Figure 7. Gene microarray and pathway analyses.
(A) Microarray results. (B) Functional analysis related to the most upregulated genes (B) and those showing no change (C).
Degradation of Ribosomal RNA (rRNA)
In order to investigate the causes of the significant downregulation of mRNA, I first examined the band pattern of rRNA in the gel after total RNA extraction. The normal pattern of 2 clear 28S and 18S rRNA bands appeared in the range of 0–100 µg/mL MOL; however, slight and significant degradation of rRNA was observed for the 200 µg/mL and 300 µg/mL MOL treatments, respectively (Figure 8, left panel). This dose-dependent rRNA degradation led to the appearance of a new band (designated as “1”) between the 28S and 18S rRNA bands on the gel. Additionally, a new band (designated as “2”) from the 300 µg/mL MOL-treated cells was observed just below the 18S rRNA band. The origin of these new bands needs to be investigated further. The data indicate that significant downregulation of most of the genes resulted from both the decrease in normal RNA patterns and the increase in abnormal RNA patterns.
Figure 8. Degradation of rRNA.
Normal rRNA shows 2 major bands, 28S and 18S, on the agarose gel. Degraded rRNAs are located between the 28S and 18S rRNA bands (designated as “1”) and below (designated as “2”) the 18S rRNA. In order to investigate the atypical band seen for 300 µg/mL MOL-treated cells in the left panel, more dilute rRNA was separated by gel electrophoresis.
Comparison of Cell Viability
The MOL extract was tested for cytotoxic effects against normal cells by using MTT analysis (Figure 9). The analysis showed the considerable toxicity associated with the soluble MOL extract in the cancer cell line, A549 (~65% for 200 µg/mL). At the same concentration, the viability of the normal cell line (i.e., COS-7) exposed to the MOL extract showed minor cytotoxicity, demonstrating that normal cells are more resistant to the extract than cancer cells. While MOL evoked death of all the A549 cells above 300 µg/mL MOL, normal COS-7 cells showed a gradual decrease in cell viability, with over 50% survival seen even at 600 µg/mL MOL. In conclusion, MOL is highly specific against cancer cells.
Figure 9. Comparison of cell proliferation between normal and cancer cells.
The average values for 3 independent experiments are shown.
Despite the recent advancements in chemotherapeutics, chemotherapy is still associated with severe adverse effects such as nephrotoxicity, nausea, hair loss, skin irritation, anemia, and infertility , . Therefore, naturally occurring anticancer compounds from natural plants, especially those with low toxicity and high potency, have important implications for chemotherapy and chemoprevention.
Natural plants have drawn much attention for their pharmacological effects in the treatment and prevention of various diseases due to their high biocompatibility, low toxicity, and potential biological activity . Among them, edible M. oleifera is known to be a rich source of various nutrients and has therefore been regarded as an important crop . Additionally, the plant has been considered a multipurpose plant that could be used as a medicinal plant; vegetable; animal fodder; and a source of vegetable oil, which is used in condiments and the manufacture of perfumes, cosmetics, and hair care products , . Among the various parts of M. oleifera, the roots, pods, seeds, and gum are used to treat rheumatism and to relieve edema and arthritis , , ; the leaves have been reported to have hypocholesterolemic , hepatoprotective , , , antimicrobial , anti-gastric ulcer , antiviral , and hypotensive  effects and have been used in the prevention of cardiovascular diseases and as antioxidant , . However, because of the importance and versatility of the plant, most of the published reports focused on compositional analysis and on its use as a dietary supplement. M. oleifera is also used as a health food and cosmetic in many countries, but its medicinal effects have not yet been well established. In particular, only a few studies have been performed on its use as an anticancer drug, and most of them are limited to solvent extracts.
Bioactive compounds from plant materials are typically recovered with different extraction techniques depending on their chemical properties and distribution in the plant . The most frequently used technique for the recovery of active compounds from plants is solvent extraction . Ethanol, methanol, acetone, and ethyl acetate have been widely used to extract bioactive compounds from various plants, including M. oleifera leaves , .
In the field of anticancer drug discovery and development process, compounds with the highest anticancer activities often have bulky hydrophobic groups within their chemical structures, rendering them water insoluble . Low water solubility leads to both formulation issues and serious therapeutic challenges. Administering the poorly soluble drug candidate intravenously might result in serious complications such as embolism and respiratory system failure due to the precipitation of the drug , while poor absorption would result from extravascular dosing . Therefore, increasing water solubility and/or developing soluble bioactive compounds with high anticancer activities have attracted increasing attention. In this study, I focused on the new water-soluble MOL extracts and examined its potential as an anticancer drug candidate.
I also demonstrated that concentrations above 300 µg/mL of the cold water (4°C)-soluble MOL extract showed a notable antiproliferative effect in in vitro experiments performed using the A549 lung cancer cell line (Figures 1, 2, 3, and 4). Additionally, the MOL extract had an wide spectrum of antiproliferative effect in different cancer cells (Figure 10). As observed by western blot and RT-PCR analysis, many oncogenes and iPS-induction genes were considerably downregulated in A549 cells treated with the extract, demonstrating that the soluble MOL extract can effectively prevent cancer cell proliferation. Although the MOL extract induced severe cell cytotoxicity in A549 cancer cells, however it was not the case anymore in normal cells. As shown in Figure 9, the MOL extract showed less cytotoxicity in normal cells, COS-7, than in cancer cells, A549, demonstrating that normal cells are more resistant to the extract than cancer cells. The reason why the difference in the cell cytotoxicity between cancer cells and normal cells is not clear at this time, but I think complex effects caused by some compounds in the extract can protect normal cells from severe cytotoxicity. Overall, these data suggest that the cold water (4°C)-soluble MOL extract may become a good candidate for anticancer therapy with high specificity and less adverse effects. In conclusion, I demonstrated that the soluble MOL extract may have be a new promising candidate for a natural anticancer drug. Further studies are required in this regard.
Figure 10. FACS analysis and Inhibitory effect of MOL treatment on the proliferation of different cancer cells.
Induction of apoptosis (A) and Colony-formation ability (B) by the MOL extract in different cancer cells was analyzed by FACS and by cologenic assay.
Interestingly, more than 99% of the genes in the MOL extract-treated cells did not show changes or were downregulated more than 2 times compared to the control, and only around 1% was upregulated more than 2 times compared to the control (Figure 7). Additionally, protein expression also indicated downregulation (Figure 6). SDS-PAGE gene analysis demonstrated that proteins of the chaperon family were upregulated, indicating that cells treated with MOL had problems in normal translation and were exposed to higher stress levels (Table 2). Because an abnormal rRNA pattern was observed by gel electrophoresis, downregulation of many genes and proteins may occur because of the increase in abnormal RNA through severe RNA degradation (Figure 8). I concluded that the MOL extract induced rRNA degradation, thus showed cell cytoxicity in cancer cells. Several experiments has been designed and conducted to explain the outstanding results of abnormal rRNA degradation, but all the efforts have failed. However, it is evident that the clear bands that the reviewer mentioned were not originated from random cleavage but from the cleavage of the specific site within rRNA or from the appearance of new rRNA, for example, mitochondrial rRNA or unidentified something else. Further studies will be required including sequence analysis of the new bands.
Table 2. Upregualted stress proteins by MOL extract.
Recently, Tiloke et al.  have reported that aqueous MOL extract has an antiproliferative effect on cancerous human alveolar epithelial cells. In the study, aqueous MOL extract was prepared “by boiling” the crushed M. oleifera leaves. They suggested that the aqueous MOL extract showed approximately 33% inhibition of cell viability in the MOL-treated group compared with the untreated group. Compared to the data, I had much greater inhibition rate of up to 90% by using cold-MOL extract (see Figure 2). The possible difference in anticancer activities between cold- and hot-DW treated MOL extract might be resulted from the heat inactivation of some bioactive molecules within M. olefeira leaves, but obvious reason needs to be clarified through further research. In addition, further studies about the anticancer effect among MOL extracts prepared with different temperatures on the cancer cells are also required.