatocellular injury initiation and progress in combination with its antioxidant activity (El Gamal, 2010).
Hepatotoxicity may be induced by a high dose of Cisplatin® (Cüre et al., 2016). One of the most significant pathway implicated in Cisplatin® toxicity is oxidative stress and may be the primary source of Cisplatin®-induced toxicity attributable to decreased GSH glutathione depletion (Bentli et al., 2013). Transaminases are the most important biomarkers specifically involved in causing cell damage and toxicity, as they are released into circulation after cell damage.
Importantly, our results revealed that i.p. inoculation of J. rubens extracts at low, high or prophylactic doses, but not P. pavonia extract doses, has successfully decreased the concentrations of serum creatinine and urea and reported a significant effect in restoring their concentrations to be close to the naïve mice comparing to naïve EAC mice and Cisplatin®-treated EAC mice. As a result, methanol extract of J. rubens may be promising nephroprotective agents that could synergistically inhibit the initiation and progression of nephrocellular injury in combination with its antioxidant activity. This effect could be due to its antioxidant activities and active antioxidant constituents.
Nephrotoxicity is Cisplatin's main dose-limiting side effect (Sastry and Kellie, 2005). Cisplatin®, as well as direct cellular toxicity, triggers an inflammatory cascade where neutrophils may induce kidney injury via different pathways such as the development of proinflammatory mediators, reactive species, proteases, and other products that may provoke tubular damage resulting in acute kidney injury (Tadagavadi and Reeves, 2017).