Glioblastoma multiforme (GBM) is a malignant primary brain tumor common amongst the young kids with range of age < 13 years, characterized by high rates of proliferation, metastasis, and resistance to chemotherapy protocols (Brada et al., 2001; Puli et al., 2006; Markiewicz-Żukowska et al., 2013). These therapeutic failures represent a great difficulty in its treatment and management, leading to short survival rate, and loss of patient’s life quality (Puli et al., 2006). Its key epidemiologic risk includes age, sex, race, lifestyle behaviors, diet, and exposure to environmental factors including pollution from different sources (Efird, 2011).

Though epidemiology of glioblastoma has reported little or no occurrence in West Africa, changes in lifestyle particularly increased urbanization and adoption of western lifestyles and diet are however a major concern (Erukainure et al., 2016). An indigenous African diet often consists mainly of vegetable leaves, unrefined grains and spices, unlike the western counterpart consisting of sweetened desserts, saturated fats, and processed grains (Erukainure et al., 2016). Almost 30–40% of all cancers have been found to be preventive by healthy life style such as weight control, exercise, and physical activities (Potter and Potter, 1997; Jemal et al., 2011). Several studies have linked consumption of dietary fatty acids in diet with reduced brain cancer. Ketogenic diet characterized by high-saturated fats and low protein/carbohydrate, has proven to be effective during therapy and potent adjuvant management of malignancies in almost all types of cancers (Woolf and Scheck, 2015). Antal et al. (2014) reported increased sensitivity of glioblastoma cells to radiotherapy after treatment with arachidonic acid, docosahexaenoic acid, and γ-linolenic acid.

Clerodendrum volubile is among the common leafy vegetables in Southern part of Nigeria and it is well established for its medicinal use (Erukainure et al., 2010). It is indigenously known as Obenetete by the Itsekiris and Urhobos in the Delta of Niger. Commonly known as magic leaf, it is also used in the management and sometimes as adjunct for the treatment of diabetes mellitus, arthritis, rheumatism, ulcers, and many other diseases (Burkill, 1985). The phytochemicals of C. volubile and antioxidant activities have been reported (Adefegha and Oboh, 2010; Erukainure et al., 2011). Erukainure et al. (2014) isolated an iridoid glycoside from the leaves and reported its antioxidant activity in rats’ brain and hepatic tissues. In our previous study, we extracted dietary fatty acids from the leaves and investigated its effect on breast cancer cells (Erukainure et al., 2016). The fatty acids arrested cell cycle progression and down-regulated matrix metalloproteinase-9 in the breast cancer cells (Erukainure et al., 2016). Furthermore, molecular studies are required to prove the proclaimed medicinal uses of the extract of C. volubile leaves.

This present study aims to report the anti-proliferative, anti-oxidative, and anti-migratory and/or anti-metastatic activity of the fatty acid rich extracts from leaves of C. volubile on U87-MG cancer cells.

A dose-dependent cytotoxic activity of the fatty acid extract was observed in U87-MG cell lines with an IC₅₀ of ∼120 μg/mL as shown in Figure 1, indicating a potent anti-proliferative effect.

Apoptosis analysis by PI flow cytometry (Figure 2) revealed high cell deaths as the concentration increased, which is an indication of a dose-dependent anti-tumor activity of the fatty acid extract in U87-MG cancer cells.

The quantity of DNA in each cancer cell population in G0/G1 and G2/M phases were significantly depleted in the fatty acids treated cells, as depicted in Figure 3. This was also observed with the simultaneous arrest of S phase of the cell cycle.

Transwell migration analysis revealed the migration of the untreated cells in all six parts of the chamber of 24-well transwell plate, as shown in Figures 4, 5A, indicating an occurrence of cell migration and/or invasion. However, the fatty acid treated cells showed little or no migration (Figure 5B), indicating a suppressive effect on cell migration and/or invasion.

Incubation of cells and fatty acid extract led to significant (p < 0.05) increase in GSH level with a concomitant decrease in MDA level, indicating an anti-oxidative activity as shown in Figures 6A–D. The significant (p < 0.05) increase as well as slight increase in catalase and SOD activities, respectively, further indicates the antioxidant potency of the fatty acids.

The fatty acids displayed a dose-dependent inhibitory effect on α-chymotrypsin activity and its kinetics as presented in Figures 7A,B.

Glioblastoma multiforme (GBM) has been recognized as a major brain tumor, and implicated in extensive invasion into neighboring brain tissue (Puli et al., 2006). This rapid invasion makes it difficult to treat especially while using surgical resection (Brada et al., 2001; Puli et al., 2006; Markiewicz-Żukowska et al., 2013). Hence, the need for an effective anti-proliferative agent that can suppress this invasion and/or migration with little or no side effects. In our previous study, we characterized the fatty acids extracted from C. volubile leaves using GC-MS to identify the fatty acid composition (Erukainure et al., 2016). Oleic acid was identified as the most abundant component, along with octadecanoic acid, n-hexadecanoic acid and 2-heptanone, 6-methyl in lesser quantities (Erukainure et al., 2016). In this present study, the suppressive effect of dietary fatty acids extracted from C. volubile was investigated on human glioblastoma multiforme cells with the aim of elucidating the possible molecular mechanism, along with its effect.

The cytotoxic effect of fatty acids on cancer cell lines has been reported in previous studies (Motaung et al., 1999). This corresponds with the observed cytotoxic effect of the extracted oil on U87-MG cells (Figure 1), thus indicating an anti-proliferative potential against GBM. This corresponds with our previous study of the anti-proliferative effect of C. volubile fatty against breast cancer, attributed to its high oleic acid concentration. The protective role of oleic acid against several cancers has been reported in previous epidemiological and animal studies (Carrillo et al., 2012). Thus, the observed anti-proliferative effect of the fatty acid extract, can be attributed to the high oleic acid content.

Novel natural therapies, targeted on induction of apoptosis in cancer cells, are gaining major interest. The role of apoptosis in cancer therapy is well emphasized in several studies. Most cancers manipulate and/or downregulates the antiapoptotic molecules, thereby evading this highly regulated program cell death (Koff et al., 2015). The observed apoptotic activity of the fatty acid extract as evident by the increased cell death (Figure 2), further indicates the anticancer potency of the fatty acids. This can be attributed to the arrest of S phase with concomitant depopulation of the DNA contents/cell population in G0/G1 as well as G2/M phases of the cell cycle (Figure 3). Cell cycle arrest has been implicated in the induction of apoptosis (Aliyu et al., 2013). Fauser et al. (2013) reported the induction of cell cycle by fatty acids.

Cell migration and invasion have been associated with the pathophysiology of cancer and in fact a major characteristic of malignant tumors and one of the key causes of cancer death (Bozzuto et al., 2010). In this study, the observed anti-migration, and invasion activity of the fatty acid extract on U87-MG cancer cells indicates a suppressive effect (Figure 5). This can be associated with the ability of the fatty acid enriched extract to inhibit α-chymotrypsin in vitro (Figure 7). Alpha-chymotrypsin has been implicated in the activation of metalloproteinases, and degradation of the extracellular matrix (ECM proteins), thus plays a major role in tumor growth and metastasis (Novak and Trnka, 2005; Mu et al., 2012). ECM acts as a physical barrier to migrating cells and must be degraded before the metastasis can occur. The in vitro inhibition of α-chymotrypsin (Figure 7) by the fatty acids therefore indicates a protective potential against ECM degradation. It can thus be suggested that the anti-metastatic action of the fatty acid extract could be facilitated by lowering the ability of glioblastoma cells to degrade the extracellular matrix components, by inhibiting the activities of α-chymotrypsin.

The implication of oxidative stress in the etiology of cancer has been reported (Erukainure et al., 2016). Oxidative stress arises due to an imbalance in the production of free radicals and the cell’s own antioxidant defenses (Erukainure et al., 2016). Often described as redox imbalance, it causes an impairment of normal cellular metabolism, which promotes malignancy, cancer initiation, and progression (Acharya et al., 2010). The observed reduced GSH level, SOD and catalase activities with concomitant increased MDA level in untreated U87-MG cells indicates an occurrence of oxidative stress (Figures 6A–D). Alteration of these enzymes has been reported in malignant cells and tumor tissues, thus suggesting an occurrence of redox imbalance in cancer cells (Barrera, 2012). The reversed levels and activities on following treatment with the fatty acids indicate an antioxidative potency against glioblastoma (Figure 6). Erukainure et al. (2016) reported a similar effect by same extract on MCF-7 cells, indicating the antioxidant protective effect of the leaf fatty acids against human invasive cancers. A remarkable association has been recognized between oxidative stress and increased uncontrollable drive of the G0/G1 and G2/M phases, leading to tumor metastasis and invasion (Pizarro et al., 2009; Chaudhary et al., 2013; Carrasco-Torres et al., 2017). This corresponds with the observed anti-oxidative activity, S phase arrest and concomitant depopulation of DNA contents/cell population in G0/G1 and G2/M phases of the cell cycle (Figure 3). This is also reflected by the decreased cell migration (Figure 5).

Fatty acids from C. volubile leaves could thus suppress tumor metastasis and/or invasion in human neuronal glioblastoma cells by the following mechanism: (1) attenuation of redox imbalance which inactivates the G0/G1 phase; (2) depopulation of DNA in the G0/G1 and G2/M phases with concurrent arrest of the S phase leading to inactivation of the cell cycle and apoptosis; and (3) Inhibition of α-chymotrypsin degradation of the extracellular matrix.

The results of the current study suggest a suppressive effect of the fatty acid extract of C. volubile leaves against tumor metastasis and/or invasion in human neuronal glioblastoma cells, thus demonstrating their preliminary therapeutic potential. This fatty acid rich extract may thus be proposed as dietary supplements for the treatment, and prevention of such ailments.

OE, NA, AN, AM, GE, and MZ: conceived and designed the project. OE, AN, and NA: performed the MTT, apoptosis, transwell migration, and cell cycle assays. OE and AM: performed the oxidative stress assay. OE, AM, AO, and MZ: performed the statistical analysis. All authors were involved in the interpretation of results. OE, AM, GE, and MZ wrote the manuscript. All authors revised the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.